Monday. Ribonucleoproteins and RNA Processing (1261

Monday. Ribonucleoproteins and RNA Processing (1261-1266)
1261
1262
REEGION ON
INFLUENCE OF 3'-UTR AND CODING
HALF-LFEM OF BOVINE
FFL. Schadacher,
ant
MCC. Neville)) Lab
Pattanajitvfllai,
Biology, Ohio Agric. Res. & Dev. Center, Ohio Sta Univ., Wooster,
LACTOFERRIN mRNA. ((S.
of
Moll
&
Dev.
Cob Health Sci Center, Denver, CO.
Dept ofPbysolooy, Umv.
R of
3e-3U
the
mNRNA destabiuaton comsess squences noted
mammary
mRNAm
lactofern(n(bLf)
ggeuedpostthairmcriptionalregulationofbLfbyNRNA degadation
raten RNA half-ife). Consuuction of expressonvectors(pcDNA) with 5'comnplet cDNAs
(
a
lac
hLn
corone
tarection allowed study of the effects of
for bovine or aman
andW
m on RNA half-ife
m of RNA segments
(COS cells) ard potein
exchange or deletion
nse
1
ando
e(ssion(COS(COMMA-(D)
mammary cel). Exchange
(KH) m ) RNAs, and deleton of
haman growth hrmnone
of
the
between bLf
33-UTRs
Ohio and
bovin
e
-l
orpbmarybovin
am!
codingcregionsegments (t334-1021)ofbLf, alloweddetenninationofeffectsofbLfmRNA
regions on mRNA
half-life in tansfected celia
and
at 0, 6,12, 18,
24
hrs
after inhibition of
of the 3'-R of
rnarcription by 5,6-dichlorobenzimidazdle-Rboside(DRB). Rplacement
hG
of bLf dramaticaly rdued the expression
of H protein, decreased
33-UTR
hGH with
the
15.6+/
the hG
mRNA, and shortenedtm
of mH RNA from 31.3+/-7.9 Ins to
the
H 3'-UTR onto bLf increared bLf protein
Susisltution of IG
expession and the amount ofbLf mRNA in the cel but decreasedthe t,, of bLf mRNA from
31.3+/-14 his to 11.9+/-2.8 hb, niggefing the coding rgion to be criticaly sensitive to
nalase degradaionn Deletion of the distal N-lobe coding region (nt 334-1021) from bLf
Tof
retducedthe t,, of the bLf 334-1021
ntivebLf or ofGH
cDNA havg
the 3'-eithero
deletionmRNAs to 5.8+1-1.1
if with bLf 3'-UR and 6.25+/-1.8 his if with bOl
3'-UTR.
m
of
and
b
Lf/C-lobe
hLf RNAs were
hLf
chimeric N-lobe
In corast, the half-ife
an! 16.2+/-1.9 her, respectively. 33-UTR
of bLf mRNA conrains strong destabilization
The
if
mabdsted
onto stable nRNAs (hOH) but is a stabilizer sn the
signals as predicted
of bLf nRNA itself, as is the distal N-Lobe coding egion of bLf mRNA, of the naucaw
sensitive the C-Lobe coding region. Elevate bLf protein expression in mammay cells
vemus native bLf cDNA
ttarsfected with bLf cDNA lstiuted with thehGH3'3-U
so ggests pnotrarucriptional regulation for Lf mRNA in mammary as for COS-1 cells.
&
Natnl.
Res. Board.
Dairy Prom
Supported by the
the
217a
level
of
GH
H
6.1 hrs as expected.
his
13+1+03
context
1263
ANALYSIS OF CIS-REGUIATORY ELEMENTS DIRECTING ALTERNATIVE
PRE-mRNA SPLICING OF THE MYOSIN HEAVY CHAIN GENE TRANSCRIPT
IN DROSOPHILA.
D.M. Standiford, M.B. Davis and C.P. Emerson, Jr.
Dept. of Cell and Developmental Biology, University of
Pennsylvania School ofMedicine, Philadelphia, PA 19104
Myosin heavy chain (MHC) protein isoform diversity in
Drosophila is generated through alternative splicing of the
pre-mRNA transcript from the single skeletalMhc gene. This
process is highly regulated with specificMHC isoforms being
expressed at certain developmental time and in specific muscle
types. We are exploring the mechanisms that enforce this
specificity by examining the cis-regulation of one alternative
exon set, exon 11, in tranagenic flies.
Based on sequence
comparisons of exon 11 betweenD. melanogaster andD. virilis,
we have constructed and are testing a model in which the
conserved, non-consensus 5' splice donors and a large intronic
conserved region direct splice specificity. Data will be
presented to show that the 5' donor plays a role in regulating
splicing for some but not all alternative exon lls. In addition
intronic conserved region appears necessary for the correct
specific regulation of exon lle. These data and others
suggest that at least two seperable mechanisms function to
regulate alternative exon 11 splicing. Data from a genetic
screen designed to identify mutations in genes for the transregulation of alternative splicing will also be presented.
the
tissue
A HETEROLOGOUS MODEL SYSTEM TO STUDY DEVELOPMENTAL
SPLICING SWITCHES IN ERYTHROID PROTEIN 4.1 PRE-mRNA
((K. Aoyagi and J.G. Conboy)) Life Sciences Division, Lawrence Berkeley
Laboratory, University of Clifrniia, Berkeley, CA 94720.
distn
Alterative pre-mRNA splicin regulates expression of functionally
isoforrs
of the stucturl protein 4.1 duing erythrid differentiation. Splicing of 4.1 pre-mRNA
in mouse is developmentally regulated such
exon 16 is compleely sipped in early
erythroid precrsors. but is
spliced in at the late eryhroblast stage. Because
exon 16 encodes pait of the critical spiectrin-actin binding domain, only in late erithroid
cells does 4.1 mechanically strengthen the erythroid membrane. This study was
undertaken to explore regulated splicing of a model 4.1 pre-mRNA in simplified
pre-mRNA into
heterologous system. Microinjection of a synthetic three-exon 4.1 mRNA
products,
analysis of spliced
Xenopru oocyte nuclei, followed by
revealed accurate alterative splicing of 4.1 pre-mRNA into two mRNA species,
srnmgly
RT/PCR
exon 16. Thus, the oocyte system recognizes the cis-acting sequences
plus/minus
which regulate spicing of mouse exon
fuuncional
aanaysis of
166 and should facilitate
such sequences via site-directed mutagenesis. Phylogenetic clues as to regulatory
sequenc were sough t by comparison of frog and mouse gene sequene arund exon
16. Isntrons of both have consensus
3' slice sites and putative brnch points, but the
splice sites appear weak and may be rsponsible for exon 16 skipping. No evidence
for other conserved cis-regulatory sequences was detected in the intrn. In contrast,
exon 16 sequences of both species contaied a purine-rich 5' domain followed by a
nt region of identity, which is also conserved in the human, dog, and chicken genes.
This extraordia
conservation suggests that exon sequences are umporannt
splicing
in
model pre-mRNAs We
regulation, as recenty described for alternative exonsotthr
also found that the efficiency of exon 16 splicing was concentration-dependent, and
could be altered by co-expression in oocytes of trans-acting splicing factors such as
SF2/ASF. Webelieve this system pprovde
wll
important insights into the mechanism
of alterative splicing which mediates a functionally important splicing switch duTing
erythrdiddevelopmedL
5
42
1264
IN VITRO ANALYSIS OF A VHS-DEPENDENT RIBONUCLEASE
ACTIVITY IN HERPES SIMPLEX VIRIONS. ((B.D. Zelus,
R.S. Stewart and J. Ross)) McArdle Laboratory for
Cancer Research, University of Wisconsin, Madison WI
53706. (Spon. by C.E. Somers)
The infection of permissive cells by herpes simplex
virus results in the rapid degradation of cellular
mRNAs. Genetic evidence suggests that the virion
host shutoff (vhs) protein, a 58 kDal polypeptide
encoded by the UL41 ORF, is responsible for the
observed destabilization of host cell mRNA. We have
used soluble extracts of purified herpes virions and
an in vitromRNA decay assay system to investigate
the mechanism of vhs action. Extracts of wild-type
virions accelerate the decay of both polysomal and in
vitro synthesized mRNAs. Extracts from vhs-deficient
mutant virions do not. Polyclonal antibodies raised
against bacterially expressed vhs protein inhibit the
ribonucleolytic activity of these extracts and can
immunoprecepitate ribonuclease activity.
Electrophoretic analysis of the decay products of in
synthesized globin mRNA reveals specific decay
vitro
intermediates resulting from ribonuclease activity in
the 3' portion of the mRNA. These results suggest
that the vhs protein is a ribonuclease and provide a
system with which to analyze its mechanism of action
and reveal why mRNA, but not rRNA or tRNA, is
targeted for destruction.
1265
1266
DISRUPTION OF THE MAJOR VAULT PROTEIN B GENE (mvpB) AND
IDENTIFICATION OF A GROWTH DEFECT PHENOTYPE IN MVP
NEGATIVE LINES. ((S.K. Vasu and L.H. Rome)) Departmt of Biological
Chemistry, UCLA School of Medicine, Los Angeles, CA 90024. (Spon. by L.H.
Rome)
VAULT RNA AND ITS ASSOCIATED PROTEINS. ((V.A.
Kickhoefer, and L.H. Rome)) Department of Biological Chemistry,
UCLA School of Medicine, Los Angeles, CA 90024. (Spon. by V.A.
Kickhoefer.)
Vaults
are
large cytoplasmic ribonucleoprotein
Vaults
particles of unknown function.
at
Dictyostelium vault proteins migrate on SDS-PAGE gels as two bands,
kDa (MvpA) and the other at 92 kDa (MvpB). An MvpB cDNA clone
one
94
was
isolated by immuno-screening
Dictyos:elium vault
Dicystelium
expression library with an antipolyclonal antibody. The cDNA was identified as MvpB as it
a
shares 60% identity at the amino acid level with the previously cloned MvpA
has been
(Vasu et al., 1993, K 268, 15356-15360). The single mvpB
disrupted in both wild-type and mvpA- genetic backgrounds. Although the mutant
that
show
loss
of
MvpA
and/or
MvpB
interferes
with
vault
they
viable,
lines are
fiuntion sufficiently to impede growth under conditions of nutritional stress.
Ovoid structures have been isolated from single mvp- mutant lines and shown to
gen
represent what remains of vault structure in these cells. Ovoid structures have
also been isolated from the mvpA- mvpB- line, M7, and characterized by electon
microscopy and SDS-PAGE. A novel protein of MW 92 kDa (MvpC) copurifies
with the ovoid structures. Antibodies directed against nornal vaults do not
the mvpB
reognize this protein suggesting that this protein might be induced
in
line.
(Supported by USPHS grant GM 38097)
are
ubiquitous, evolutionarily conserved,
cytoplasmic
particles of unknown function. The 13-MDa ovoid
ribonucleoprotein
vauft
has bifold
and each half can open into a
particle
symmetry,
flower-like structure, which contains eight petals surrounding a
central ring. Vaults purified from rat liver consist of four protein
species (210, 192, 104, and 56 kDa) and a 141-nt RNA polymerase
lIl transcribed RNA (vRNA). The 104-kDa protein constitutes >70%
of the total vault mass. We have characterized the distribution of
the vRNA by fractionation of cellular extracts. Whereas all of the
major vault protein is associated with the vault particle and pellets
amount of
with the microsomal fraction (100,000xg), a significant
the vRNA is present in the
fraction. These results
demonstrate that not all of the vRNA is associated with the vault
particle. We have determined by UV-crosslinking that the vRNA in
the S100 fraction is associated with protein in two different RNP
complexes. In label transfer experiments we have identified a 50
kDa protein present in one of the vRNA complexes. We are
currently purifying the vRNA binding proteins. (Supported by
USPHS Grant GM38097).
S100
218a
Stress Response Genes (1267-1272). Monday
1267
1268
VARIATIONS IN FERRITIN SUBUNIT SIZE CORRELATE WITH SURVIVABILITY OF YEAST TO HEAT AND OXIDATIVE STRESS. ((E.C.
Thomborrow, M. Donovan, and A.A. Infante)) Department of Molecular Biology and
Biochemistry, Wesleyan University, Middletown, CT 06459-0175.
CLONING OF STRESS-INDUCIBLE GENES OF S. porbe. ((M.
Morimyo, E. Hongo, K Mita, H. Hama-Inaba, and I. Macbids))
National Institute of Radiological Sciences, Chiba 263, JAPAN
S.cerevsuiae
porbe which
of
To elucidate the repair mechanisms S.
exhibits extreme resistance to radiation, stress-inducible genes
were cloned. Firstly, cloning vector, pYMM5, was constructed
by ligation of pBR322 vector with ars gene of S. porbe, URA3,
and reporter gene lacZ carrying multi-cloning site on itt
upstream site. Secondly, the fusion S. porbe DNA library was
made by ligation of BarHI and CIAP treated pYMM5 vector
with MboI digested and fractionated S. porbe DNA. Cells of a
wild-type strain were transformed with these plasmids by Liacetate or electroporation treatment at a frequency of 106
transformants/g.g DNA. Thirdly, transformants exhibiting pale
blue color on Xgal plates were obtained at about 1/200, and
were replica-plated on Xgal plates. They were treated with
various stresses such as UV, X-rays heat and oxygen radicals.
Those colonies which showed darker blue color after stress
treatment, were obtained at about 1/100. Plasmid DNA were
extracted from 71 candidates and were classified into 26
groups by determining DNA sequences of S. porbe region
flanking to multi-cloning site. Many of them were new genes
and were induced by various stresses, indicating that they
were involved in the later cascade of stress-induction.
is
a
facultative anaerobe, capable of metabolism by both fermentation
Weatem
and reapiration. We report a protein in S.cerevisie that is recognized
blots specifically by an anti-homse spleen ferritin antibody. The molecular weight of
the putative ferritin subunit, however, appears to change depending on the mode of
on
yeast metabolism and during heat ahock. During normal fermentation the yeast ferritin
migrates on SDS-PAGE at 42 kD. When the yeast are respiring, ferritin migrates at
a poaition consistent with a 19 kD protein. Further, if fermenting cells are subjected
protein is no longer observed
to a one hour mild heat shock at 38 C, the
at 42 kD; rather, its migration is shifted to 19 kD. Additional studies have shown
ferritin
that these transitions in ferritin migration rate correlate to the ability of the yeast to
survive severe 52 C insult. Studies show that respiring cells-which contain the 19 kD
ferritin-survive (as measured by colony forming units) 80-90% better than fermenting
a 5
stress at 52 C. Prior to the 52
cells-which contain the 42 kD
C stress, if fermenting cells are heat shocked at 38 C for one hour, causing a ferritin
from
0-10% to 80-100%. Thus,
shift from 42 kD to 19kD, their survivability increases
a
ferritin-foUowing min
a close correlation between the presence of the 19 kD
and the coils' ability to survive a 52 C heat stress. Similar results have
been obtained using hydrogen peroxide as the stress. Compared to cells with the 42
kD ferritin, yeast possessing the 19 kD form of the protein have a greater survival rate
when subjected to mM quantities of hydrogen peroxide.
we
have shown that there is
ferritin subunit
1269
1270
CARDIAC MYOSIN LIGHT CHAIN GENE IS MODULATED
ACTIVATION OF
INVOLVE THE
COILED-COIL STRUCTURE.
and
OF HUMAN HEAT SHOCK TRANSCsls?ON
ABIIITY
DNA-BINDING FROM
TRI-TRANDED
AN INTRAMOLECULAR TO AN
TRANSMON
DD
adVRoelly))
of RIaU.milty
R.
Zo,. IL B.e,. 0.
UUaiveyof Miami SdioolofMddciic Miam, 3311i.
Bioby.i.o,yBipbysU.
M.iacut b.hag.
BY STRESS (EDUARDO MASCARENO) ANATOMY & CELL
BIOLOGY DEPARTMENT, SUNY at Brooklyn, NY 11203
(spon. by M.A.Q.Slddlqui)
The response ofanimals and humans to stress is reflected at
the cellular level by an increment in heat-shock gene
expression. Studies in our laboratory have indicated that
the promoter of the MLC2 gene is, among others, the target
for stress-.related transcription activator proteins, the heat
shock factors (HFSs). We have observed that the heat shock
responsive sequence elements (HSEs) located in the
proximal promoter of MLC2 gene, binds the HSFs in primary
cardiac cells in culture following stress induced heat shock
recombinants
MLC2
promoter/reporter
treatment.
transfected into primary cardiac cells, respond positively
upon heat shock treatment but not when HSEs were deleted
from the promoter. These results, therefore, strongly
suggests a role for the HSFs in targeting stress responsive
genes such as cardiac MLC2, indicating a new and novel
molecular mechanism of stress-mediated modulation of
gene
Hat
d
mot
(39-42C)
..y
ds,q,a
MAY
FL
ad
of1 bum
ofg.hs.
.sock
hYdrpbobic r_ (b,c
Het
FACTOR
INTERR1LBCULAR
aM'.
I.
zsppa or
12
be
1-3).La
a
cti_.
and
oci
by bet
Xc
umd
lumA..I
HSFI
baugb
t.e
-dhd
genesa
iza
n
mbiliz id by,
.sracbo
haS-o-tr5m
bHSFIr
(370C). HSFI
a
oeU
ba
binding bity. HHSFIexp x d Xapoocyso.n6
a_cripa
ad
HSE
.,qis
ass..,
containing dee
to bc
pp
ima
in
M_.5
& b.p70.
6..srcio.
S6.
(Hem
E_at)
DNA-
oNA-DNAbiding l ias
" .g.imi o. B.. dc.
DNA-bading form upon expomm of oocyI. to bed ock (35-37C
HSF DNA-bindg sivity l., ad .a&hHSFI anibody doe not f..ogaie 1.wp.s HSF. w. emplyed
anIy.i of
a yq d
for m.,.ppag fgio
tam requid for dke .ala...ac of dh .am aaeic sdo.
hHS
ins
e
of .asil deikdi
bht -reg,atd DNA-bnding biity .ad trknerbti of hHSFI. lig a
kg bHSFI
Xeopw ooyt., duhe dierese, goa. contain" iZI-3, we identifd dat wm crit for
of
of LZ anaurm by amag6gdy. Xcg
..ive moomer atda
on-beat hbock en aer .um (200C). Disrupt
to a teric,
..
hydr.pbobic rdue aboliabectd beti
wid susitu of zpper reidu
ek
s ame
tn..f.sien
.A
hydopob.c
nos-hat
owe u -der
acdie
an
.eil. T1
iy
involog
vin
eracbo.
ll
ma
tedt d.
and
LZ
1.uve
wh
may
Tl
sadwia...
oodd.
shz*
ve
qpd-ryI_ige
.6e
g
am
HSF
naata hckF
amays widhub..
u ,i.ntA r
ofbHSFIHSE DNA-badig .biity.W
LZ12t dasg.kbdfor HSE DNA-bind
,guio
bHSFI
form
r
was
*
s.
=.ad
by
baiied by
csdid-coiL
o
f HSFI byf.iibatingcoop. tivce badagof_asIc
T,ri.rtio emb DNA-bindingfbasi.o
cead
hHSFi DNA-bidcg
wbs.
with
to thU HSE motif.to
bindng
boundto LcxA bid-" ia
resultg
cbbmr_
sithtUe DNA-bindag do.a of LcxA. b.seai
tik
d wi tU.iagrit of dbHSFILZ
bert-reguladflab. Sagk cido
coadiauivy oCigom.,, aWd OA-bidag.LteA bound_blyto DNAonly *die butast s_ i our
DNA-bidig
LZ of bHSFIm
of
Tbarfore. dU hydmopbobic intac
D
NA-bndingabiky.
doai bat-mp,6d
may
expression.
DNA-
U.e
doa.
nots,..
great
apte..
a
am
doma w
ab.iti.a
i
a
g
rader.d
y
a
a.
tUe Uee
confer
oa
b_..gA
1272
1271
HEAT SHOCK AND DEFLAGELLATION INDUCE A SHORT-LIVED
mRNA IN CHLAMYDOMONAS. ((J.A.J. Burrows, P. Liggit, and E.J.
Department of Biology, University of Nevada, Reno, NV 89557.
HSP70
Baker))
Using 3' UTR probe specific for an hsp70 mRNA previously shown to be
inducible by light as well as heat shock (Gromoff et al., Mol. Cell. Biol. 9: 3911,
1989), we find that this hsp7O mRNA is also inducible by flagellar amputation
(deflagellation). Whereas induction of,the hsp70 mRNA in response to heat
shock is not blocked in the presence of cydoheximide (a primary response), its
induction in response to deflagellation is prevented, implying the existence of
two different induction pathways. This finding allows us to study the posttranscriptional regulation of this mRNA in two different cytoplasmic
environments. The hsp70 mRNA is short-lived under both conditions. Heatshock
at 370C induces peak levels of hsp70 mRNA within 15 minutes. The mRNA
decays rapidly back to low basal levels within 2 hours, exhibiting a half-life of
15-20 minutes. Unlike most short-lived mRNAs, inhibition of protein synthesis
does not significantly stabilize the induced hsp7O mRNA. Hsp70 mRNA induced
by deflagellation affains peak levels within 30 minutes, and also returns to basal
levels within 2 hours with a half-life of 15-20 minutes. The relative instability
of polysome-bound hsp70 mRNA is reproducible in extracts prepared from heat
shocked or deflagellated cells. Analysis of poly(A) metabolism of the
heatshock-induced hsp7O mRNA reveals that unusually rapid poly(A) tail
shortening precedes its degradation, and this rapid shortening is independent of
ongoing protein synthesis. Within 40 minutes of heat shock, >50% of the induced
mRNA is poly(A)-deficient. The measurement of poly(A) shortening rates for
deflagellation-induced hsp70 is in progress. Poly(A) tail shortening has recentlY
been shown to play a key role in the post-transcriptional regulation of hsp7O
mRNA in Drosophila (Dellavalle et al. Mol. Cell. Biol. 14: 3646, 1994),
suggesting that regulated poly(A) metabolism may be a highly conserved
CEREVISIAE
70 kDA MOLECULAR
THE ROLE OF SACCHAROMYCES
CHAPERONES IN PROTEIN TRANSLOCATION. ((Nancy M. Wang and
William J. Chirico)) Department of Anatomy and Cell Biology,
Science Center at Brooklyn, Brooklyn, NY 11203
SUNY-Health
a
property of this mRNA.
70 kDa heat shock proteins (Hsp7O) have been shown to act as
molecular chaperones in many cellular activities including protein folding,
One function is
protein assembly, and the uptake of proteins into organelles. proteins
into the
to facilitate the post-translational translocation of
which 70 kDa
by
mechanism
the
To
reticulum
explore
(ER).
endoplasmic
heat shock proteins facilitate translocation, we chose to study their interactions
(ppaf), a precursor of the a
with prepro-a-factor in vitro.
cerevisiae, can be post-translationally
mating pheromone of
translocated across the endoplasmic reticulum membrane in vitro. A
biochemical dissection of this process would be greatly aided by the
the entire coding
availability of sufficient amounts of pure ppxf. Therefore,
in the
region of ppaf was cloned into an expression plasmid that resulted
placement of six histidine codons at its 3' end. Histidine tags allow purification
of proteins from E. coli in one step using nickel affinity chromatography.
in an E. coli SecY
resulting plasmid was
The
mutant to preserve the signal sequence of the overexpressed protein. The
conditions. Upon
histidine-tagged ppaf was purified under
and
dilution into
containing yeast microsomes, it was translocated
glycosylated. However, the amount of translocation decreased by 50%a when
yeast
Ssalp,
If
refolded histidine-tagged ppaf was used as a substrate.
level of
cytosolic Hsp7O, was present during the refolding reaction, the original
Ssalp or Ydjlp, a 45 kDa heat shock
translocation was restored.
presecretory
Prepro-a-factor
Saccharomyces
temperature-sensitive
transformed
denaturing
reactions
Furthermore,
protein
that modulates
Ssalp,
formed stable
complexes
with
histidine-tagged
ppaf. Together these results suggest that 70 kDa chaperones facilitate posttranslational translocation by binding presecretory proteins and maintaining
them in
a
translocation-competent
conformation.
Monday.
Stress
Response
Genes
(1273-1274)
1273
EXPRESSION OF HUMAN HEAT SHOCK PROTEIN 27 kD IN BOVINE
ENDOTHELIAL CELLS ALTERS CELL GROW1H
((RS Piotwicz', LE
Weba&, E Hickey2, and EG Levin')) 'Dept. of Mol. and Exp. Medicine, The
Scripps ResearchInstitue, La Jolla CA, 92121; 2The Univ. of Nevada, Reno, NV,
89557.
Heat shock protein of molecular weight 27 kD (HSP27)
exhibib
ehancdexp
and phosphoylation in endothelial celis exposed to activaing agents, ic stimuli
el
have bon
or stessfl conditions. Bovine pulmonm y erial endo l
tsuzsfbcted with either the complete human HSP27gum or a mutageniard gme which
lacks key phosphoyLion sites (seine residues 15, 78 and 82). The enogeo
bovin
analog is sightly smaller (MW 25 kD), emsts as ree isofns ad fracionates witn
.
staining of the
a hypotonic rleasate and an NP40 homog
Immunofuoe
bovine
HSP25
show
puctatio
associated with
plasma membme along with
diffuse cytosolic staining. Both exogenous gene products co-localize wihi the
HSP2S. The wivdtype human HSP27 is phosphosylated in respons to
endogenous
similar
agonists which rsult in edogenuHSP25 phosphorylion, genati
phospho-isoforms. Tritiated thymidine inporon into thiten clon exp
wildtype human HSP27 was measured and compared to the meain obtained fom a
panel of eleven vector control cloes. On average, the HSP27 exprsing clones
in
amomunt of triated thymidine inrpona-ed.
of celis expressingthe mutgenied human HSP27 (which can not
be phosphorylated) grw at significantly siower sates and exhibited masked decreases
in thymide incoposation. These data suggest a causive relaionship betwee HSP27
expreson/phosphorylton ad the enhacd growth of endodheal cels in response
to agents (immune andthrombogenic fctors) and even (wounding) which activate the
a
three-fold
In contsast, culs
219a
1274
HEAT SHOCK PROTEIN (HSP27) IN MELANOCYTES. (T. Dizon,
D.A. Reilly, M.D.
) Departments of Plastic Surgesy
and Dermatology, University of California Davis Medical Center,
Sacramento, CA 95817. (Spon. by R.R Isseroff.)
Melanocytes found in the epidermiis are responsble for an individual's
skin color. Because of their superficial location, these cells react to such
How thes cells react to
V light and heat.
of our invetigaton. In particular we have
protein expression after exposure to an
environm ental streses as
such sesses is the topic
focused on heat shock
environmental
stress.
Heat shock
proteins
are
synthesized within human
fibroblasts and keratnocytes after exposure to stress. It has been
serve to protct the cel from
hypotheszed that these protei
environmn insults. Using indirect immunofluoresence, we determined
the cytoplasmic presence and location of low molecular weight, 27Kdprotein (HSP27) in unstrssed cells. Using sodium arsenite as an oxidative
stress
inducer, HSP27 was then observed to translocate from the
cytoplasm to the nucleus. In contrst, in the unteated control cells,
HSP27 remained in the cytoplasn. Isoclectric focusing gel analysis was
used to identify two different isofonns of HSP27. There is also an
increase in the amount of HSP27 after stress. The precise role of heat
shock proteins in meanocytes has yet to be documented, however given
our initial characteization of these proteins we are one step closer to
understanding this most remarkable cell the meanocyte.
endoelium.
Sperm and Spermatogenesis II(1275-1278)
12T5
12TS
IMIJNOCYTOCHE3ICAL LOCALIZATION OF SPE-4, A PROTEIN REQUIRED
FOR SPERMATOGENESIS IN C.ELEGANS.((P.Nichele Arduengo, O.K.
Appleberry, and S.W. L'Hernault)) Dept of Biology, Emory
University, Atlanta,GA 30322
IDENTIFICATION OF MULTIPLE PROTEASES IN GERM CELL-CONDMONED
MEDIUM (GCCM) THAT AFFECT SERTOU CELI (SC) SECRETORY FUNCTION
((G. R. Arvinda, C. Pineam B. egon, C.W. Badin ad C. Yan aimn)) The Populaion
Couil, 1230 York Avenue, New York, NY 10021.
Germ odl (GC) developme in the testis ivelves exensve pwiapaton ad coordnaion
of poteme
ad prowe inimbito that allow the traocaton of GC from the bas to the
adluminal crmps
and the rease of m re spermatoa into the seminifeus tubul
Spermatogenesis in C. elegans uses unusual organelles, called
the fibrous body-membranous organelle cosplexes (FB-NO), to
prepackage and deliver macromolecules to spermatids during
their formation. The FB-NOs are segregated non-randomly into
the spermatid during the second meiotic division while
non-essential components are discarded in a structure called
the residual body (RB). Mutations in the gene spe-4
(spermatogenesis defective) disrupt FB-HO morphogenesis and
cytokinesis during spermatogenesis. Consequently, spe-4
mutants arrest spermatogenesis as terminal spermatocytes that
contain four haploid nuclei; spermatids are never formed. The
spe-4 gene is predicted to encode a novel 465 amino acid
transmembrane protein. The ultrastructural phenotype of spe-4
cells suggests that spe-4 might reside in the FB-MO, but it
could possibly be found in the plasma membrane also. To
localize SPE-4, polyclonal antisera were generated against an
114 a.a. hydrophillic portion of SPE-4 fused to GST. Preliminary results reveal sperm specific punctate staining of
wildtype sperm during all stages of spermatogenesis. This is
consistant with FB-MO localization. Additionally, in wildtype
sperm, signal is excluded from the residual body, as predicted for a protein located within the FB-NO. Future
studies will look at SPE-4 localization in the six
alleles currently available.
lu1n _ile
mntining the integrity of the bkod-1seis bwsier. Wehaveexmined if peimmy
clture
of GC roel
_e any protes using ILSI-colagen
substrate ins liquid-phse
aay. When GCCM was prpeduo poedure involving trypization 26kDap---n
tha ws purfied to appwent hemngncqity by sequential HPLC frm GCCM which yield a
w
a
sdendnt
inhibitio
on
Seatli
el
(SC) testin
film
secretion wa
subequendy
shown
to
trypsin
to
oine
trypsin by GC and that typin at doses betw
sewetion
protease
10 pg ad I
>g/ml indeed inhibited SC testin
pdnmy cultures of SC, conclude that this
aoigated from the typsn used for the GCCM prepuatoL To further examine
in
an
is
Witro
bioasy system using
whetdr other factonwe
GC from ahult rat tese by
ent
we
GCCM that afect SC
echicsl procetur withdwo
soeerery function,
the
we have
ueof ay enzymatc
prepued
_tatments
codition at 32 'C for 20 hi while nuntemning cdl viabiity
of gter tha 90% at the end of the culture pesiod. When a 10-lite batch of GCCM was
frcionated by prepswative anion-exchange HPLC, three peas of ptease activities were
detcted togete with three peas of biocativity that modulate SC testin secretion. Two of
these protease activities were co-eluted with fractions that modulated SC testin saretian
whras the third out did not ovesap. One of thee proteaes designated GC-1 that cx
stidmle SC testin sewemoa hs been puried to sptw hemnogeneity by sequentia BPLC
using C8, C18, ad CCI8 reveed-ph solums which displayed appun Mr of 30,000
on SDS-polyacrylnide gd ude
ucig conditions. In smmary, GCCM prepared by
mechaoical
without eazymatic teatme
centais multiple biological factors
affec SC testin eetion, seme of wich also edhibit prose acivity when daemined
supported in pst by
NIH gr
aay. Tis wkw
HD-13341.
ad cultured
seun-free
a
cedures
vitro
a
177
1278
EPrITlLIAL(E)- AD NEURAL(N)-CADHERIN PES SION IN
THE DEVELOPING RAT TESTI AND OVARY. ((J-C Wul, L-FH Linl,
MJ. Wheelock2, KR. Johnson2, and RM. DePhilipl)) lDeptment of
Cell Biology, Neurobiology & Anstomy, Ohio State University, Columbus,
OH 43210 and 2Depa1ment of Biology, University of Toledo, Toldo, OH
43606.
GENETIC CONTROL OF GERM LINE RENEWAL IN THE
DROSOPHILA TESTIS ((G. Hime, M. Fogarty, M.T: Fuller))
Deparm nt of Developmental Biology, Stanford University School of
Cadherins have not yet been localized at contac
stes betwee Seoli cells
ublot analysis were
scopy and
ncen
in the testis. Imnlh
used to exploe the exWession of epitheial(E) and neural(N)-ca rin
during the period of SaWfi cel (blood-tsts) barrier fomation. Throughout
the frst four week of postnal developmet, E-cadherin was assocated
win
of the
seninHers tubules, q il
inaecular ree testis, and intstitl cells pre ed to be Leydig cells ESertoli
between
at
conta
of
cells In
cadhein was not detcted areas
contrast, N-cadherin was llizedin de bal reon of seminiferous
tubues of 3 and 4 week old esds,
and rcatnin. N-cadherin
a-,
and the three catenins wae also locaHzed at contact sies between Serooli
in tess and
cells in culue. Cmpison between cadhn l i
ovary reveald similarities that my eprs conmon featues of function.
Germ cels in both orans expressed E-adherin, while supporting cels in
both orgns (Serli cdls and ganuksa cells, resctvely) expressed Ncadhein Interstital cells in both gonads also expressed E-cadhrin. These
results suggest roles for N-cadherin in junction fnation betwen Satoli
vent and
cells and granulosa cells and for E-cadherin in germ cell
interstial cell
egdon.
with germ
cells
ces
as were
-,
be
through direct protlin sequencing (NH2-IVGGYTXAAN). Since
falled
confirm the synthesis of
_mmunoprecipitation experments mig PS1S5-tn
porcine
Medicine, Stanford, CA 94305.
Adult germ cells in Drosophila are constantly being replaced as they
differentiate, by division of a small number of stem cells. In adult
males the 5-7 germ line stem cells per testis are located in close
physical association with the somatic hub cells at the apical tip of the
testis. As the stem cells divide, one daughter remains associated with
the hub, and maintains stem cell character, while the other daughter is
displaced from the hub and differentiates into a spermatogonial cell.
As the germ cells mature they move down the testis, such that cells at
more advanced stages of spermatogenesis are located further away
from the hub. Function of the shut off and 406 genes are required for
continued renewal of the male germ line. In adult shat off males, cells
at advanced stages of spermatogenesis including mature sperm are
present, but cells at the early stages of spermatogenesis are missing.
In newly eclosed
406 males, all testes are rudimentary in morphology, resembling
agametic testes, but most contain a small number of sperm bundles
and no early gonial cells. In wild type males, the hub cells are the
only cells in the testis apical tip that express fasciclin III, a cell
adhesion molecule. Using an antibody to fasciclin HI, we have
detemined that the hub fasciclin Im staining dis as in shat off
testes at about the same time that the germ line stops renewal, and in
406 mutants the hub becomes disorganized after the larval stage. This
may indicate a role of the soma in stem cell maintenance.
Sperm and SpermatogenesisII (1279-1284). Monday
220a
1279
LOCALIZATION AND REGULATION OF CONNEXIN43 IN THE RAT EPIDIDYMIS.
((D.G. Cyr8, D.W. Laird', andL. Hermo")) Maurice-LamontagneInstituta, MontJoli,QC,bDepartment of Anatomy and Call Biology, McGill University, Montreal,
QC, Canada.
Gap junctions are composed of complementary membrane proteins, connexins,
which form a pore connecting the cytoplasm of adjacent cells. While gap
junctions are present in the epididymal epithelium, little is known about the
proteins which make up these junctions ortheir regulation in this tissue. The
objectives of this study were to determine the presence and regulation of
connexin43 (Cx43l inthe ratepididymis. Immunolocalization was done with antiCx43 antisera and either horseradish peroxidase or immunogold detection. Our
resuits show that Cx43 was localized between principal and basal cells but not
between adjacent principal cells where gap junctions have been characterized
morphologically. At the electron microscope level, Cx43 was present at sites
along the plasma membrane where basal cells interdigitate with principal cells.
In order to determine if Cx43 was regulated by a testicular factor or specifically
by testosterone, rats wereeither orchidectomized or orchidectomized and given
a testosterone implant and sampled 7 and 14 days post-surgery. The
testosterone implant (18.6 cm) raised serum concentrations of testosterone to
levels present in the epididymis. Cx43 immunostaining between principal and
basal cells in orchidectomized rats was stil detectable despite the absence of
testes. Furthermore, in the initial segment and caputepididymidis, but not in the
corpus or cauda epididymidis, staining was also present between adjacent
principal cells. In epididymides from orchidectomized rats which received
testosterone, Cx43 immunostaining was similar to controls. This is the first
report that gap junctions are present between principal and basal cells. These
results also suggest that the intracellular targeting of Cx43 to specific cell to cell
interfaces is segment specific and androgen-dependent. Supported by MRC.
1281
AUTOPHOSPHORYLATION OF THE C-KIT RECEPTOR IN RAT TYPE A
SPERMATOGONIA. ((M. Dym, M.C. Jia, G. Dirami, J.M. Price, S.J. Rabin, I.
Mocchetti, and N. Ravindranath)) Department of Cell Biology, Georgetown
University Medical Center, Washington, DC 20007.
expression and activation of the c-ldt receptor in rattype A spermatogonia was
from 9 day old rats and subjected to sequential
enzymatic digestion. The mixture of testicular cell types was then separated by
sedimentation velocity at unit gravity and the isolated type A spermatogonia were
characterized by light and electron microscopy. They exhibited spherical nuclei
containing several nucleoli and associated chromatin clumps and organelles
generally in a perinuclear location. Syndtesis of the c-kit receptor by the
spermatogonia was established by hybridization of total RNA with a specific cDNA
for mouse c-kit receptor. Two mRNA transcripts migatng at 4.8 Kb and 12 Kb
were observed. Localiion of the c-kit receptor in the isolated cells was
determined by immunocytochemistry. Specific staining for c-kit receptor was
observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the
presence of the ckit receptor protein in the spermatogonia was confirmed by
Western blot analysis using the same antibody. The antibody recognized the ckit
receptor at -160 kDa. In an attempt to determine whether this receptor has a
functional significance, we examined the effect ofkit ligand on the phosphorylation
of the c-kit receptor.
The c-it receptor appeared to be constitutively
autophosphorylated on tyrosine at low basal levels and upon stimulation with kit
ligand, the amount of phosphorylated protein increased significantly. These
observations indicate that kit ligand induces autophosphorylation of the c-kit
receptor which may lead to the activation of other celular target proteins
responsible for spermatogonial proliferation and/or differentiation.
The
examined. Testes were obtained
1280
GERM
Y IN THE ADULT RAT PROM SPERMAIOGONIA TO
CELI -MORPHOMI
SPERM((LR.
IPm,M. S ,dbegand LD. Russell)) Depwqn tof Phbysolo
of Stmcaral Biology, Souten lifioisn Univs, Carbondale, EL62901.
LaboraIty
One of the most amazing cell tran
isdIaracsized bya prolifraavephase(
occurs
during
a
apeI
oeeisTs process
genetc rM
division phase (melosis of spmuocyes), ad adiffean pe (spermatids).
present stIdy forth to chaacteize the entie 7.5 week
prces in
ae
usig quantitative exesion of cell mopolo
coreatd with functona evens
om
se, cll surace
volumes and surface
mch that
stuctr
were
areas
zed to de
for viualy al
cell
rat
ouoldbe
pm
ua
d
rmaie
cell
bcelular
paramet of adult rat germ cells, including sperm. Over three thousand electron
were tab
consuct monges from four nimasat min ofthethurees
miropa
ofthe ermogenkc cycle (Russell et aL, 1990). Viealy al germ cell components
of tbeir
dynamic properties associated with specific phaa
For example the
devw
dsow
surfae of smooth ndopasmic reticulum, expresd per
increased subsamty
(about x25) in gam cel development fom type A rmagonia diplosene
It
halved during the two meiotic divons, buticea slighty
spermatids
step 6 of
md declined slowly
16-17 (is
sta I-I). accelerated decra occurred in sbsequet step reuting in overll 52fold depki ofthis orlnele fm its peak at pachytee (930pm*e) its nair t step
19.
in smooth endoplasmic
temendous membrn
and numae
orpnelks occuring during late
paled therise in lipid ineloogat
spermatids, sugesting that lipid of spermatids is a reservoir for degraded smooth
endoplamnic ticulum and other membanowuscompoen Membra processed to lipid
reservoirs prior to permiation would not overwhelm th Setoli cell's ability o degrade
membranous componts at the end ofsrmio
also explains the pid cumulation
in Setoli cells post spmmit. The nature ofthe methdolo
fored ustoqutify germ
cell subcellular components and necessitated that several structures, yet unnamed, be
characteized and desnibed The data provide a srucura basis for physiological events
relating to germ cell development Suppored, in part, by Brazilian Reseich Council (CNPq).
ar
an
cel,
sraOCY
in
young
unl
er
ep
An
to
reclum
other
rm
It
all
1282
IMMUNOCYTOCHEMICAL LOCALIZATION OF GERM CELL RNA-BINDING
PROTEINS p48/p52 TO SPECIFIC STAGES OF MALE GERM CELL
DEVELOPMENT AND THE CHROMATOID BODY.
R. Okoa, R. Korley5, M. T. Murrayb, N. B. Hecht' and L. Hermoa,
McGill University, Montreal, Canada&. Wayne State University,
Detroit, MI 48202b. Tufts University, Medford, MA 02155c.
Sequence-independent zaRNA-binding polypeptides, mRNP3 and
URNP4, are associated with a pool of stable nontranslated
poly(A)+mRNA in Xenopus oocytes. Proteins homologous to URNP3
and mRNPA (p48/52) have been identified in male mouse germ
cells. Western and Northwestern blots of testes and isolated
germ cells indicate that p48/52 are present during meiosis but
reach highest levels post-meiotically at a tine when many mRNAs
are stored [Dev. Biol. 158:90-l00 (1993)]. Here we analyze the
distribution pattern of p48/p52 in the rat testis by LM and EM
issunocytochemistry. p48/p52 izasunolabeling was found to be
predominantly cytoplasmic and germ cell and stage specific. It
began in early pachytene spermatocytes, progressively increased
during meiotic development, reached a peak post-meiotically in
round spermatids, and gradually declined in spermatids
undergoing nuclear condensation and elongation.
A
proportionally high concentration of cytoplasmic
was
found in association with the lacunae of the
granulofilamentous chromatoid body. The pattern of synthesis
of these mRNA-binding proteins together with their presence in
the chromatoid body suggests their role as germ cell specific
mRNA stabilizing and/or storage proteins. Supported by MRC of
Canada and NIH.
isosunolabeling
1284
LEYDIG CELLS OF
TESTIS
BUT NOT
IN GERM CELLS.
S. Igdoura,
THE
L.
Hermo
and C.R. Morales,
Anastasiades,
Department of
McGill
Canada
Montreal,
Anatomy
University,
Biology,
a-MANNOSIDASE
IS
II
IN
EXPRESSED
SERTOLI AND
RAT
ADULT
Cell
and
2B2.
a-Mannosidase
processing
a
is
II
transmembrane
modification of
and
Golgi enzyme involved in
N-linked
oligosaccharides.
study, mannosidase II was immunolocalized in the
epididymis using light and electron microscope
Paraffin sections of the testis and
immunocytochemistry.
present
and
were
epididymis
staining
specific.
appeared
against the catalytic
light microscope, the Golgi
antibodies
In the
II.
cells was immunostained.
The
pattern of the Sertoli Golgi apparatus was stage
At
VII-VIII
of
the cycle the Golgi apparatus
stages
as
a
distributed
not
deeply
the
of
microscope
and
Sertoli
of
the
cycle,
incubated with
mannosidase
of
apparatus
Leydig
stained
cell
to
anastomotic network
the
lumen.
and
extending from
At other stages of the
Electron
supranuclear.
imunocytochemistry revealed iunogold particles
saccules of the Golgi apparatus.
Mannosidase
localized
in germ cells.
In the epididymis, a
reaction
was
basal
over
over narrow cells of the initial
caput, corpus and cauda regions.
type specific expression of
within
the
testis and epididymis suggesting
mannosidase
specialization in the processing of N-linked
oligosaccharides.
Supported by MRC of Canada.
was
segment
and
noted
clear
results
thus
II
functional
exclusively
cells of the
demonstrate cell
BCL-2 AND BCL-X GENE EXPRESSION DURING CHICKEN
SPERMATOGENESIS. ((X. Vilagrasa, C. Mesquita and J. Mesquita))
Molecular Genetics Research Group. Faculty of Medicine, University of
Barcelona, Casanova, 143, 08036 Barcelona, Spain.
The bcl-2 gene product has the ability to block apoptosis and may confer stress
spermatogonia and Sertoli cells,
which are unaffected by temperature elevation and other stresses. We have
determined RNA levels of bcl-2 and bcl-x during spermatogenesis by Northern
hybridization and PCR amplification using bcl-2 primers that amplify a region
of 362 bp that comprises the C-terminal coding region (aa 190- 232) and 227 bp
of the 3' region of the gene. The bcl-x primers amplify a region of 617 bp that
corresponds to the whole coding region of the bcl-x gene (Boise et aL, 1993,
Cell, 74:597). For Northern hybridization experiments the PCR products were
used as probes. Bcl-2 and BcI-x transcripts are present in chick embryo testis
and in 5-6 week-old chicken testis, where
of the basal layer of
cells, the spermatogonia, occurs. Later in spermatogenesis, when most of the
cells are meiotic and postmeiotic, the bcl-2 and bcl-x mRNA's are no longer
detectable. When the chick embryo was exposed to increased temperatures
(42°C) for variable periods of time (5-24 hours) the expression of bcl-2 and
bcl-x genes in the testis persists elevated. Bcl-2 and bcl-x are developmentally
expressed in testis cells which are particularly resistant to hyperthermia and
-resistance to certain testicular cells, such
us
niultiplication
other stresses.
Monday. Sperm and SpermatogenesisII (1285-1290)
221a
1286
1285
ZATIONOF RETINOIC AACID RECEPTOR-a TRANSCRIPTSIN THE RAT
EPIDIDYMIS. ((K.M. Akmal and K.H. Kim)) Department of Genetics and
Cell Biology, Department of Biochemistry and Biophysics, Washington
State University, Pullman, WA 99164.
DIFFERENTIAL
As spermatozoa traverse the epididymis, they undergo physiological,
morphological, and biochemical changes which resuit In their maturation.
The epkiddymal epithellum provides the appropriate microenvironment
for these processes. Abnormalities in the epididymis have been reported
in vitamin A-deficient animals. Although the mechanism of vitamin A
and
The development of spermatozoa in mice procedes normally at
is severely inhibited by higher temperatures. To examine the effects of
LOC
heat stress
action on the epididymis remains unknown, Itis postulated that the effects
3S-methionine, and the proteins extracted with 4% trichloroacetic acid
(Hlt)
44°C
1288
LIGANDS FOR THE IGF-I1CATlON-INDEPENDENT MANNOSE 6-PHOSPHATE
RECEPTOR MODULATE GENE EXPRESSION IN SPERMATOGONiA,
PACHYTENESPERMATOCYTES AND ROUND SPERMATIDS. ((J.K Tsunra and
DA. OBrien)) The Laboatories for Reprodudive Biology, Depts of Pediabis and
Cell Biolgy & Anatonw, Univerity of North Carolna. Chapel HE, NC 27599
MANNOSE
and round
levels in both pachytene spem
This elec is completely abolshed in the presenceof mM ManS-P,
that this change in gene expression is mediated specifically by the
IGFIU+I-MPR. Nolthem analysis and irnmunohistochemrstry have shown recently
tha
spermtogonia and eary spermatocytes contain markedly higher levels of
ceels at ater stages of developnent
K; VCIMPR mRNA than se
Spermatogonia were treated wIth Scm, IGF-II and two IGF-II analogs to determine If
spermatWds.
indicag
modubete
IGFIVCI-PR
expression
marked,
these celis. Spermatoonia
dose-dependn
inceaes
l8S
rRNA
which were 3-fold highwr han changes observed in round spermatids and may
reflect hb her cell-surface levels of the receptor in thesecelis. Quantitative RT-PCR
wasused to determineretie leves of c-fos mRNA In spenratogonia after treatment
levels
the IGFNUC'I-MPR) or [g"
IGF-ll (an analog that
IGF-11,
IGF-11 (an anabD VW binds to only the IGF-I receo). Both native IGF-11 and [Leu27]
IGF-11 was not
IGF-II mncreasTo-fos mRNA levels to sinilar lvls whle jArgS
able to increase c-fs mRNA levels. Thus, MLP-bearing glycoproteins secreted by
enic cels and IGF-11 Increases
Sectoli cels aier gene expresslon in sperm
of
with
[LuzY
c-its mRNAspecificaly through the IGFIIICI-MPR. The secretion
by Seroli cells may act as knportant cues for the reguiation of
PR
IGFIIIC
spermatgenic cell gene expression durig spernatogenesis. Supported by NIH
spermatid
grant
HD2"8
6-PHOSPHATE-BEARING GLYCOPROTEINS ARE ABUNDANT IN
((D.A. O'Brien', P.L. Magyar', D.E. SIeat2 and P.
for Reproductive Biology, University of North Carolina,
'Laboratories
LobeI2))
Chapel Hill, NC 27599. 2Center for Advanced Biotechnology and Medicine,
UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ 08854.
MOUSE TESTIS AND BRAIN.
6-phosphate receptor(lGFIICI-MPR)
medates the uptake and targeting of mannose 6-phosphate beanngg copotems
P-berg
(MBP-gp) and triges second messenger cascades alter bind
reported previously that Seytlcel condtd medium
growthbtks or IGF-11.US
(Scm) and MP-gppurfioed om Scm are able to elicit dose-dependent increases in
of
c-fos mRNA in spemaogenic celi
(pedominantly
steady-state
spermatocstes and spemats). VWk nowreport that Scm ecits dose-dependent
ls able to
the
treated with Scm demntae
oigands
andthe Melon Foundation.
et
209:156)
glycoproteins (M6P-glycoproteins) in paraffin sections of several mouse tissues
using standard avidin-biotin immunoperoxidase methods. Binding specificity to
M6P-glycoproteins was verified by inclusion of 5 mM M6P in the incubation
buffer, which completely abolished sCI binding. M6P-glycoproteins were not
detected in liver, pancreas, stomach, duodenum, large intestine, salivary glands,
spleen, lung, kidney, thyroid, adrenal, pituitary, ovary, uterus, prostate, seminal
vesicles, heart orskeletal muscle. However, M6P-glycoproteins were prominent
in vesicular structures in the seminiferous epithelium and in large neurons in
several regions of the brain and spinal cord. M6P-glycoproteins in the testis
particularly abundant in Sertoli cells and in pachytene spermatocytes.
Adjacent sections were immunostained with a monoclonal antibody to LAMP-1,
lysosomal membrane protein. The distribution of M6P-glycoproteins and
LAMP-1 was distinct in both brain and testis. Multiple M6P-glycoproteins were
detected with 12Sl-labeled sCI on protein blots from these tissues. Our previous
studies also indicate that Sertoli cells secrete several M6P-glycoproteins
(O'Brien et al., 1993; Biol Reprod 49:1055). These results suggest that M6Pglycoproteins serve unique functions in the testis and brain, where they retain
the M6P recognition marker and do not appear to be localized exclusively in
and NSF DCB-9118681 (PL).
lysosomes. Supported by NIH HD26485
were
a
(DAO)
1290
H IT PROMOTER. EVIDENCE FOR MULTIPLE CIS-ACTING TESTIS
ELEMENTS. ((SA. Wolfe, J.M. vanWert, and S.R. Grimes)) Research
Service (151), Overton Brooks Veterans Administration Medical Center,
Shreveport, Louisiana 71101-4295; and Department of Biochemistry and
Molecular Biology, Lousiana State University Medical Center,
Shreveport, Louisiana 71130-3932.
SUGGESTS
proximal promoter
of the testis specific histone Hlt
gene
from the
human, monkey, mouse, and rat were compared for nucleotide
homology. The HIt promoters are highly conserved from nucleotide -107
to nucleotide -24 upstream from the tracriptional initiation site of the
sequence
((J.M.
AN IMPORTANT ROLE IN
Research Service (151),
Center, Shreveport,
Biochemistry
and
Overton Brooks Veterans
71101-4295,
Louisiana
Molecular
Spi
Analysis
synthesized using the rat H It promoter sequence containing TE 1, TE2,
and the centrally located SpI consensus (5'-GGGGCGGGG-3) does
bind to SpI as determined by supershift assays using polrclonal anti-SpI
was
not
antibodies, This
workc
was supported by the Veterans
N.I.H. Grant ROIHD29381-01.
Administraion
and
Grimes))
and Department
Medical
of
LA
71130-3932.
The
testis-specific
histone H lt
is
gene
transribed
spermatocytes during spermatogeness.
cloned and sequenced and its
mammalian
species with regard
unique
to
rat
to
sequence
of the
expression
specific
revealed the
gene
assays
does
not
and TE2,
Nuclear
elements.
sequence
appear
to
codon, and
promoter
that
contains
to
Veterans Administration
sexually
extracts
the
bind
other
two
located
proteins
Within the
from
coding
S I nuclease
start
ste
Northern
66
blot
nucleotides
analysis
of
the
indicated that histone H It mRNA is
using nuclear
to
revealed
93% identity with the
human
trsnscription
translation start
of the H It
binding
GC-rich
box.
genes
and 61% identity with the
and accumulates only in testis from
binding
lt
designated TE1
and human H
shows
protection analys
upstream
has been
that of
protein
these conserved
region, the
primary
It
region compared
H1/CCAAT
the
H
to
elements,
promoter
between the H 1/AC box and
only in
The
conserved elements and
promoter
promoter contains two elements that bind to nuclear proteins from the
testis, TEi and TE2. These two elements are spaced approxmately 10
nucleotides apart and flan the GC-rich element. Binding to the TEl
element has been characterized using methylation interference.
croshnling experiments indicate that a complex of testis-specific
proteins that bind to this element have a molecular mass of
approximately 180 kDa. The GC-rich element, common to all of the cell
cycle dependent histone H 1 promoters and the H lt promoter, contains
of a 39 bp oligonucleotide that
S.R.
and
Administration
Biology, LSU Medical Center, Shreveport
testis bind specifically
sequence.
SAL Wolfe,
H.R. Panek, P.S.Merchant,
vanWert,
rat nucleotide sequence, and they are essentially identical from the
H1 /AC box through the TATAA box. In addition to the four conserved
elements that are common to cell cycle dependent HI promoters, the H It
consensus
HIT PROMOTER ELEMENT
TESTIS-SPECIFIC TRANSCRIPTION.
A CONSERVED MAMMALIAN HISTONE
binding activity. Studies of the
WV-
(sCI)
A soluble form of the cation-independent mannose 6-phosphate receptor
al., 1993; Anal Biochem
isolated from fetal bovine serum (Valenzano
was biotinylated and used to localize mannose 6-phosphate-bearing
1289
ANALYSIS OF A HIGHLY CONSERVED REGION WITHIN THE HISTONE
The
protein synthesis in various testicular cells, seminiferous
on
tubules from adult mice were cultured at 320C, 37°C or 440C, labeled with
1287
mannose
PROTEIN
ON
Synthesis of the testis-specific
analyzed by acid-urea PAGE.
in pachytene primary spermatocytes was
isoform of histone Hl
slightly reduced by incubation at 37C, and sharply reduced by incubation
at 440C. In contrast, 440C slightly reduced synthesis of transition proteins
To determine whether heat stress
1 and 2 in elongated spermatids.
inhibits translational initiation or elongation, the distributions of a variety
of mRNAs in extracts of cultured tubules were analyzed in sucrose
gradients and Northern blots. 440C greatly reduces the size of polysomes
translating the Hlt mRNA, the lactate dehydrogenase C mRNA which is
translated in pachytene spermatocytes and round spermatids, and the
sulfated glycoprotein 2 mRNA which is translated in Sertoli cells.
slightly reduces the size of polysomes translating the transition protein 2
and protamine 2 mRNAs in elongated spermatids. In combination, these
results suggest that the initiation of translation in pachytene spermatocytes,
round spermatids and Sertoli cells is more sensitive to heat stress than in
elongated spermatids.
grant HD25094).
F-Iwcation-independent
SHOCK
HEAT
were
mediate vitamin A signal transduction in the epididymis, its expresslon
pattem was Investigated using a rat RARa cDNA to perform Northem blot
analyses and a rat RARa cRNA to carry out in situ hybridization analyses.
Northem blots revealed two major RARa transcripts, 3.4 kb and 2.7 kb,
expressedin the epididymis at approximately equal levels. Results from
the in situ hybridization analyses demonstrate variation in expression of
the RARa along the ingth of the epkidymal duct. RARa is expressed in
the initial segment, then increases to its highest ievelsin the proximal
caput. RARa expression then decreases to low levels in the corpus and
caudal regions of the epkdidymis. These results suggest that RARa may
play a role in spermatozoan maturation in the proximalepididymis
The
OF
32°C
ofvitamin A may be mediated by two nuclear retinoid receptor families,
retinoic acid receptors (RARa,p and y) and retinoid X receptors (RXRa,
yi. These receptors may act as heterodimeric transcription factors
P and
in the regulation of gene expression. To determine whether RARa couid
(supported by NIH
EFFECTS
((L.M.
SYNTHESIS IN VARIOUS TESTICULAR CELL TYPES.
Cataldo and K.C. Kleene)) Dept. of Biology, University of Massachusetts,
Boston, MA 02125.
TE
N.I.H.
mice.
Protein
from various tissues show
elements.
site,
This work
Grant
testis-
These elements overlap
SpI
this element
and
mature
but testis
Spl
supported by
ROIHD29381-01
the
222a
Sperm and Spermatogenesis II (1291-1296). Monday
1252
THE 5 FLANKING REGION OF THE MOUSE GLYCERALDEHYDE 3-PHOSPHATE
DEHYDROGENASE-S (GAP" GENE CAN DIRECT SPERMATOGENIC CELLSPECIFIC GENE EXPRESSION. ((J.E. Welch, P.R. Brown, E.H. Gouldng, and E.M.
Eddy)) Gamete BologW Secton, Labary of Reproductve and Devebpmerntal
Toxicology, National Institue of Environmental Health Sdenoes, National InsiuAtes of
Health, Research Trangle Park, NC 27709.
1291
ALTERATION IN HISTONE H4 PRaOTER GENE FUNCTION IN SPERNATOCYTE
AFTER GOSSYPOL TREATIENT. ((C.S. Tang and N.Y. Yang))
Departmnt of Anatomy, Physiological Sciences and Radiology,
North Carolina State University, Raleigh, NC 27606.
This research project is to gain an understanding of the
machanim of action of a male contraceptive drug - gossypol (C)
at the genomic level in rat spermtogenic cells. After C
treatment for various times (8 to 19 rk), the spermatogonial
cells wera allowed to rest for 2 to 4 wk. The function of
histone H4 promoter gene (H4PG) in the repopulating pachytene
spermatocytes (RPS) was investigated. The sequances of the
oligonucleotides for HIPG binding site I and II were
synthesized by an ABI-392 DNA synthesizer. The complementary
strands of oligonucleotide were annealed in Tris-saline
containing EDTA, and than labeled with a-32p deoxy-GTP by
filled-in reaction. RPS and the control pachytene
spermatocytes (CPS) were obtained by centrifugal alutriation
and subsequently they were used for the preparation of nuclear
protein extracts (NPE). The NPE interaction with site I or II
was studied by an electrophoresis nobility shift assay (EISA).
EKSA of CPS-NPE revealed 5 mjor gel shift band group for sits
I and II. After 2 to 4 wrk recovery from 8-, 12-, and 19-irk of
G treatmnt, RPS-NPE failed to shift two bands (c and d) in
site I. Results suggested that band c and d formed by site I
represent binding activity involving transcription factors
(e.g., H4TF-1). G treatmnt could affect the factors for
interaction with the H4PG site I. No effect was dmonstrated
by EKSA of RPS-NPE on H4PG site II (supported by Rockefeller
Grant RF 90034).
Many enzymes involved in spermfatogenc cel metabolsm are dIfferent from the
metaboic enryfes preaent in somaft cels. This Is parduiarly true of enzymes
wtich funcion In the hIgy conserved glycolyt pathway. The gene encoding one
such s
glcoy enzyme, gcerbyde 3-phosphae dehydrogenase
(GspdLs), has been cacerzed In our labo y. Wib GAPD-S Is cbsely reated
to the somatic GAPD enzyrme, Gapd-s gene exprion Is detectable only In
spermatds, durIng the post-melotic phase of apermatogenesia. In orderto detefrrne
what regions of the Gapd-s promoter are responsible for thls haploid gene expression,
we inroduced DNA construct contairing the Gapd-s pnomoter and the E. co# Pgabclosdase gene into the mouse genome by drect pronuciear Injecion.
Identification of the regions drecting haploid gene expression Involved a series of
constructs (A,B,C,D) contairing progressive 5' deletions of the Gapd-s promoter.
Haploid cell-spedcic expression of P-galaclosidase was detected in rnice Integting
constructs wlth 1350 bp (A), 626 bp (B), or 336 bp (C) of Gapd-s promoter secquence
5' to the Initiation of transcrption, but no reporter gene expression was detected in the
testes of two founders contairdng 162 bp (D) of Gapd-s pmoter. ThIs would Indicate
that the 175 bp region present In the C construct, but rrissing from the D construct
Is required to specify haploid cell-specIfic expression. Whie no sequence motifs
reported to be involved in spermatogenic cel-sapecifc expression could be lderntfed
in this region, possible binding sites for CCMT, AP-3, E2A, ets, and GAGA
transcription factors are present. The presence of an ets binding site is inrguing
since members of the ets gene fanrily are expressed at high levels In the testis (Rao
et at., Science 244.66-70, 1989) and may serve to regulate Gapd-sgene expression.
1293
1294
WITH A RAPID TURNVER
O?TESTEN uRaIM
OF JUNClIONAL COMPLEX
(JC) IN THE TESTIS (Josehne Onma, Prendu Mathw
md C. Ya Cbhg) bem Population Council, 1230 York Avenue, New York. NY 10021.
THE EXPRESSION
the tests, the JC betwe Stoli cells (SC) and Satoh-gSm cels must be disrupted md
In
rgsneted in a prcidy coordinated famion to allow the pag of dveloping germ cells
(GC) from the bsa to the adluminal cmparment while mintaining the integrity of tbe
blood-testis barrier (BIB). Testinsha reody boen down to be a cotmp
of the JC in the
testis mad odbr tissues. Whe
miniferous tibeles (SI) isolated from 60-daysd
enlturd in vitro, ST e_psd epificandy les test mRNA compad to 10-dayold rats
ratswere
possibly
becue of
a
extensive
if GC have ay effec
ae
tisue
on
restuctuing
in the
tatis
in the younge
rats.
To
pesion. OC md GC-conditioned medium
testmi mRNA
(G0CM) dpreaeby mseadcal proarewidloomyynzaQatic tent_ weacn.sltured
ormboad with SC. Both GC ad GCCM isduced virully so effect en the SC tesinamRNA
levd indicain the age-related dages in tesis level observed in the ST mny be related
not
to
ofustn is indeed craWed with qaid JC tumover,
total RNAs
md kideys of pre-. neo-. sd post-nal rats. Testes
isoklaed from rat
from 3- ad 20-days old rat expressd 3-8 fold
testi
adult rats.
pattn of
expemon ws dlar in the kiddwy whe pro md no-natd rats exprsed sigpificmdy maze
treted with loidnne,
Whben at
testin
post-natal md adult
ic drug known to dirpt JC between SC, ad SC-OC, the testicular testin
matiper
mRNA levd iwesaed by umch 30-fold withn 24 hr. perked at day 6, mad declid by 15
days consie with the posuate tee the expresion of testin is corated with the rate of IC
turnover. Since testis is a namber of the cathepin superfanily. without my apparen proteaw
activity, we heve also ained the expession of multiple catepsin sNAs in tese same
samples. Cathpsin B. H, L. md S showed respo to dte loidmine _ret wherei
C md D showed a sigh incae se demea, raspectivdy, in thsir mRNA in the
c
tdasarId the uniquere
of tein folowing JC
following loaidsaine
damnge We postulate thett depti of GC by1ondne induoss testi aeeo by SC
in attempt to untoais the inteity of the BIB. In cedusica the expesson of tesdt in
mad during GC devdopment.
the testis is a senstive masker to imoitor the staten of the
Tibis work was supported in pwt by NIH gmts HD- 13541 mad DK-0M13.
GC.
To
exuaie
whether the
expression
were
a
tes
mere
thm
rats
rat.
s
were
an
no
n
tstis
In Mab Mous Gem Clls Cu-Zn Sup
de Dsu
i Und
Both T p on and Tri
Contol Ih ulil Tra
from
L
Dofferertlal
Two P e
((W. Ou, C. M
Hmo, aid N.B. Heft')) 'Deatmt of Biolgy, Tuft U dsraY, MedIcd, MA
02155, Depminet of Anatofy, McGU UniersIy, Montreal, Cada. (Spon by
N.C. Mibum)
Cu-Zn supeadde dlsm
(80-1) is a ewzme
is widely sped in
eukeryt-lc coa Nd perfrm a vIal role in protectIg cefll agaonat fe radical
dwnage In mouse bt, tee dfeir sizes d OD-1 mFNAs of about 0.73,
staes of
0.80 nd 0.93 kB are detected. The 0.73 Kb mRNA is omd In e
mae germ cob and in go somatc tisues The mRNAs d 0.80 Kb and .93 kB
are aekuIvely detacted in posmidc grm cals F4ie H dlgeilorm and
norn blot aly reveal thatththree soD-I mRNAs e doved from two
a po met bcts
transsdt
whIch dwerby abot 114 ucodds. RNae protction assap demonstate
tW the addIonal ucleoodas prest in hFe post-"maltc mRNA ae soey In
the 5! uriranslatad region (IT. Usn a probe dWerIfdfim the 5S UTR d the
tip 8 SOD-1 mRNA, we have establd that the 0.93 kIS mRNA
oignats from mnaornaive upetmr promoter contguouB wth the somatic
SODI promoter. Polysomal gradIent sanlysIs d the re mouse test SOD-1
mRNAs res tha the 0.93 kB SODI mfRNA is prIwly nonpolysomal, while
the 0.80 and 0.73 kB SOD-1 mRNAs are mostly polome associed A faster
mIgratIg form of the 0.93 kB SOD-1 mRNA is pest on poloms as a result
nl
These data d NA
tat male germ coa
d par
transcrbe two sIze clas of SOD-I mRNAs by UOINIng two fferent pmotrs
post-mnltc SODI mNAs udWgo adyaton dc g and one of the postmelctIc SOD-1 tancrpts is hInIy stored s klIt is "t d at the end d
an
1296
IN-SITU EXPRESSION 0F a-IN5IN IN HUMAN AZOOSPERMIC MALES
W1TH SERTOLI-CL ONLY HISTOLOGY. ((CS. Ndrbg, DJ. Lamb,*
S. Kim,* S. Maislos,* M Glinz,* S. Pursel S. Rizvi, LS. Ross, LILUpshultz,*
of Urolo
ad Y. Cho)) Depae
and Genetcs, the University of linois at
of Cell Biow and Urology,
Chiag, Chicag, IL 60612, and *Dea
Bayklr College of Medci, Hou. TX 77030 (Spon. by R. Bcaker)
In situ hyWiizon present
the
powerul method to
mechaoimS regulati
S. and the abeant functon of
mechanisms in human infertlty. Unti recenty, the fixatves and techuiques
t
commonly used to prpare teis issue for in situ hy
sverely disoted
a
cellul
demnstrate
spe
thedelicattstcula
specfic gen
have
andthwarted
it
exopessed during
peviously
shon
using
ema
ientify
to
of
tiand
cell- and
stae
diffn
We
ammble timage anlysis system in the
an
in
Depawtment Pirofesionl gphicslanguagn an Amig 3000 platmn.
an a-inhibin indin model in rat Serti cel cultur, that Zamboni's and
pfixaes provd suprior siga
deten of mnNA and
slgul-to-nol ratio compred to Boin's, p
adh+
%),hyde(l%),
Art
Carnoy's.
and
denstt
Zambooi's
human
rded%)
+
ot-inhibis
exprsion
of whom dislayed Seol-cel only
evaluationsfor
eevated file stmlat
in tess of
histoy.
infertlty
t-Inhibibn
We sough
me,
az
to
a
Bght tstis biopss fm 6 males
with normal
horme (PSE)
fixadve.
buman
high
normal
lwels wae hybrid
ad moderat
in
situ
y
with a
scdtions diqslyed expresson of
inhibin in Serol cels, but the intesil exrson of a-inhibln varied beteoen
patents with diffi
PSH levds. In siu expression of ct-inhibin in hnman tests
may
prvide senivc
of dinica subtp of
infetlty.
227 base
pa
human
a
(Supported by American Fow
pmbe
All
a-
male
bsdon for Urologic Disease Grant NI94 -04)
1296
EXPRESSION OF TiE I EDIATE EARLY GENE NUR77 IN-SlTU IN
MURINE AND lNF T[LE HUMAN ISTIS. ((C.S. Ni erb , DJ. Lamb,*
L Las, S. Kim.* S. PurwlL S. Rizvi, LS. Ross, LL Tpsliushz* and Y. Cho))
and Geneucs, the Uvrsity of Minois at Chicago,
Depart s of Ur
ag., IL 60612, and *Departlnts of Cdl Biology and Uroloy, Baylor College
of Medicine, Houston. TX 77030
We invesdgaed a putative role for nur77 during murine and human spem ic
difSe itio by probing for its expr n using in situ ld oeL Like othr
imadiate eawly gunes, nw'77 ws Iadefied in thu search for gem rapidly
exprssed as cells in G, re-ene the cdl cyce in response to growth factors. NurT7
cDNA hus boe shows t be bigMy ho
to sequencs of membes of the
sterdthyroid hmone recptor g superfaily, nd a human homeoog s
identified
in
ests
cDNA
nd
libraries. Using a sucrose buffrd
peviousy
proem
parafomaldhyde fixative optmizd for both mRNA ra_nto and histogic
prservation, and a user-r ammed ima anaysis system on a SUN SpaclO
pladformusing theDLgraphics lguage. we dputed therelatvecell- ad stae
spcfic expressin of murine =wr77 duig mmine mausis. We foud
nurT7exreio to be rtiaced to the round smatd in sages IV tough IV
imth low lves of Intbstitial expression. Eight
dintg use
estds bbopse fro 6 azoosPermck mals un
ing evaluati for infility with
nrmal high normal nd moderately devated folce stmuatn hormonme levels
wer t probd in siu wth the humn nur77 qence Of note highly abundant
,sr77 expei was noted in the int tum of these paten who uniformly
paten with
displayed SerH-i-ol only histology. Expesn vari bet
dfg clinical pfiles. Nur77 may ths be a adtia reulat i spermagnic
differeti
whose abant apaeon reults in hmn male Inft lity.
(Supported by American Foundaton for Urologic Disease Grant N194-04)
Monday. Sperm and Spermatogenesis II (1297)
223a
129T
IDENTIFICATION OF NUCLEAR PROTEINS B1NDING TO THE
C4MOS NEGATIVE REGULATORY ELEMENT (NRE) ((W. Xu and
G. M. Cooper)) Dqeatent of Pathology, Harvard Medical School, DanaFarber Cancer Institute, Boston, MA 02115
The expression of the c-mos proto-oncogene is highly regted such
that its eoxpssion is almost restricted to male and female germ cells, which
appears consistent with its role beng a meiotic cell cycle regulator.
Previous experiments in our laboratory indicated that an upstream negative
regulatory element (NRE) was responsible for the suppression of c-mos
transcripton in somatic cells. In order to find trnscription factors
imvolved in interaction with the NRE, we used gel shift assay to identiIy
nuclear proteins that bind to the NRE in a sequence-specific manner. One
protein factor identified in seval somatic cell lines and tissues appeared to
bind to a region (box 2) previously demonstrated to be required for NRE
activity by site-directed mutageness. This box-2 binding protein was
absent in nuclear aetracts of male germ cels, consistent with its role as a
somatic cell repressor of c-mos. Sequence immediaely upstream of box 2
was similar to a negative regulatory sequence of germ cell specific
phosphoglycerate kinas (PGK-2) gene. This sequence was required for
repressng c-mos expression in NIH3T3 cells and for the binding of the
protein to box 2. The box 24inding protein may thus be a general somatic
cell repressor of the transcription of c-mos and other germ cell specific
genes in somatic ces.
Fertilization I: Gamete Recognition and Binding (1298-1301)
1299
STRUCIURE AND FUNCTION OF gp55 - AN mZP3-DERIVED GLYCOPEPTIDE
THAT CONTAINS THE COMBINING-SITE FOR SPERM. ((E. S. Lischer and PM
Wassarman)) Roche Institute of Molela Biology, Roche Research Center, Nutley, NJ
07110.
During fertilization in mice, sperm fust bind to mZP3 (.83K Mr), one of 3
n undgo the oome
th mabe-up the mouse egg zona peucida, and
1298
ACMVE mZP3
SYNTHESIS OF
EXPRESSION
ENCODING
VECTORS
((S. Mortiio1,
TRANSFERASE.
2DtpL.
AND
a1-3-GALACTOSYL-
Litcb&l, D.H. Joziasse2, 1.J. Shspe3,
E.S.
Amstrdn,
Medicinal Cem., Vrije Univ.,
Ceter, Jobns HpBoins
mZP3
TRANSFECTED WrIH
STABLY
Mod Biol., Roche Rea. Center, Nutey,
Waan1)) 1Roche InsL
P.M.
CV-I CELLS
BY
Me Netherands,
ad
NJ 07110,
and 3ncology
Sb., Balimo
mZP3 (-83K Mr) is
of 3 glycoproteins tat consattte the
egg zx
pelncida Dring ferlzatd, sperm
to mZP3 0-linked migomcaides that have
in a-lika wil t penuhimate sugr, at thek nonreducing end (1J2).
EC
ativEC-mZP3 iDts the medim
saby rsfected wItb the mZP3
bhve been repnted forCV-l acel ansected with exjespfn
rvecm
(3). Similar
Howev,
(4).
sequence
cont
ng the compite mZP3 coing
since CV-1 cels
not
have functous al-3-pglacoytrnsfer (al-34T) ge (5), dte results suggst
lHe, eatbisd stybly hfeed CVthat mZP3 may nolreqe a-al
lines wit
used previousy (3) ud
cells
tbe mZP3 pe
CVmZP3 (-71K Mr) into medium. However, unlie mZ3 and EC-mZP3, CV-mZP3 in
Med.
MD 21205.
one
eacion
mouse
cels
considerably
do
a
for ac
we
y.
con
these
secrete
speem
whether the
binding and
lack of
acsosome
tmiml a-al an CV-mZP3 acounts
CV-mZP3 wenr
c
CV-1
ideosfifed
wer
mZP3/0;1) ino
AR-inducing
the
Tbhs
usro.
m.73 e_gues Ithe pseneofa-at na itsO-liS
O
dcide.
Ned. Aced. Sci.
(1) Bleil, J.. Waarman,
(1988).
Wassman, P. (1990). Dcwlopswat 108,1. (3) Kinlocb, R. et el.
P.
115, 655. (4) Beebe, S. et
(1991). Proc. Nad. Aced.
264,
Ccs
el.
Sa.
USA
Dev. Bfol.
88.
ckones
(CVad
activity of
7401.
(6)
USA 85, 6778. (2)
(1991). J.
48.
A
beaviy
a
specific
size-
s may
be
glycopeptide
resuts rvea
tat
ies
ci
(5)
GaiU, Swanso, K.
Jozise, D. et et. (1989). J. Diol.
151,
and bind to
mZP3 (3); these o
from
compble
al-3-GT
binding
tha the
on
orinlly suggested.
was
k do
Proc.
(1992).
sem
provide fiher cvidence
resut
ad
lo sereterecombinantglycoprotei
Purified CV-mZP3/GT exibited both
modum.
activity
ceDls qxesming
foud
mZP3
both
exocytosis (1.2). Sperm recogize
mZP3 with papin and pifW by HPLC for furter anlys
that
(i) In addin to peventng speam fm biding to ovulated eggs, gpSS induces sperm to
the
AR
in
Removal
of As- (N-) lned oligopclsaies
viro. (ii)
mdergo
gpS55
(glycopeptdase F digestio) reduces its Mr by -35K ad signifantly increass its pI.
However, gpSS lacking N-linked iigoach'ide retins both sperm binding and ARinducing activity. (iii) Cleavage of internal P-galactosidic linkages of gpSS
odeswrin'_( _ _ _ ~~~digestio) ees its Mr by -15K, buthas no effect
on its sperm binding and AR-inducing activity. (iv) Removal of sialc acid from gp55
(ne_xaminidse digestio) gnificantly incases its pL but has no effect o its sperm
ke ionct mZP3, gpSS
binding and AR-inducing activity. Tbes results suggest
polypepdide ad 0-lnkd o
uppboth sperm binding and AR-inductio
(1) Wasuan, P. (1990). Dcvelopment 108, 1. (2) Wssarman, P. (1993). Semu,rs
Dev. Biot. 4, 189. (3) Litcher, E., Wamnun, P. (1993). Trends Glycoci. Glycotech.
To detrmine
inacvity,
for its
expoessiovecoitrc enting the
wih
a1-3-GT coding sequence(6).
drsdc_aractorlPsd, ad
bovin
reaction (AR)-induction assays.
fom of
of
the
inative in
a
smaller
(-55K Mr), derived by poteolysis from the C-tminal half of the mZP3 polypeptide,
binds to em d. ike intat mZP3, pivents the srm frm binding to ovulated egg in
vitro (4). Tbee findings suggest that
0-linbed oh
recogized by sperm
are present on the -55K Mr mZP3 glycopeptide (gpS5); i.e., gp55 poesses the
combining-site for sperm. Here, we extended our analysis of the rlationsip between
isoled by digesion
glycepepti
gpSS ohigosacch-ides ad biolgical activity.
seete
reas
cel
(AR).
class of SetIr- (O-) lind o._Igosachuid
bind
enI
5. 369. (4) Rosier
14290.
T., Waainan, P. (1992). Dev. Biol.
154.
309.
1301
13W
EVIDENCE FOR
5SkD
SPERM
RECEPTOR ACTIVITY OF A
PELLUCIDA
ZONA
EXPRESSION SYSTEM.
Depatmet of Cell
Biobgy,
EXPRESSED
PROTEIN
((Wilkins,
Baytor
B.,
Prasad,
College of
RECOMBINANT RABBIT
IN
S.V.,
BACULOVIRUS
A
and
Medicine, Houston,
Dunbar,
TX
ELS.))
77030.
XENOPUS LAEVIS EGG JELLY CONSISTS OF A FIBROUS
GLYCOPROTEIN MATRIX TO WHCH ARE BOUND GLOBULAR
PROTEINS. ((B.S.Bonnell, D.Reinhart, and D.E.Chandler)) Department of
Zoology, Arizona State University, Tempe, AZ. 85287.
ZP proteins which ad
as receptors for
homoogous sperm have been described for
species and those proteins have been shown to be important in the
of lmmunocontraceptive vaccines.. However
deviopment
obtaining such proteins
in quantity and of a purity suff8entto allow detailed charaderizatIon of bological
a
number of
a
and Immunolgical
the
activity Is frequently complicated by the extrme hetergeneity
proteins due to extensive post translational m
wo have expressed a cDNA encoding the rabbit 55kD ZP protein
of
native
baukvr
expression
a
as
to
near
used to
to
system.
The
soluble, paetlally glyc
tions. Therfore
using a eukaryotic
resuitant recombinant protein, BV5S,
ated protein (BV5S) and has been
is
purified
homogensity by lectin affinity chromatography. The purified protein was
immunize guinea pigs or was biotinylated and tested for it's ability to bind
sperm directly. Both the guinea pig antiseum (GP.a-BV5S ) and FAB
purfied from the serum (FAn-a-BV55 ) were shon to recognize naive
rabbit
and recombinant rabbit
and
ELISA
55kD
protein
by
one and two
dimensional immunoblotting
sperm in hi
techniques. Blotinylated BV55 bound to capacitated rabbit
binding
exriments. The
blotnylated protein localized
to the anterior
poxtIon
head hI the acrosomal region. Occasional iabeHng of equatorial
seen, and these sperm were presumed tobe acrosome readed. GPeiBVS5 ihibited rabbit sperm binding to rabbit eggs hi vbo In a dose dependent
nnmre. However FAe-a-BV55 failed to block sperm binding, suggesting that the
Inhibitory effect of thisserum was due to stearic hindrance, rtherthan biocking of a
of
the sperm
segments
wa
igsnd
specIfic sperm
the devopme
for
puAtiv
rabbit
werit was
spwm
sugest that BV55 is a poeti immunogen
vaccine. and that it's role as a
ecepor waants fuher hIvestigation.
by a gat to BSD lfom fte CONRAD pmgm.
site. These data
of an
Wpoed
Eggs from Xenopgg levi are imvested wih tree jelly layers klown as J1, J2, and
J3. In this study, we have found, through rotary shadowing of whole egg jelly
(WEJ) on ceaved mica chips, that these layers consist of a fibrous matix to which
globular proteins are bound. In addition, WEJ, run on a SDS-PAGE gradient gel
is shown to consist of- 700
kDa
staining
450 kDa
with Alcian
blue, and prominent
with
PAS,
a
630
90 and 100 kDa bands
ain
WEJ,
sepated into J1, J2, and J3 and then
by SDS-PAGE, showed that the PAS staining bands where found
pred
in J3, whereas the Alcian blue stained band was prominent in J2 and
J1. Soubilized egg jelly was separated on a Sephacryl-500 column into fractions
which wer analyzed by SDS-PAGE and rotary shadowing The 700, 630, and 450
kDa facons were found to be linear fibers whereas the fraction containing the 90
and 100 kDabands was found to be globulr in foxm Further SDS-PAGE analysis
showed that eggs, incubated for 1 to 16 hours in buffer, released low molecular
weight proteins inluding the 90 and the 100 kDa bands into the medium; in
contras, the high molecular weight glycoproteins remained associated with the
egg. These results suggest that egg jelly consists of a stable matix of high
molcula weigt gyopoteins to which diffisble low moecular weight globular
proteins are bound. Release of these proteins dring jelly hydration may either
enhance or inhibit sperm passage and egg frlizability.
staining with
run
sil
Fertilization I: Gamete Recognition and Binding (1302-1307). Monday
224a
1302
1303
SIT
TEROGENOUS DISTRIBUTION OF LECTIN BINDING
XNOPUS LAEVIS EGG JELLY. ((N.M. Mozingo and J.L
Molcular
Davis, CA 95616.
Secon
of
from the X
anuran
and Cellular
Biology, Universty
IN
Hedrick))
of Califonia,
X.
evis
d
lization.
successively
tie
th iso
of 30 lec
be essetial in elizat.
-lein
as
with
jelly coat layer was xamminedeEenyme-l
Te reacvi
lectn
wer
then
led
A).
FITC-
in conjunction with confocal micr
These stdies
of lectin binding sites among
heteogenous di J stri
within the thee jelly layers.
3 is a thick layer which has a flooclent
appearane
at its outer aspect. Several lectn bound well to the outer
poronof
including LcH, PSA, jacain and GS1. However, none of the
lectis tested bound to the
3. isarelativel thin
portion Jof J2
which non-uniformly stained with WGA. This lectin
very intense, band of fluoresce
at
J1112 inefrface while the
li
y. Using ELLA we found that most of the
remainder of stained
showedme reactivity to al
lecti
howeve, two
jelly lay,only
bound
lay
lectins sowed jelly
first, TKA,
to J and
slectivity.
13 while MAA bound only to JI and
MAA produced
inen
fluorescencetaining pattem att heJ12 interac which extended wel into
effect of jelly-lecn binding on ferdliztion is currentiy being
J1.
ininggrant HD0 7131.
determined.
NM was
orted by NIHa
labeledlectins
ficttion
can
and
as
of
Molecular
microbicides
tive
ep
((11,. Hedrick,
Secto
CA 95616
tract
C;ellular
and
by cros-
prm
c,
STD
uad
-like matrix ae d
reby
entrappig the cel within a visous
To test this hypothei we are quantify
prevening; infection and coio
the binding of frty
to spem seminalplama, seum
ant d
an ELLA and detminig the viscosity
ofloelctins on
scretiom
sperm wwe bound to 96
plates, fioowed
pathogen
oeci
gell
cervic
ed
usn
by vaying
concentratons
we
biotdnylae
of
m
sem
or
lctin,
thn alkaline
stae
y.
revealed
an
ner
the
er
an
1344
The rate of
hydrolyis
The maximal and
reader.
was
determined
m
using
a
lctin binn
kinetic mode
plae
con
Seval
lecins, suh as WGA, jaci
ConA, and LcH
emae
sperm,
reproductive trat secretions aN
stronl
whereas
bound to male and
sWGA and TKA bound
Viscosity
was
detmined
using
a
only
the male
trct
sretions"perm
conslate viscoseter at
Hr
rate.
non-Newtonian viscosity. Semen vscosity increasd lneary
with ConA
concenn and a four fold increas in viscosity was obtained at
Semen
ezbibited
6.7 mg/mt ConA. As oberved
cross-flinked
pically,
sminal
Thee
sperm wer
plm glycptin
aspport
obsevtin
i
micides nd SI
proposal that lectins can funcion
glcoproin aelaeatemmtrix Sup
entapping
in par by a fom Lectin BioPharma Inc. to the Universy of California
the
icelsinacross-linked
gffi
by
1305
TOPOLOGY OF THE SEA URCHIN EGG RECEPTOR FOR SPERM.
((K.R. Foltz)) Department of Biological Sciences, Division of Cell,
Molecular and Developmental Biology, University of California, Santa
Barbar,
The
and
Davi
are
may
Some of
AS
Mah
We propose that lectis
srounded by a thick jelly cot which
is essential during
jely coat contains the distinct layer,
added to the eg as it
ignated Jl, J and 3, which are
transits the oviduct. We xammined
lectin bning
propets of the
g
a s
individ ual jelly coat layes
as
ep in identifyinjel ly
which
Egs
ANDR
LECTIANS ANDRISOBI
MLI.ieduWit
LJA.
Biology, University of California
CA 93116.
egg receptor
for
INTERACTION OF THE SEA URCHIN EGG RECEPTOR FOR SPERM
WITH THE ACTIN-BASED CYTOSKELETON. ((R. J. Belton Jr. and K
RR. Foltz)) Department of Biological Sciences, Division of Cell, Molecular
asa Developmental Biology, University of California, Santa Barbara, CA
sperm
has been purified, cloned and sequenced from
Strongylocentrotus gpvWjurga [Foltz et al. (1993) Science 259, 1421-1425;
Ohlendieck et al. (1993) J. Cell Biol. 122, 887-895]. Although it is known to
be a transmembrane glycoprotein, the exact topology of the sperm receptor
is unknown. Several topologies are possible; in addition to the presumed,
single transmembrane domain (residues 1003-1019), there are two
additional, slightly hydrophobic domains in the 258 C-terminal residues.
These hydrophobic domains span residues 1043-1061 and residues 12251238. Because aknowledge of the topology of this protein is crucial to
structure-function studies, we have used several approaches to determine the
actual topology. First, antibodies directed against defined epitopes of the
931069
used in immunocytological studies of intact eggs.
Antibodies directed against epitopes on the N-terminal side of the first
hydrophobic domain labeled the membrane in permeabilized and nonpermeabilized eggs while antibodies directed against epitopes located on the
C-terminal side labeled egg surfaces only when the eggs had been
permeabilized. Thus, the C-terminal epitopes appear to be located on the
intracellular face. Further, we used in vitrotranscription and translation of
various receptor cDNA constructs in the presence and absence of
microsomes followed by protease protection assays. The results indicate
that the 258 C-terminal residues are intracellular. Therefore, based on the
data of the two independent methods employed, we conclude that the sea
urchin egg receptorfor sperm is a type I transmembrane receptor spanning
the membrane at residues 1003-1019. Supported by an NIH Shannon Award
,and the California Cancer Research Coordinating Council.
Fertilization of the sea urchin egg initiates an extensive reorganization of
the egg cortical cytoskeleton. At the point of sperm entry, a fertilization cone
is formed, consisting of actin microfilaments that extend out around the
sperm, while cortical actin filaments elongatethroughout the egg microvilli.
a transmembranous glycoprotein
Yhe sea urchinfor egg receptor for sperm is interactions
at fertilization. We
responsible species-specific gamete
that
the
for
is
spern associated with the actin-based
egg receptor
propose
egg cytoskeleton and that it also may be involved in thereorganization of the
egg cytoskeleton following fertilization. We have shown that the sperm
receptor co-purifies with the actin based cytoskcleton, and that recombinant
sperm receptor protein co-sediments with a crudefilamentous actin
preparation. Using recombinant GST-sperm receptor fusion proteins, we are
the actinattempting to determnine how the sperm receptor interactsbe with
based cytoskeleton, and what other components may involved. Egg
with
cytoskeletal proteins were isolated, solubilized and incubated
recombinant receptor protein attached to agarose beads. The interacting
followed
were
eluted
SDS-PAGE
from
the
beads
and
by
analyzed
proteins
Our preliminary data indicate that two proteins, of
by silver staining.
79K and 32K, are candidates for mediating the
caloulated molecular weights
interaction. Currently, we are assessing the specificity
receptor-cytoskeleton
of the interactions using competition assays and are attempting to identify
these proteins via antibody cross-reactivity and protein sequencing.
Supported by an NIH Shannon Award and the California Cancer Research
Coordchating Committee.
1306
1307
KINETICS OF MOUSE SPERM BINDING TO SOLUBILIZED ZONAE
PELLUCIDAE. ((C.D. Thaler andRA. Cardullo)) University of California,
Dept. of Biology, Riverside, CA 92521.
CHARACTERIZATION AND LOCALIZATION OF FLUORESCENT
ZONAE PELLUCIDAE ON MOUSE SPERM ((Q. Chen andRA. Cardullo))
Dept. of Biology, Univ. of Califonia, Riverside, CA 92521.
sperm receptor were
Mammalian
sperm first bind
to
the
egg at
the
zona
pellucida (ZP),
a
glycoprotein matrix surrounding the egg.
This species specific adhesion event
additionally initiates a signal tranaduction cascade resulting in acrosomal
exocytosis. Unlike many signalling systems, the biochemical interactions of the
ligand (ZP3) and the sperm surfiace receptor have not yet been charcteized.
In this study, we quantified binding of soluble rnI-ZP glycoproteins to mouse
sperm and present data defining the biochemical interactions. Live, capacitated
mouse sperm displayed a bimodal binding pattern with a transient binding event
at -40s and a second binding peak at 10-20 min. Fixed sperm also bound
ZPs, displayed similar overall binding as live sperm, and allowed us to quanty
binding without iitiating exocytosis. Both capacited and
'25I-
non-cpadtae
sperm bound similar amounts of 'I-ZPs at equilibrium, suggeting that
n are required for eps other than
spem uface change during cra
binding to the ZPs. 12iI-ZP binding to fixed sperm was specific and saturable.
Specific binding could be displaced by unlabeled ZPs and non-specific binding
was les than 1%. Using apaed sperm that were fixed, equilibrum biding
was attained within 60 min at 37°C, and had a t,, of 14 min. Initial shdies of
saturation binding indicate a KI in the nanomolar range and a B of 14-20k
fixed
sites per sperm. These data will allow us to identify the ZP
and complementary rcptors involved in primary and secondary
binding. Funded by NI HD27244 (RAC) and a Lalor Fndn.Fellowship (CDT).
total ZP binding
components
Theinitial adhesion and signalling events between mammaliangametes involve
ligand (ZP3) within a rigid matrix (the zona pelhicida).
animmobilid
revealed that
cAbe broken down
events: (1) ligand-receptor adhesion, (2) acivation of effector moleules, and
(3) opening of Ca2 chann. Undesanding theseeventa would be sided
labeled and used toqantify the
gready if ZP3andcould be fluorescently
mobilit of the ZP3 receptor on the mouse sperm suface. We
distriion
have covalently labeled intact zonae pelucidae (ZP) with
isothiocyanate (TRITC) at pH 9.0 for 2 hours. TRITC-ZPs were sohilized
and separated on anHPLC size excluion column revealng 3 distinct peaks.
i
Biophysical modeing
has
this
into 3 distinct
The relative positions of the three peaks were identical to those using
ZP2, and ZP3 indicaing that all 3
labeled ZPs corresponding to ZPI,labeled.
were able to bind
gycoproteins wre fluorecnt
aed
TRITC-ZPs
sperm and initiate acrosomal exocyosis
as
determined
using
a
TRITC-ZPs retain adhesion and signal
properties similar to native ZPs. In addition, we have used
unsduction
video microscopy to show that solubilzed TRITC-ZPs are
digitaly enhanced
to the anterior head of the sperm overly" the aerosomal veside.
localized
Taken together, these data demonstrate that fluorescent zonae will be usefil
tools for studying ZP3
loction and dynamics. Supported by
HD27244 and the Whitaker Foundation.
Coomassie Blue assay.
Thus,
roeeptor
NIH
Monday. Fertilization I: Gamete Recognition and Binding (1308-1311)
225a
1308
1309
A SPERM MEMBRANE PROTEIN THAT BINDS IN A SPECIES-SPECIFIC
MANNER TO THE ZONA PELLUCIDA IS HOMOLOGOUS TO VON WiLLEBRAND
FACTOR. ((D. M. Hardy and D. L. Garbers)) Dept. Pharmacology and Howard
Hughes Medical Inst., Univ. of Texas Southwestem Med. Sch., Dallas, TX 75235.
The molecular basis of species-specific adhesion of spefmatozoa to the egg
extracellular matrix (zona pellucida, ZP) remains unclear. We previously Idenified
sperm membrane proteins that bind in a species-specific manner to ZP. The Mr
105,000 and 45,000 subunits of one of these proteins (p105/45) were pufified from
pig sperm membranes based on the ability to bind directly to ZP. Degenerate
primers designed from tryptic peptide sequences were used to amplify a PCR
product encoding part of the protein; this PCR product was subsequently used as a
probe to clone overlapping cDNAs that represent the entire coding sequence of the
p105/45 mRNA. The 7785 base composite sequence contained a 7431 bp open
reading frame; the sequences of eight tryptic peptides from p105 and five tryptic
peptides from p45 were present in the 2477 amino acid deduced sequence,
confirming that the open reading frame encoded p105/45. The deduced sequence
predicted a 2418 residue N-terminal extracellular region, a single transmembrane
domain, and a 37 residue C-terminal intracellular segment. Dot matrix analysis
identifed amino acid sequence similarities among four extracellular domains of
approximately 400 residues each and a fifth 110 residue extracellular domain.
These domains are homologous to the D-domains of von Willebrand factor. The
putative extracellular sequence also contained a region of repetitive sequence rich
in proline and threonine residues; these properties are typical of mucins. Northem
blotting and in situ hybridization detected expression of the p105/45 message only
within spermatids. Thus, p105/45 is a spem-specific ZP-binding protein that has
structural characteristics similar to two different classes of molecules that
participate in cellular interactions; these properties strongly imply a function for
p105/45 in sperm adhesion to the zona pellucida. Supported by a grant from the
USDA (37203-9024).
TRANSCRIPTION AND TRANSLATION OF SP56, A MOUSE
SPERM FERTILIZATION PROTEIN, IS RESTRICTED TO
SPERMATOGENIC CELLS
((L.H. Bookbinder, A. Cheng, and J.D. Bleil)) Department of
1310
INTERSPECIFIC COMPARISION OF AN ABALONE FERTILIZATION PROTEIN. ((W.J. Swanson and V.D. Vacquier)) Center for Marine Biotechnology and Biomedicine, University of California, San Diego, La
Jolla, CA 92093-0202.
Abalone are large marine archeogastropod molluscs. There are seven species on
the California coast. Fertilization is external and the species have overlapping
breeding seasons and habitats. We have been studying the molecular mechanism of species specific fertilization in abalone. Here, we present an interspecific
analysis of an abalone sperm acrosomal protein. Abalone sperm possess a large
acrosomal granule containing two proteins. One protein, 16Kd lysin, dissolves
the vitelline
The second acrosomal protein, of 18Kd, coats the acrosomal process of the sperm, suggesting it is involved in fusion or egg activation.
The 18Kd protein was purified from 2 species, and shown to have
effect
on dissolving the vitelline envelope The cDNA sequence of the 18Kd protein
envelope.
no
was
obtained
from 5 species. The
sequences are
extremely divergent, ranging
from 27%o 87% amino acid identity. Analysis of the nucleotide substitutions
demonstrates that the divergence of the 18Kd has been promoted by positive
Darwinian selection. In one comparison, the frequency of amino acid altering
substitutions (Dn) is 4.7 times higher than the frequency of silent substitutions
(Ds) (normalized for the excess number of amino acid altering sites so that
Dn=Ds if divergence were neutral). One of the purified 18Kd proteins forms
rod-shaped crystals.
Molecular Biology, The Scripps Research Institute, 10666 N.
Pines Rd., La Jolla, CA 92037.
Torrey
Sperm-egg recognition in mammals involves species-specific binding of
sperm head plasma membrane to ZP3, one of three glycoproteins in the egg's
extracellular matrix, or zona pellucida. We have identified a mouse sperm protein,
sp56, which has many of the characteristics expected of the sperm protein
responsible for recognition of ZP3. sp56 is a homomultimeric, peripheral
membrane protein, confined to plasma membrane overlying the sperm acrosome.
sp56 is the only mouse sperm protein having specific affinity for ZP3 and ZP3s
functional domain oligosaccharide, the part of ZP3 recognized by sperm.
cDNA encoding sp56 has been isolated. Primary sequence of sp56,
derived from this cDNA, indicates that it is a member of a superfamily of protein
receptors. Monoclonal antibodies and molecular probes developed from sp56
cDNA have been used to demonstrate that sp56 expression is restricted solely to
spemiatogenic cells of the mouse. Both sp56 mRNA and polypeptide accumulate
in round spermatids, which are early haploid cells of the spermatogenic pathway.
These tools have also been used to demonstrate that presence or absence of
sp56 on sperm from different species can account for species specificity of spermegg recognition.
1311
ANALYSIS OF CELL FUSION IN CHIAMYDOMONAS.((N. Wilson,
G.Huang, G.Fletcher, W.Snell)) Department of Cell Biology and
Neuroscience, UT Southwestern Medical Center, Dallas, TX 75235.
We are interested in understanding the molecular mechanisms of cell
fusion between gametes. In Chlamydomonas mt+ gametes, fusion
occurs at a well-defined site, the tip of an actin-filled, microvillus-like
fertilization tubule. Formation of this fusion organelle is triggered by
signals induced by flagellar adhesion. To identify adhesion/fusion
proteins we have begun to study fertilization tubules using both genetic
techniques and cell fractionation. Screening of 11,000 insertional
mutants yielded 7 fusion-defective clones that underwent normal
flagellar adhesion, but did not fuse to form zygotes. Thin-section
electron microscopy and fluorescence microscopy with phallacidin
indicated that 2 mutants, 6D1 and 60A8, formed fertilization tubules
that did not bind/fuse with the mating structures on mt- gametes,
suggesting a defect in adhesion/fusion proteins. In a parallel approach
we have employed cell fractionation to obtain fractionshighlyenriched
in fertilization tubules as indicated by fluorescence microscopy and by
immunoblottingwith anti-actin antibodies. SDS-PAGE combined with
surface biotinylation has identified several proteins as candidates for
fertilization tubule surface proteins. Studies are in progress to compare
wild type and mutant tubule proteins and to generate monoclonal
antibodies against tubule proteins that function in gametic fusion.
(Supported by NSF EBM-9318708).
Invertebrate Development (1312-1313)
1312
1313
AN EXPRESSED RNA HELICASE GENE IS EMBEDDED IN MANX, A GENE
REQUIRED FOR DEVELOPMENT OF THE ASCIDIAN TAILED LARVA. ((E. L
Pedersonl, B. J. Swalla2, M. L. Just 3, and W. R. Jefferyl,3)). iCell and
Development Graduate Group, University of Califomia, Davis, CA 95616,
2Department of Biology, Vanderbilt University, Nashville, TN 37235, and
3Bodega Marine Laboratory, Bodega Bay, CA 94923.
EXPRESSiON OFTHE hG4WGENE IS RKEURED FOR SPECIFYINGTHE LARVAL
BODY PLAN IN ASCIDIANS. ((B. J. Swalls1 and W. R. Jeffery2))
1Department of Biology, Vanderbiit University, Nashville, TN 37235, and
2Sectlon of Molecular and Cellullar Biology and Bodega Marine Laboratory,
University of CaliHomia, Davis, Bodega Bay, CA 94923.
The manx (uro-1 1) gene was identified by a subtractive screen for cDNA
clones expressed during development of the tailed ascidian Molgula oculata
but not the closely-related tailless species Molgula occulta. Manx encodes
a zinc finger protein that is required for development of the tailed larva.
The manx gene encodes 2.3 and 1.9 kb mRNAs, which differ only in the
length of their 5' leader sequences. Several overlapping M. oculata
genomic clones were isolated that encode the entire manx gene. The
manx gene contais 9exons and spans about 6 kb of DNA. The first exon
In the manx gene is separated from the downstream exons by a 2.7 kb
intron. Surprisingly, this intron contains a single copy gene encoding a
putative DEAD box-family RNA helicase with high sequence homology to the
yeast and mammalian p68 genes. In contrast to the yeast and nmamallan
p68 RNA helicases, the ascidian p68 gene lacks Introns. Northen blots
indicate that manx and p68 have the same developmental expression
patterns in M. oculata embryos, but both genes are down-regulated in M.
occults embryos. However, manx but not p68 mRNA acccumulation is
enhanced In hybrids produced by fertilizing M.
eggs with M.
.
oculata sperm, suggesting the two genes can be regulated ind
The unusual structure of the manxlp68 gene complex suggests that the
p68 gene may have been retrovirally inserted before the divergence of M.
oculata andM. occulta.
occuvta
The zinc finger gene manx (uro-1 1) was identified in a subtractive screen
cDNA clones expressed during development of the tailed ascidian
Molgula oculata but not the closely-related tailless species Molgula occulta.
M. oculata embryos differentiate a brain sensory organ, a notochord, and
tail muscle, whereas these tissues are lacking in M. occulta. The missing
tissues are restored when M. occulta eggs are fertilized with M. oculata
sperm, suggesting they are specIfied by zygotic genes that are inactive in
the tailless species. The manx gene is expressed in presumptive
notochord, neuroectoderm, muscle, and tail epidermis in M. oculata but
not M. occulta, and manx mRNA accumulates In the same restored tissues
In hybrid embryos, suggesting a role in specification of tailed larval
features. Hybrid embryos were treated with manx antisense
phosphorothioate oligodeoxynucleotides (ODNs) before first cleavage and
their phenotypes were examined after hatching. Treatment with an
antisense ODN corresponding to the +4/+21 region of the manx gene
reduced manx mRNA to undetectable levels, but had no effect on muscle
actin mRNA accumulation. The corresponding sense ODN did not affect
manx mRNA levels. Antisense ODN-treated hybrids failed to develop the
brain sensory organ, notochord, and secondary tall muscle cells, but these
tissues developed normaily in sense ODN-treated hybrids. Similar results
were obtained with antisense ODNa in other regions of the manx gene.
The results indicate that manx expression Ia required for restoration of
tailed larval features in hybrid embryos, suggesting that the manx gene
plays a fundamental role in specfyng the larval body plan in ascdans.
for
226a
Invertebrate
1314
Development (1314-1319). Monday
1315
ASCIDDANS
WWll
EVOLU'rONARY CHANGES IN MUSCLE AC71N GENES IN
ALTERNATE MODES OF DEVELOPMENT. ((T. Kusakabebl2, B. J. Swells3, N.
1
Bodega Marine Laboratory, University of
Satoh2. and W. R. Jefferyll)).
Califoria, Davia, Bodega Bay, CA 94923, 2Department of Zoology, Kyoto
University, Kyoto 606-01, Japan, and 3Department of Biology, Vanderbilt
University, Nashville, TN 37235.
CELL CONTACT DIRECTED SPINDLE ORIENTATION IN EARLY DEVELOPMENT
((S. W. Wang', F. J. GrUn' & W. H. Cr,k Jr.15)) 'Bodeg
Laboratory,
MarIn
Cdlornla,
UniversIty of
and Aquatic Scince
DavI,
Bodega
Bay, CA 94023.
estmet of Fisheries
Uniesiy of Foidda, Genevl, Ft 32806.
Sdyoh Ingendls
During saly ceavages
In
eby
mitoti c spindle orIeta
er between blastomr
and change a predictabe arnwe
dll
aat0angle
succesive mitoels. From e through cevge, sp
orent9
In
Molgula occulta are closely-related ascidians with
Molgula oculata and
different modes of development. M. oculata embryos develop into tadpole
(urodele) larvae with striated tail muscle cells, whereas M. occulta
embryos develop into tailless (anural) larvae lacking differentiated muscle
cells. The muscle actin genes MocuMA 1 and MocuMA2 were isolated from
M. oculata genoric library. MocuMA has no introns and is expressed
specifically In larval muscle cells, whereas MocuMA2 has four introns and
resembles adult muscle actin genes in other ascidian
5' species. Despite
lacking introns, MocuMA 1 is a functional gene because its flanking region
is sufficient to drive in vivo expression of a microinjected lacZ fusion
construct. Muscle actin mRNA was not detected in M.
embryos,
suggesting that the gene(s) corresponding to MocuMA is not expressed.
Two different intron-less genes similar to MocuMA were isolated from an
M. occulta genomic library. These genes have nucleotide substitutions and
insertions that could make their products non-functional. Hybrid embryos,
produced by fertilizing M. occulta eggs with M. oculata sperm, express
MocuMA1 mRNA in vestigial muscle cells, suggesting that trans-acting
factors regulating larval muscle actin genes have been retained in M.
occulta. Muscle actin mRNA could not be detected in embryos of five
other anural species, although transcripts were observed in closelyrelated urodele species. Analysis of structural changes in the larval
5' flanking regions, may elucidate the
muscle actin genes, including their
basis for lack of expression in M. occulta embryos.
an
occulta
embryoswas pall
which
blatomere
The e
of
Drosophila abdominal hypodermal muscles (AHM) are remodeled
larval intersegmental muscles that survive the destruction of
neighboring larval muscles during metasmorphosis. These
larval muscles then lose their contractile apparati and
Subsequently, they
become strap-like bands of cytoplasm.
regenerate into the muscles responsible for the emergence of
pupal
case
and
later
for the inflation of
the
the adult from
the wings. We have devised a selection scheme for the
isolation of Drosophila mutants with defective AHN in order
This procedure has been
to study the regeneration process.
applied to the isolation of mutants on the third chromosome.
We have been successful in isolating several recessive
mutants that cannot emerge from the pupal case as homozygotes
unless the operculum is opened manually. These flies are
also unable to inflate their wings after emergence. We are
currently screening these mutants for defects in abdominal
musculature by examination of abdominal "pelts" and have
observed abnormal AHM. Isolation of
ultimately permit genetic dissection
regeneration process.
such mutants will
of this muscle
tothe
lay
hI
produced
four paralle quartts. Then,
ceavage
respec
44'ange
nrxmaldevWopnt
to
with
orientatIon
mitotic
embryos.
leave
We
bt rath
s
regions
of ths
pealle pairs
hI
to
the
substrate.
the
ret In
embryos sugges
where th
ed
spinl
poles
during
Grant
eauly
deparKewlt
but
wIh
(ceroom"es)
cll-cd ontact during
en
Sea
hI
se
of mitoic spndles
nor age
(Supported by NOAA
as
were not
d tod orinwtation
an
property
Itrrnc
conunctIon
thmes
Furthr
at a
wer separated into lkiear halflike tiose observed for Intct
In 4-cll lkiea
liner emyos
regresse
hI
arage
sM
ori
reore
Is
mrtherelcel contact related.
d nonmranulated
cytopimic
eeuthhg
parll
prepartion
orietatI
sugges
of
(3W emnbryonic cleavege)
foor 5e eavage, spinles
previou ceavage plan, rathe than parall
In
When 8-cl
embryos,
8-cd lina
Ue
recbig the
axle of the embryo, r
a common pln which we
a linea embryo wth the 16
the
Is
I specific
res, vieated
wea affected hI
coy.
embryos, subequnt spidle
#NA36RG)7)
early
ACTIVE NUCLEI FROM
ISOLATION ((R.OF A.TRANSCREAONAILY
and K. Q. Tran)) Dept of Chemistry and
Acey
90840, and ((J. C.
Biochemistry, Cal.ofState Univ.,andLong Beach, CAWorcester
Polytechnic
Biotechnology,
Bagshaw))Worcester,
Dept MABiology
01609
Institute,
the fators regulating gene expressin durng the
Attempts at und ofandig
mpered by theiabiltyto isolate
eaiiy development
acie Arma
nuclei freehaveofbeen
yolk pladets. We report here
procedure for theisolation ofactive nuclei from both encysted embiyos and
pH 7.5, 10 mM
swimming naupl Shrimpare mgenized in 10mM
MgQ2, 10 mM NaCl, 0.l%(vtv) NP40 andoentrifuged to obtain a crude
buffer without
pell The pellet is ruspended in homogenizato
nuckl
gradients.The nuclei band at the
detergentandfiactionated on buffered Prcoll
as
of
well asLDH
is
platet
yolk
Percoll/buff
and SDH activity. The nuclei in
at 32p UTP
into RNA for up to 30
l sCi of 32pminutes at 30D C in the following buffersupplmented with 30
UTP: 30 mM Tris, pH 7.6, 75 mM KCG, 2.4 mM Mg(OAc)2, 20 mM
2mM
20 mM DTT, 200 &M
400isM CTP, 400
aM GTP, 100pM UTP, and 0.4 U placental ribonuclease inhibitor. Moreover,
the ransciptioal activity of the isolate nuclei mimics the in vivo activity during
i.e., nuclear actiiy peaks dwing the first 24 hrs of
early development,
then deases signifiany. Preinicubation of the nuclei with
development and
D
activity while
Actinmycin completdy abolishes their
with a-amanitin causes a 30% decease in activity. Southern blot
I
RNA from nuclear nm-on assys indicates the pesene of
analysis of the and
tubulin gene
Supported by NIH-M 08238
rRNA,
sioy
Tns,
rliable
free
inter&fe.pTepeatio fiee
(NH4)2S04,
histone,
1318
orbtrton
To detemin
ble
ing
4-cd
tdbydesocl
c
wer
hI a near
erey. The enuing ceavage
ARTEMIA.
DROSOPHILA MUTANTS WITH DEFECTS IN REGENERATION OF ABDOMINAL
MUSCLES. ((S.L. Tobin and A. Wilson Malaska)) Department of
Biochemistry and Molecular Biology, University of Oklahoma
Health Sciences Center, Oklahoma City, OK 73104.
the blasme.
of
and asked
each
spiNe
pene that fome
a
1317
Using G-50 Sephedex molecular exclusion and DEAE-Sephadex ion-exchange
we reported the presen
of a soluble, developmentally
regulated,nlalothionein-lhe zinc bining poten (ZnBPII) in Artemia. We
report here that ZnBPII isactually composed of fourindividualzinc binding
activities Forty eight hr naupii were collected and homogizdin 50 mM
Tris,pH 8.0, 0.1 mM DTT, 0.5 mM PMSF and 10pg/ml of SBTL The
soluble proteins were obtained by highspeed centrifugton,incubated with
on a G-50molecular exclusion column. The
109Cd, and thenfrac
column eluate was monitored for 109Cd and endogenous Zn to locate metal
binding activities. Peak fiacons peaning to ZnBPII wee pooled and
applied to an FPLC Mono Q ion-exchange column.The column was eluted
with alinear gradient of Tris, pH 8.0 (10 to 400 mM) Four endogenous zinc
binding activities (ZnBPHab,c,d) are cleaiy resolved by this procedure and
coelates dircty with the exogenous 109Cd binding activity. The individual
aceamide andfa ated by SDS-PAGE
pteinswere allated with i
using a10-20% liear polyacrylamide gradient Each of the preparatin was
judged tobe homogenous usng silver stain deectio Theindivdual poes
all had a caculated relative molecular mass of 6700, cactistic of
metallobhionein. Inteeting, itappearsthat the four proteins arediffeentially
expressed during development Supported by NIH-GM08238
spinle of the par
perdel to the
spidle orintatwon
1316
chromatography,
ne
bastomer Thus,
dcvage
tuinsic propertes nhd udu st
w
soClatloMM
how
spindeb
mic1 .
confocal
lawser
ceseaseavage
scumingbryoe
and
to the
with respec
MMTAL BINDING
PURIFCATION OF METAILLOTONEIN-LIKE
PRaOTINS FROM ARTEMIA. ((J. L. Brook, B. G. Harpham and R. A.
of
and
Biochemistry,
Cal.
State
Univ., Long Beach,
Acey)) Dept Chemistry
CA 90840
wh
7"
ATP,
MnCI2,
transcripts.
1319
CLONING AND CHARACTERIZATION OF GASE GENES, A GROUP
OF PUTATIVE SERINE PROTEASE GENES EXPRESSED IN THE
MIDGUT OF DROSOPHILA MELANOGASTER. ((Q. Wu, C.M. Cheney,
and J.E. Sadler.)) HHMI, Departments of Medicine & Genetics, The
Jewish Hospital of St. Louis, Washington University School of Medicine,
St. Louis, MO 63110.
Serine proteases participate in a variety of essential biological events,
such as food digestion, blood coagulation, complement activation, and
To further explore the function of serine proteases in
fertilization. four
novel putative serine protease genes, Gasel-4 (for
development,
gastric serine protease), were cloned from a Drosophila embryo cDNA
library. Gase cDNAs encoded a group of chymotrypsin-like or elastase-like
enzymes that consisted of a signal peptide, a short activation peptide, and a
domain. Expression of the Gase genes was
carboxyl terminal protease The
developmentally
regulated. transcrpts of the Gase genes were abundant
in late embryos (12-18 hr) as well as in larvae and adult flies, but completely
absent in pupae. Northern analysis and in situ hybridization showed that
in the gastric
Gase mRNAs were localized in the anterior midgut,
were
hybridization to polytene chromosomes, Gase genes
caeca. By
99C8, and
to
several
66C5,
65A1-2,
25B1-2,
mapped Similar positions including
99F6-7.
chromosomal locations were reported previously for the
Jonah genes which also are expressed in the midgut, but the structrues of
Jonah proteins have not been reported. Two of the original Jonah clones
were analyzed and shown to contain typical serine protease gene sequences
that were different from Gasel-4. These results indicate that Gase and
Jonah genes belong to a multigene family of over 20 genes, and that the
members of this gene family encode serine proteases that may function in the
digestive system of Drosophila.
especially
Monday. Invertebrate Development (1320-1324)
1321
1320
SCREENING FOR GENES INVOLVED IN THE IMMUNE SYSTEM AND TUMOR
FORMATION IN DROSOPHILA. ((D.A.Kimbrell, ARodrigue, C.Wu, X.Zhou,
n)) Deparmn of
E.Yonters,S.Meller.,Jhen, K.MaLtin. LMann, K.Hale,
Biology. University of Houston, Houston, TX 77204, HHMI and Institute of
Molecular Genetics, Baylor Colge
Medicie Houston, TX 77030.
H.BelW
The Drosophilh immune
effectively respond to boceril
oo ponertsVt
and c
llular
by the production of
proteins
i als orltical
. Thas
systemn
to
aggregation of hemocytes. We have
hashumoral
ifction
swteib
and the mobilizaonf hemocytes for
tumor formatio, whichls hallmarkd by the
the
screend enhancer detectorattai to find two typesof genes relaed to e
iriection, B
procese": A -genes induced or decrease in expression by
- genes exp
immune system
sse in
tissues. i.e. hernocyts lymph glnds (Ih
bacterial
hematopoltc organ) and fat body (majorsite ofantibectrial proteinsyrhesis).
For type A, we devised n
a ew variation of enhancer detection in which
slings of
eachstrainareassayed _ r o
wiIh
folyforthelevelof
response ogaiacoeidase
Sains
that show an increase or decrease inB
and without inection.
genes. 2,600
upon infecton are caKides forlines identifying knm e
stocks were screened for type A and/orB, and 3 type A strains have been found.
and
the
in
Twosirains areki esd in Mcx_seexpresin both
stages upon infection, and one strain is developmeally resticted to the laval
hrval
adult
stage. For type B. embryos andVorlavae were screernd and 18 strains with
hemocyte, lymph gland and/or fat body expression were identified. We
characterized the reporter gene expression of many of these strains throughout
development and are using these to study the developmental origins and
relationships amongimmune systemtissues. Two Pelnemnts map to the third
chromosome and the remaindermap to the second chromosome. P element
mobilization of all strains tested far confirm that lethality resultsfrom the
insertion. Geneticcomplementation toest and restriction mapping of pasmnid
two of thesestains carryinrtions into the sane
rescued DNA show that at
ximately 100 base pais apart. These two enhancer detector
locus and are
so
stmis
are
associed with
foffation
((S.W.
Chem, J. Hau,
L Berg
and GM. Weael))
Biology and Cell Biology & Bio.
Department
of Molecular
eaist, Brown University, Providence RI
THAT ALTER PATTERNS OF GENE EXPRESELEGANS. ((C. Xie, Y. Jia and F. Aamodt)) Department
Biochemistry and Molecular Biology, Louisiana State University Medical
SION IN C.
Center-Shreveport, Shreveport, LA 71130-3932.
developed a method for isolating mutations in Caenorhabdiis ecantibody or histochemical staming patterns. The basis for this
method is a new procedure for making C. eLgans permeable that does not kill
the eggs contained within the uterus of gravid adult hermaphrodites. We have
used this procedure to isolate mutations in genes that affect the expression of
and pag-S, will be
a mec-71acZ fusion gene. Mutations in two genes, pag-i
chromosome IIL
described. pag-i
is located between lon-i and dpp-17
Mutations in peg-i cause 8-16 fold higher expression of the mec-71ecZ
the amount of
and an
not
increase
does
wnc-861acZ gene. A pag-i mutation
mec(B7lacZ mRNA but it does increase the amount of polyadenylated message
we believe that the peg-i product is involved in deadenylation of mRNA
in preventing polyadenylation. peg-S is on the right arm of the X-chromosome
between the right end point of mnDfS and mnDf20, which lies between sne-S
and wnc-7?.
A mutation in peg-S results in expression of the touch neuron
the
specific genes mec-71ecZ, mec(B4lecZ and mec-7 in the BDU
lineal sisters of the ALM touch neurons. The pag-S('lsO) mutation also causes
a reverse
kIinisr Uncoordinated phenotype and axonal guidance errors. The
pag-S gene product may be involved in spedifying or interpreting spatial inforWe have
that alter
on
gene
so
or
neurons,
.
We are
A
CHAIN
GENE
Davalos',
defects in hypodermal morphogenesis.
differentiation.
1324
elegans
A.
DURING
SEA
Z. Rosenttal'
URCHIN
and
of
Hawaii.
The spreading and rearrangement of epithelial cells is a widespread phenomenon
development. Our laboratory is using digital time lapse,
04-dimensionalO, and confocal microscopy to characterize the morphogenesis of
the embryonic hypodermis of C. elegans as a prelude to a genetic analysis of this
model embryonic epithelium. The cells of the dorsal hypodermis, which initially
comprise a strip two cells widenlnning the length of the anterior-posterior (A-P)
axis, interalate to form a single row. Concurrent contralateral nuclearmigration
also takes place in these cells. Based on analysis of unc-83 (e1408) embryos, in
which nuclear migration is blocked, these processes are separable. The nuclear
migration defects can be phenocopied by treatment with nocodazole without
perturbing intercalation. Ventral hypodermal cells undergo epiboly, ultimately
enclosing the embryo as they meet at the ventral midline. We have used 4-d
microscopy and laser ablation to investigate the role specific hyp-6 and hyp-7
precursors play in enclosure. The bilateral pairs of cells derived from ABpraapp and
ABplaapp seem tobe particularly important for the advance of the ventral margin.
Ablation of the C blastomere or its progeny indicates that dorsal and ventral
morphogenetic movements areregionally autonomous, at least within large sectors
along the A-P axis. We are currently engaged in screening a bank of zygotic
embryonic lethal mutations, as well as existing chromosomal deficiency strains, for
MUTATIONS
mation in C.
LMUNfN
Page',
Biological Sciences, California State
Hayward. 2Kewalo Marine Lab, University
Laminin is a multi-domain basement membrane glycoprotein
composed of three chains; Bi, 82 and A. A cDNA representing a
region of laminin A chain was isolated from aS. purpuratus
lambda ZAP cDNA library. Sequence analysis indicates this cDNA
(WL 23)represents 1307 bp encoding a region of the globular or
"G" domain at the carboxyl terminal region of laminin A chain.
The G domain is composed of five homologous repeats
(Gl-G5).
WL 23 represents G2-G3. A homology search using the deduced
amino acid sequence of the
cDNA showed 30-40% sequence
similarity with other laminin A chain genes in the carboxylterminal G domain. Other sequence features are more highly
conserved including invariant cysteine residues which form the
loops of the "fingers" in the G domain. Northern blot analysis
indicated that the 11 kb mRNA was present in unfertilized eggs
and remained at near steady state levels through development
to the 72 hr pluteus stage embryo. Inhibition of zygotic
transcription in 4-8 hr embryos by actinomycin D suggests
steady state levels require continuing transcription. Northern
blot analysis using RNA isolated from polysomes of embryos at 4
hours after fertilization indicates that there is active
translation of the maternal mRNA at 4 hours post-fertilization.
A polyclonal murine antibody was raised against a fusion protein and Used to invest- igate temporal and spatial accumulation of the laminin A chain protein. Developmentalimmunoblot
analysis on total protein extracts indicates the presence of
immunoreactive protein of 400 kD in eggs which increases with
the formation of a basal lamina during blastulation. Immunofluorescence microscopy reveals reactivity confined to the basal
lamina of blastula, gastrula and pluteus stage embryos.
in animal
a
by transformation resu
TE
((L.
THE CELLULAR BASIS OF EPITHELIAL MORPHOGENESIS IN THE
EMBRYONIC EPIDERMIS OF C. ELEGANS. ((J. Hardin, A. Malik, and E.
Williams-Masson)) Dept. of Zoology and Program in Cell and Molecular Biology,
University of Wisconsin-Madison, Madison, WI 53706
The extracelular matrix (ECM) plays an important regulatory role during
endoderm cell differentiation and gastrulationin the early sea urchin embryo. Most
of the ECM molecules identified in the sea urchin embryo are stored in the egg
within secretoryvesicles. Fertlization initiates series of regulated secretory
events of these ECM vesicles that contribute to formation of the nascent basal
lamina and the blastocoel matrix Herewe describe an ECM molecule of
osregatus with a contrastng pattem of expresson that was identified by
L1tec
an expression cDNA screen using antibodies to the embryo ECM. We found that
the ECM 18 protein is not detectable in eggs or early embryos eventhough its
mrRNA of approximately 6kb is present matemnally and thoroughout early
development. During gasnuabon, mRNA accumulatedseveral fold over matemal
levels and was enriched approximately 3 fold in the endoderm. Using antibodies to
reombinant ECM 18 proteins in either westem blot or immunofluoreseence
assays, no protein was detectable in eggs or early embryos until just prior to
gastrulation. Dunng gastrulation the protein accumulated to high lvels throughout
the basallamina/blastocoel matrix. Partial characterzation of the ECM 18 DNA
sequence shows a repeat motif withhigh cysteine content (10.5%). No sequences
similar to ECM 18 were found within theGenbank. These results suggest that
ECM 18 is anewly described ECM molecule whose expression is rgulated by
translational control. Since this extracellular matrix protein is expressed just prior to
gapulation and selectively by invagnating endoderm cells ECM 18 may participate
gans
OF
1323
0291. (Sponsred by AW. Colem )
in the regulation of endoderm
EDCX8SSICN
DEVELOPS(NT.
en n'Det.5ept.
BUniversity,
of tumors.
1322
UNUSUAL EXPRESSION OF AN EXTRACELLULAR MATRIX
MOLECULE DURING GASTRUIATION IN THE SEA URCHIIN EMBRYO.
of
227a
presently attempting to
cone
peg-I
and
pag-S
228a
Cell Lineage (1325-1330). Monday
1325
1326
LIPOCORTIN-1 IS EXPRESSED IN MICROGLIA AND THEIR
PROGENITOR CELLS IN CULTURES. ((S. Fedoroff, andJ.A. McKanna))
Department of Anatomy, University of Saskatchewan, Saskatoon, Canada
and Depatment of Cell Biology, Vanderbilt University Medical School,
Nashville, Tennessee.
A MATHEMATICAL MODEL TO TEST HYPOTHESES ABOUT THE
DEVELOPMENT OF
IN CULTURES. ((JP. Novak S.
1800 mnonte Ste.-Julie, Varennes, Que., Canada J3XlSl
Fedoroff))
and Department of Anatomy, University of Saskatchewan, Saskatoon,
Sask, Canada
S7N OWO. (Spon. by GD. Burkholder)
In
rat embryos lipocortin-l (LC-l) is expressed at E12.5 in primitive
Disaggreged cells from newbon C3HJ armou nu llimonin cultur
or microglia, depending
culture
astroglia,To oligodendrogliadevelopmental
pathways of astrogia
conditonns.
we
a
mathemacal
microglia quantitadve peimentaldataw
Neopallial cells were planted atS 103 cells/cm2/l.5 medium
(modified MEM, 5% horse serum, 10% medium condidoned by LM
fibroblasts which contains CSF-1, and 5% mediumconditioed bySTO
fibroblasts which contains IL-6). The resulting cell colonies were
immunostained for OFAP (astroglia marker), and CR3(miicrogia markr),
and cel nucki, were stained with Hoechst 33258.Th total cell number and
numbers of varioustypes of colonies were counted after 1,3
days. A
of a common astrocytemathematical model based on the assumptiontotal
numbr of unbaeled,
yielded good agreement
microgiaprogenitor
CR3+ and GFAP+ cells, total number of CR3+ andGFAP+ colonies and a
glia
in the floor plate of the hindbrain and spinal cord.
LC1§ primitive glia give rise to LC-1l cells which have the characteristics of
(McKanna,
Res
491,1993).
36:
We
now report
microglia.
Neurosc.
ttat
LC-1+ cells appear to he pronitors of the microglia (Mac-I+) that are
induced by CSF-l andLL-6 tissue cultures initiated from
Po mouse
neopallium
We counted LC-1+ cells in 3, 5, and day neopalluaa cultures
anddeermined the number of LC-l1 cellsthat were also Mac-lI or GFAP+.
In 3 day cultures 67.8% of LC-l cells were LC-lI+Mac-l- and 31% were
LC-l+IMac-l+. In 5 day culturs 50.7% were LC-l+/Mac-l- and 46.9%
forming araphe
29.5% were LC-1+/Mac-1- and
cells were also LC-1+.These
results indicate that microglia precursor clls express LC-1 before they
receptor
d
t
CR3
immunoreacts with Mac-l antibody and is
express the
characteic to microglia. e observations that microgia can form from
the primitive glia ofthe floor plate and thatin neopqalial cell culures the LCl+/Mac-l- progenitorcells can give rise to LC-l+/Mac-l+ mic glia,strongly
support the notion that microglia are of neuroepithelial origin and may
originate in more than one site in the CNS. This wok was supported by
grant MT4235 from MRC Canada and by the anadi Network ofCentres
of Excellence, NeuroScience Network
LC-l+/Mac-l+. In 7 day cultures
66.5% were LC-l+/Mac-l+. All Mac-l+
were
1327
DORSAL AND VENTRAL CELLTYPES CAN ARISE FROM COMMON
PROGENITORS IN THE CLOSING NEURAL TUBE((K.B. Artingerl, S.E.
Fraser2 and M. Bronner-Fraser1)) lDevelopmental Biology
Center, University of California, Irvine, CA 92717 and 2Division
of Biology, California Institute of Technology
To challenge the developmental potential of dorsal neural tube
cells and test whethersingle neuroepithelial cells can give rise to
the full range of neural tube derivatives, we grafted a notochord
lateral to the dosing neural folds. This results in juxtaposition of
dorsal and ventral cells types, by inducing floor plate cells and
motor neurons dorsally. Clonal analysis with the vital dye
lysinated rhodamine dextran (D) showed that both "dorsal"
and"ventral" neural tube derivatives can arise from a single
precursor. Cells as diverse as sensory ganglion cells, presumptive
pigment cells, roof plate cells, motor neurons and floor plate cells
were observed in the same done. The presence of such diversity
within single dones indicates that the responses to dorsal and
ventral signals are not mutually exdusive; even in the dosing
neural tube, neuroepithelial cells are not restricted to form only
dorsal or ventral neural tube derivatives.
IREQ, MICROGLIA
form
determine the
1329
HOMEOBOX CONTAINING cDNA EXPRESSED IN ALVEOLAR
TYPE II CELLS DURING LUNG DEVELOPMENT ((Hamdan, H.,
Metter, J., Horowitz, S., Dodds, S., deLenos, R., & Minoo, P.)) Dept. of
Pediatrics, USC School of Medicine, Los Angeles, CA 90033
Immaturity of alveolar type II (AT2) cells which includes aberrant expression
of SP-A, a protein comnponent of surfactant, has been described in premature
neonates born in advance of complete in utero development and may involve
absence (or inactivity) of AT2 cell-specific transcriptional factors. Cklning
and analysis of a human SP-A genomic clone revealed the presence of helixturn-helix-like binding motif in the 5' upstream domain of this gene. Using
degenerate oligonucleotides, and AT2 cell cDNA, we amplified and cloned a
DNA fragment of approximately 150 nucleotides which contains a
homeodomain. Screening 105 phage clones from a fetal lung cDNA library
with this fgment yielded 8 cDNAs, suggesting high abundance of
complementary sequences expressed in fetal lung. DNA sequence detmination
of one clone, 12A2 revealed 98% homology to a thyroid transcription factor
TTFl Analysis of mRNA complementary to 12A2 demonstrates: 1) expression
of 12A2 occurs in AT2 cells and not adult lung fibroblasts; 2) 12A2 cDNA
hybridizes to one major RNA band at 2.3 kb which is expressed during lung
development and two minor, larger bands which appear to be developmtally
regulated with the highest level of expression found in association with
completion of fetal lung development. Priser extension/rapid amplification of
5' ends of the mRNA, RACE is underway to clone and characterz the
developmentally regulated minor
transcripts. We hypothesize that 12A2 may
play a role in cell lineage and differentiation of AT2 cell during lung
development. Supported by NHLBI 48298.
and
modeL
x
mi
and 5
for
number of mixed CR3 andGFAP+ colonies (typically within
±i15% with
one exception of +28%). Differendal and cumulative distributions of the
colonies according to specific cell counts and bivaiant distributions of the
mixed colonies (CR3 plus GFAP+ and CR3 plus unlabeled cells) were
well represented. A model assumingmonopotential progenitors yielded
reasonable agreement for the total cell and colony counts and for colony
distributions; however, the CR3+ andGFAP mixed colonies were
underested by 70%. Overall compason withobservatons favors the
hypothesis ofa common CR3+ and GFAP+ progenitor. Supported by grant
MT4235 from MRC Canada.
1328
EMERGENCE OF SALIVARY GLAND CELL LINEAGE DIVERSITY
SUGGESTS A ROLE FOR EGF RECEPTOR SIGNALING. ((E.M.
Durban and P. G.
University of Texas Houston, HSC, Dental
Branch, Division of Oral Pathology, Houston, TX 77225.
Nagpala))
Diversity of
cell
lineages within glandular
is generated
progenitor cells.
not understood.
Differentiative transitions of mouse submandibular salivary gland
served as a model to assess the role of epidermal growth
factor (EGF) receptor signaling in generation of cell lineage
protection analyses revealed temporal fluctuations
diversity.
in EGF receptor mRNA levels coincident with crucial differentiative
cell lineage transitions. EGF receptor mRNA levels were highest
between Days 2-S when proacinar cells are maturing and striated duct
cells emerge; all differentiating cells exhibited EGF receptorspecific immunoreactivity during this period. Following these
differentiative events, EGF receptor mRNA levels declined sharply
and immunoreactivity became confined to ductal cells.
A second
increase in receptor mRNA occurred between Days 11-16 coincident
with emergence of granular convoluted
cells which were
strongly EGF receptor immunoreactive. Reductions in EGF receptor
mRNA levels and intensity of immunoreactivity were observed at DAY
22 upon completion of GCT cell emergence. Thus, temporally distinct
patterns of EGF receptor expression correlate with identifiable cell
lineage transitions in the developing SSG suggesting a role for this
signaling pathway in the emergence of cell lineage diversity in a
organs
by differentiation of committed
postnatally
Fundamental aspects of this process are
(SSG)
RNase
(GCT)
glandular
A
and
organ.
Supported by grant DE07766.
1330
PERMANENT CELL LINES FROM MOUSE VON EBNER GLAND. ((L.S.
Chen and M.L. Snead)) The University of Southern California, Center for
Craniofacial Molecular Biology, School of Dentistry, CSA 142, 2250 Alcaar
Street, Los Angeles CA 90033
Yon
Ebner's glands (VEG) are small tubulo-alveolar serous salivary glands
whose secretory ducts open
into the troughs at the base of the taste
bud containing
papilla. Morphological and biochemical data
indicate that their anlages acquire functional acinar structures late after birh, at
the
when taste buds in the cicumvlae papillae divide and mature. Von
Ebner's gland secretion is serous
polypeptides such as acid
phosphatase, amylase, lingual lipase, and members of the lipocalin superfamily
of VEG-proteins. The anatomical organization, biochemical properties and
neurological control of this gland strongly suggest that Von Ebner's secretion
play a role in chemical discriminaton, perhaps similarly to the functions of
define the role of VEG
accessory glands of the olfactory epithelium. To
cell products, we are developing permanent cell lines from the Von Ebner's
mice bearing the MHC-promoter coupled to the
glands of
circumvallateexclusively
timne
including
better
transgenic
transforming
"1
antigen of polyoma
virUS
(Immortomice"). Using RT-PCR
with amplimers unique to a VEG-specific messenger RNA, we demonstrate
that isolated VEG.derived acinar cells grown in monolayer, maintain a
dominant feature ofthe intact gland, namely, the capacity to transcribe a VEGcells have the potential to
specific messenger RNA. These
the execution of experimental strategies designed to understand VEG's
functions difficult to execute due to the physical limitation of the gland in situ.
VEG-derived
pennit
Monday. Cell
ineage
(1331)
229a
1331
MEGAKARYOCYTIC DIFFERENTIATION IN HUMAN LEUKEMIC
CELL LINES: ALTERATIONS IN TRANSCRIPTION FACTOR ACTlVITY ((Karen M. Hudson and Michael A. Lieberman)) Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine,
Cincinnati, OH 45267.
The mechanism of differentiation of a pluripotent bone marrow stem cell into a committed multinucleated megakaryocyte is poorly understood. To investigate the role
of transcriptional regulation in megakaryocyte development, several experimental approaches were taken utilizing human kukemic cell lines as a model system. The human cell lines (CHRF-288-11, K562, HEL) all differentiate exclusively through the
megakaryocytic lineage when treated with the appropriate concentration of phorbol
ester. Studies with the CHRF-288-11 cell line demonstrated that a number of transcription factors were up-regulated in response to phorbol esters. Gel moblity shift
assays and Northern analysis indicated that the transcription factors AP-1, NF-1, and
ETS-1 were all up-regulated during differentiation, although the timing of the regulation differed between them. These changes were also observed with either the K562
or the HEL cell lines. GATA-1, a hematopoeitic specific transcription factor, does not
change its level of expression or activity during CHRF-288-11 cell differentiation. Similarly, the level of expression of the transcription factors SCL, MYC, MAX, and SP-1
were unchanged as the cells differentiated. GATA-2 was found not be expressed in
CHRF-288-11 cels as determined by RT-PCR. These data suggest that regulation of
transcription during megakaryocyte differentiation may be occuring through changes in
the levels and activity of the factors AP-1, NF-1, and ETS-1. The relationship of these
factors to that previoudy identified (SKI, Oncogene 9:1407-1416,1994) is currently under investigation. This work was supported in part by grants from the NIH (HL-51555
and T32-ES07250)
Pattern Formation (1332-1335)
1332
1333
E(PRESSION OF A HOMEOBOX GENE, msn 2, iS AFFECTED BY ABERRANT
PaK 3 IN SPLOTCH MUTANT EMBRYOS. ((Y. Takahashi1, and J. A. Weston))
Institute of Neuroscience, University of Oregon, Eugene, Oregon, 97403.
1 Present address: Department of
Bioscience, Kitazato University,
Sagamihara, 228, Japan.
MESODERM INDUCTION IN THE MAMMALIAN EMBRYO. ((C.
A. Burdsall, and R. A. Pedersenl,2)) lLaboratory of Radiobiology
and Environmental Health, 2Departments of Anatomy, Radiology
and Obstetrics, Gynecology and Reproductive Sciences, University
of Califomia, San Francisco, CA 94143.
The Splotch (Sp) gene in mice codes for a transcription factor, Pax 3.
Mouse embryos homozygous for the Sp mutation produce aberrantly spliced
Par 3 mRNA and display spina bifida along with failure of the embryonic
neural tube to clse. Alfthough Par 3 mRNA expression Is localized in the
dorsal porton of the deveoping neural tube, it is not known what role Pax 3
plays in regulating neural tube development. We have observed that a
homeobox gene, msx 2, is co-expressed with Pax 3 in the dorsal half of the
neural tube. In order to study a possible regulatory relationship between
Pax 3 and msx 2 expression during neural tube formation, we have
examined max 2 expression pattems in embryos homozygous for the Sp
mutation. In El 1.5 Sp embryos, Pax 3 mRNA is localized in the dorsolateral
portion of the open neural tube, suggesting that the region-specific
expression along the dorsoventral and mediolateral axes of the neural tube is
maintained in mutant embryos. In contrast, msx 2 mRNA expression, which
Is normally evident in the El 1.5 dorsal -hindbrain, is below the level of
detection in the exencephalic hindbrain regions of Sp embryos. Likewise,
the level of msx 2 mRNA expression in the spins bifida regions of Sp
embryos Is markedly lower than in corresponding regions of the dorsal
neural tube of wildtype embryos. These results suggest that, during
development of the dorsal neural tube, Par 3 lies on an upstream regulatory
pathway for msx 2 expression Supported by PHS Grant DE-04316.
In amphibian embryos, peptide growth factors have been shown to
specify mesodermal fate in embryonic tissue. To test the role of
growth factors in mammalian mesodermal differentiation, we developed an in vitro system in which fragments of undifferentiated
epiblast (embryonic ectoderm), obtained by manual dissection, were
cultured in a serum-free, defined medium with addition of bFGF
and TGF-01. Treated explants adopted a complex morphology
(rather than a simple, flat epithelial morphology as in controls) and
by immunofluorescence regions within these explants stained
positively for vimentin, a marker of primitive streak mesoderm.
In situ hybridizations demonstrated that in 2/3 of treated explants,
small regions within the explants expressed brachyury, a marker of
streak-stage mesoderm and later dorsal mesoderm. These results
indicate that peptide growth factors may play a role in mesoderm
induction in mammalian embryos. (Supported by NIH Grant No.
HD26732, USDOE Contract# DE-AC03-76-SF01012 & and American
Heart Association Postdoctoral Fellowship).
1335
1334
ALTERNATIVE SPLICING YIELDS TRANSCRIPTS FOR BNP-l, NANNALIAN
TOLLOID HONOLOGUE *TId, AND A NETALLOPROIEASE CONTAIsN/ A
UNIQUE HISTIDINf-RICH DONAIN. ((Y. Takahara G.E. Lyons and
D.S. Greenspan )) Departments of Pathology and Anatomy,
University of Wisconsin Nedical School, Nadison, WI 53706
Bone morphogenetic protein-I (EBP-1) is a metalloprotease found
in extracts capable of inducing ectopic bone formation. In
humans, it has a domain structure similar to that of Drosophila
dorsal-ventral patterning protein tolloid (Tld), but is shorter.
We have found that, in humans and mice, alternatively spliced
transcripts encode both BNP-1 and longer protein which we have
designated mammalian tolloid (oTld) due to a domain structure
identical to that of Tld. A third alternatively spliced product,
in which a novel domin is inserted near the BNP-1 C-terminus,
was also found. Low levels of RNA transcripts for *Tld were
found in all adult human tissues surveyed, while BNP-1
transcripts were found in all adult tissues except brain. This
difference in distribution of expression in adult tissues is
mirrored in embryonic mouse tissues where in situ hybridization
found high levels of *Tld transcripts but no detectable BNP-l
transcripts in floor plate of the neural tube of the developing
CNS. The third alternatively spliced form was not found in adult
human tissues. Northern blots showed high levels of all three
forms in human placenta. In situ hybridizations found punctate
signal for all three forms, in 17.5 day mouse placenta,
localized to trophoblast giant cells throughout the labyrinth,
with higher levels of expression, especially for BNP-1, at the
Junctional zone near the aternal interface.
,
a
,
WITHDRAWN
230a
Pattern Formation (1336-1341). Monday
1336
1337
EXPRESSION OF RETINOIC ACID BINDING PROTEIN I IN THE
DEVELOPING CHICK OTOCYST. ((J.-L. Sanne and D. K. Wu)) NIDCD, 5
Research Court, Rockville, MD 20850.
A NEWLY ISOLATED QUAIL ZINC FINGER GENE EXPRESSED IN THE
DORSAL NEURAL TUBE.
Barembaum and Bronner-Fraser))
Department of Developmental and Cell Biology, University of
Irvine, CA 92717.
The inner ear, derived from the otic placode, undergoes tremendous
morphological changes during development. We
retinoic acid (RA)
may piay
in this developmental
are
interested in the role
Exogenous RA has
process.
been shown to induce supemumerary hair cella in organ of Corti cultures from
mice (Develop. 119:104-1053, 1993). RA also inhibited proliferation and
induced differentiation of chick otocysts in vitro (Develop. 110:1081-1090,
1990). RA is thought to act through nuclear RA receptors (RAR; RXR), which
belong to the steroid/thyroid hormone receptor family. However, the amount of
RA reaching the nucleus is thought to be modulated by a mechanism involving
cytoplasmic binding protein for retinol (CRBP) and RA (CRABP I-il). Whole-
mount in situ hybridization was performed to study the distribution of mRNA
ear (stage 11 -18). CRABP I
encoding for CRABP I in developing chick inner
0 0-11). When the otic placode
mRNA was not found in the otic placode (stage
invaginated to form the otic pit, CRABP I was expressed mostiy in the dorsal
and anterior region of the otic pit. After complete invagination of the otic pit to
form the otocyst (stage 18-19), CRABP mRNA was found in the entire otocyst
except the most ventral portion. At embryonic day 2.5 (stage 18), positive
hybridization signals were also found in neural tube, neural crests, branchial
arches and limb buds as reported by others. The expression of other retinoic
acid receptors and binding proteins, as well as the effects of RA in the
developing otocyst are currently under investigation.
((M.
California,
Genes containing zinc finger motifs constitute a
family of DNA binding
eWe
proteins, some of which have been found to be important
are interested in determining whether zinc finger genes are involved the
a
cDNA
of
neural
tube
and
neural
crest.
We
have
sceened
hlbrary
development
made from quail neural crest culture RNA with a degenerate
oligonudeotide
probe corresponding to the H/C-link region of C2-H2 class of zinc fingergenes.
Among the 60 positive plaques
picced, we have studied one in detai. This
cDNA contains 14 zinc finger domains and aKRAB domain. Several other
cDNA clones with regions of high sequence identity were also found, indicating
that this gene belongs to a cosely related group within the C2-H2 zinc finger
family. Whole mount in situ hybridization shows that the gene is expressed in
the dorsl neural tube as early as the four somite stage and at least as late as
stage 25 with the strongest expression located in caudal neural tube. Signal can
also be detcted in the ecoderm of stage 14 embryos but not older embryos.
The limbe also express the RNA as early as stage 16, and later the message is
localized distally. Other places where signal is found in older (stage 19-25)
embryos are the branchial arches and the tail. This gene is an interesting
candidate
for involvement in dorsoventral patteming during neural
tube development, and perhaps in cell proliferation in other regions of the
in
in
in
molecule
embryo.
1338
1339
PHOTORECEPTOR PATTERNING IN EMBRYONIC AND ADULT
GOLDFISHRETINA. ((D.L. Stenkamp and P.A. Raymond)) Dept. of Anat. and
Celi Biology, Univ. of Michigan School of Medicine, Ann Arbor,MI 48109.
The photoreceptor layer of the teleost retina consists of an array of repeating
mosaic units, each a precisearrangement ofspecific cone photoreceptor typea.
This mosaic may develop as a consequence of lateral interactions among
quentially committed cones,with an early-differentiating cone type acting as a
"founder, as in Drosophilaretina Using in situ hybridization to identifyspecific
P-CATENIN LOCALIZATION DURING XENOPUS EMBRYOGENESIS:
ACCUMULATION AT TISSUE BOUNDARIES. ((F.Fagotto and
B.M.Gumbiner)) Cellular Biochemistry and Biophysics Program,
Memorial Sloan-Kettering Cancer Center, New York, NY 10021
photoreceptortypes bytheir opsn mRNA,
have studied rod and cone cell patare detectable first, in a
nasally-positioned patch of retina in stage 24 (85 h) embryos. A wave of rod
differentiation then proceeds centrally along the choroid fissure, and cieumferentially around the eye. By stage 26 (120 h), scattered cells throughout the eye
also expr rod opsin. A slightly later wave of conediffentiation also begins in
a nasal patch and progresses similarly, with red conesfirst, at earlystage 25(95
h), followed by blue and green (100-105 h). Regular spacing ofblue cones in rows
is evident even at the earliest times. We have also studied the order of cone commitment in peripheral retina of adult goldfish, where new retina (including cones)
continues to differentiate. The distance between the proliferative germinal zone
(BrdU-labeled) and the nearest cone labeled with one of the opsin probes represents time between cell birth and cell commitmnent; this distance would be
smallest for the cone type which iffeentiateS first. Our results indicate that the
blue conemay be first to differentiate at the retinal margin. The observations suggest that mosic formation in embyonic goldfish retina may include cellular mechanisms distinct from those which continue to generate cone mosaic in the adult.
we
terning in the embryonic goldfish retina. Rods
1340
OVEREXPRESSION OF P-CATENIN CAUSES THE FORMATION
OF A SECONDARY AXIS IN XENOPUS LAEVIS EMBRYOS.
((Kadtleen A. Guger and Barry Gumbiner)) Departmnt of Cellular
Biochemsitry and Biophysics, Memorial Sloan Kettering Cancer
Center, New York, NY, 10021.
p-catenin is a protein known to associate with the cytoplasmic tails of
members of the cadherin family of cell adhesion molecules. Recently,
it has been shown that injection of Fab fragments directed against pcatenin can cause the formation of a duplicate axis in Xenopus lwvis
(McCrae et al. 1993, JCB 123:473-484). To further explore the role
played by P-cnin inXenopus development, P-catenin was
overexpressed in early Xenopus embryos by mRNA injection.
Injection of mRNA into the vegetal-ventral blastomere of 8 or 32 cell
embryos was found to cause duplication of the dorso-anterior axis and
was also found to rescue axis formation in UV-ventralized embryos.
Lineage tacing experiments revealed that cells receiving D-cat mRNA
do not contribute to axial sucues. In this respect, overexpression of
0-caienin is simlar to the ectopic expression of certain members of the
Wnt gene family (Sokol et al. 1991. Cell 67:741-752., Smith and
Harland. 1991. Cell 67:753-765.). Like Wnts (Olson et al. 1991.
Science 252:1173-1176.), overexpression of P-cauenin was also found
to increase gap junctional communication in cells of the ventral animal
cap. These findings ae intriguing, particularly in light of the fact that
P-cat shaes homology with armadillo (McCreaetal. 1991. Science
254:1359-1361.), a protein which is Inown to act downstresm of a
Wnt-signaling event during patterning in the epidermis of the fly
(Pfeiferet al. 1991. Development 111:1029-1043.). Data from this
that a similar signaling pathway nmy play a role in the
specification of the dorsal-ventral axis in Xenopus.
study suggests
p -catenin is a cytoplasmic protein associated with cadherir
adhesion molecules, and has beenimplicatedin axis formationin
Xenopus (McCrea, Brieher and Gumbiner, J. Cell Biol. 127, 1993,
447). We have studied Its distribution in Xenopus embryos by
on frozen sections.
Consistent with its
immunofluorescence
function
cell adhesion, it is present in every cell. However,
variable amounts have been found in various regions and
different tissues of the embryo. No simple correlation appears to
exist between the levels of P-catenin with the expected strength of
adhesion. High levels of p-catenin were found in regions
undergoing active morphogenesis, such as the marginal zone of
blastulae and gastrulae. This suggests that high expression of pcatenin could be involved in dynamic adhesion events.
Surprisingly, p-catenin also accumulates on plasma membranes
that probably do not establish direct or strong contacts with other
cells. In particular, high amounts of P-catenin are found
transiently at boundaries between tissue anlagen and at the
intersomitic boundaries. This unexpected pattern of P-catenin
that this molecule participates in
expression raises the
developmental processes, perhaps independently of its classical
role in cell-cell adhesion.
in
possibility
131
EXPRESSION OF ECTOPIC XWNT8 OR TREATMENT WTH LICL
IN ALTERED INVOLUTION OF THE DORSOANTERIOR
RESULTS
MESODERM IN XENOPUS. ((J.R. Fredieu, D. Maier, M. Danilchik, and L.
Christian)) Dept of Cell Biology and Anatomy, Oregon Health Sciences
University, Pordand, OR 97201
The centl nervous system (CNS) of Xenopus is derived from an
layer of cells which have been provided cues as to their relatve
ectodennal
position along the anterior-posterior axis by underlying mesodermal cells.
mesoderm
is positioned beneath the neural ectodem during the
flse
morphogenetc movements of gastulation. Misexpression of the protoin the dol anterior meoderm of early Xenopus
oncogene
gastrulac causes this tissue to adopt a more ventml fate followed by aloss ofof
the most anteior portions of the CNS. In similar manner, the
embryos with LiCl just before gsrulaon reslts in the loss of forebrain.
of staged control, plasmid Xwnt8-injected, and
Midsagital optical sections
wee examined by confocal microscopy to delineate
embryos
LiCI-teated
the extent of dorsal mesodermal involution during gatulation.In these
misexpion of Xwnt-8 in the dorsal mesoderm of
studies, we fnd thatasthewell
as the exposure of eary gastulse to LiCl retards
gastnlae,
Xenopus
the involution of the dorsal anteior mesoderm, thus preventing it from
its normal position beneath the most anterior neural ectoderm. in
reaching
addition, the extent of meodensa involution correlates dicty with the
extent of anterior CNS dfentainsuggesting that normal mesodermal
involution is critical for the
tansmission of neural
pattening signals.
We are
embryos as a model system to
using these
characterie the nature of signals involved in neural paterning in the
Xwnt-8
a
currendy
embryonic frog.
comct
teratogenized
treatment
Monday. Pattern Formation (1342-1347)
231a
1342
1343
REGULATION OF HOXA13 EXPRESSION DURING LIMB REGENERATION IN
THE AXOLOTL. ((D.M. Gardiner, B. Blumberg*, Y. Komine, and S.V.
Bryant)) Developmental Biology Center and Department of
Developmental and Cell Biology. UCI, Irvine, CA 92717 and *The Salk
Institute for Biological Studies, La Jolla, CA 92037.
important in the regulation of outgrowth and
pattern formation during development of the vertebrate limb.
Although leas ia known about the expreaaion of homeobox genea
during limb regeneration, it is likely that they play an equally
important role aa limbs regrow and pattern is reformed. We have
iaolated and identified axolotl homologs of 17 different homeoboxcontalning genes expressed by cells of regenerating limbs. Nearly
half of the clones represent genes belonging to the HoxA complex,
which are thought to be involved in pattern formation along the
proximal-diatal limb axis. We have analyzed expression of the 5'
most member of this complex, HoxA13. This gene ia expressed in
developing limb buda and ia reexpressed in the diatal-moat celia of
regenerating limbs. Its expression pattern is consistent with the
hypothesis that is functions in the specification of distal limb
structures as has been suggested for developing mouse and chick
limbs. HoxA 13 reexpression is induced within 24-48 hours after
amputation and is an early molecular marker for dedifferentiating
limb stump cells. Retinoic acid acts to down regulate HoxA13
expression, coincident with the change in positional information of
blastema cells from distal to proximal.
expression is more
distally restricted in anterior regions and extends more proximally
in posterior regions, and thus is asymmetric with respect to the
anterior-posterior axis as has been observed in developing limbs.
Homeobox
are
genea
HoxA13
1344
HOX D EXPRESSION IN DEVELOPING AND REGENERATING
AXOLOTL LIMBS ((M.A. Torok, D.M. Gardiner, S. V. Bryant )) Department
of Developmental and Cell Biology, University of California, Irvine, CA
92717
Hox complex consists of an evolutionarily duplicated group of
homeobox-containig genes that are expressed along the body and limb
axes in overlappig regions. Hox genes are expressed durig and thought
to play role in the pattern
formmtion of developing limbs. Urodeles
have longbeen studied for their abilty to regenerate lost tissues, wicluding
ims bs
their limbs. Amputation of axolotl,
results in a blastema from which theregenerate is formed. Blastema
issue was used to make a cDNA librry from which seventeen homeobox
genes were isolated. (Gardiner and Bryant, 1994, in press). Three of the
genes identified by the library screen show significant nucleotide
sequence identity to theHoxD complex genes HoxDll, D10, and D8.
in pettening of the
HoxD genes are believed to be involved
anterior/posterior axis of the limb. This work details the temporal and
spatial localization in the axolotl limb of these three genes. HoxDll, D10
and D8 have three, one and two transcripts, respectively, as detected by
Norther analysis. Al tascripts studied to date appear exclusively in
then meschyme and are developmentally regulated. Retinoic acid, which
has been implicated altenrg the patter of gene expression in
regeneratng and developing vertebrate limbs, was analyzed and was
found, insome cases, to alter the expression patter of the HoxD genes.
One of the HoxDll transcripts is expressed only in response to retinic
acid treatment and one of the D8 trancripts is upregulated by such
treatment. The implications of these results for pattern formation will be
explored.
1345
DLX-3 LS IVOLVED IN VERTEBRATE LIMB DEVELOPMENT
AND REGENERATION. ((L. M. Mullen, M.A. Torok, S.V. Bryant
and D.M. Gardiner,)) Department of Developmental and Cell
Biology, University of California, Irvine, CA 92717
TME DRO6OPHIL
TER
18-WHEELE
ENCODES
A
LEUCIE
((X William,
RECETR-LIKE
RICS
1. Departmet of Biological
Blslen2, ofEldon1))
X University
Dusman,
Notre Dana,
Notre
Dana,
46556.
sciences,
(219) 631-4161. 2. Howard Hughes
nstitute, Baylor
of
77030.
Medicine,
Houston,
Colleg
REPEAT
MOLECULE.
t.
IN
Medical
Tx
Distalless (Dli) is a homeobox containing gene expressed in the
developing head andlimbs of Drosophila embryos. Six vertebrate
genes related to DlU have been isolated in mouse, frog, zebrafish,
newt and human, and comprise the Dlx family. We have isolated
an axolotl homolog of Dlx3 from a regeneratinglimb cDNAlibrary
and studied the expression ofthis gene throughlimb development
and regeneration. In other species, the Dlx3 homolog has been
shown to be expressed in embryonic ectoderm andlimb primordia
(mouse) and in the otic vesicles (developing zebrafish). By whole
mount is situ hybridization analysis, we find Dlx3 is expressed in
the epidermis of developinglimb buds as well as in that of
regenerating limb blastemas. Dlx3 expression during limb
regeneration is markedly upregulated approximately 6-8 days
following amputation (medium to late bud stage) and is seen as an
anterior-posterior band in the terminal epidermis.
Immunohistochemical analysis shows the protein to be in cells of
the limb epidermis as well as in differentiated limb muscle cells, a
result supported by RT-PCR from musde fractions.
18-whe.ler(1lw) UREA ia expressed
unique
9w aRK& expression patterns (detected
through whole mount in situ hybridization with digoxygeninUTP-labeled19w cDOA fragment) were observed in ebryos mutant
for the segmnt polarity genes wlnglesa(vg), patched(ptc),
naked(rkd), engralled(en), or azmadlllo(arm). Escaping
show
bypcorphic
high frequency
leg, antenna,
and wing deformities, presumaly resulting frcm improper
aversion of appendage imaginal discs. 19w is exprossed in all
larval imginal discs. Expression of 18W protein in nonadhesive Schneider-2 cells prmotes cell aggregation to a
degree
in similar experimnts with To}L and other
data indicate that the 18w
receptor-ligand molecules. Theseand/or
protein functions as
cell adhesion molecule,
receptor
ccmunication
during various
mediating intercellularincluding
pattern formation and imaginal
events,
developosntal
cell determination. Recent experimnts have shown that 1lw
be involved in the tranalocation of Dorsal-related
isnity factor(Dif) protein into the nuclei of larval fat
body. Dif is a cytoplamic protein found in larval fat body
that quickly accumulates in the nucleus upon injury or
with lipopolysaccharide (1p, 1993). The potential
injection
role of 19w in the wg->en and/or the*n->wg signalling
pathways, imaginAl cell determination, central nervous systm
developmnt, and the larval ine response is currently under
investigation.
13"
134T
DEVELOPMEL
CONPOCAL
Hughes
BlUrERPLY WINGS: A STUDY BY LASER SCANNING
Institute,
apposed layers of epithelium,
arranged in precise linear arrays (1).
and is
Each
develops from two
wing
butterfly
Laboratory
1525
Wisconsin,
Paddock, J. Gates and S.B. Carroll)) Howard
of Molecular Biology, Bock Building,
Linden Drive, Madison, WI 53706, USA.
((SW.
MICROSCOPY.
Medical
University
The
ON
thousands
of
scales
overall wing pattern, which usually
surface of the wings of most butterfly species.
wing in the imaginal discs
the
butterfly
processes
pattern
have
described, and are remarkably conserved between
recently
and flies (2).
As a first step to an understanding of the
which
by
specific colours are assigned to individual scales, we
have undertaken
structural study of scale cell development in the pupal
the butterfly, Erseia..xsenn
wing epithelium
using laser scanning confocal
microscopy.
Tne scale forming cells are first detected as large polyploid
between
the pupal epithelial cells, and eventually
interspersed
The scales
precise rows in the pupal wing epithelium.
small
subsequently develop and grow out from the scale cells; first
single colour to the
scale
diffen
on
upper
lower
and
that
been
a
of
protrusion,
which
shape.
preparations
protrusions
thickens
This
where
and
is
and
best
bundles
eventually
eventually flattens to form the charactersitic
viewed in fluorescent phalloidin-stained
polymerised
parallel arrays
of
form
actin are detected
the scales flatten
as
in
out.
the
It
of the mature wing
in
appear much laser on in the
wing
disc
field
cells in the imnainal
some
scales
but only a subset of them
lateral
pupal epithelium, perhap
inhibition.
using a mechanism akin
(1) Nijhout
(1991) The Development And Evolution Of Butterfly
Pater.
Smithonian Press. (2) Carroll S. et al. (1994) Science 265:109-114.
first
wherea scales
imaginal discs,
might
sugest
that
all
discs.
kDowledge of their future identities
to
note
that
the
sigs
pattern
have
to
H.P.
Wing
in
pattern.
segmntal
a
Abnormal
a
adult
flies
of
a
seen
a
ny
HOW DO SEA URCHINS GASTRULATE?
A
BIOMECHANICAL STUDY. ((L. Davkio&', R. KelO2, M. A
R. Kohi, and G. F. Oer2)) 'Gra Group in Biophysics,
Cell
Biolgy,
A
Bioogy, 31ntegraive
and
Uniesity of
Calfrni, Berkeley, CA 94720.
Formabon of th
early
rchen-on
mechanical deformation of th
in th
epithl
as
shoe
urchin involv
a
comprising th
vegea
plabt. What the m tat dri this invagiratn?
We hav developed a meail mode which can simulate four
m of re gene , and which allows us to define what
nmati
mechanc propertes the cabt and of ft
fte
nee to be in order to reproduce observed mnbryonic
is
of
extraelular
th
The four models are: (i) apicl
(N) apical ctotrng of a -ubpopulat1on
of vegeta
geometriss.
plat
clls,
of vegea
pate
(ib) swellg of Poneogaycan gel
apical by
plae cals, and (iv) anular conrbacbton within a subpoplation of
cells,
a
secred
veget
out differnt range of
vege pat cells. We hawhichmapped
each mec
can operate. For
mateil stiffneseswiftin
and col tractoring meaim requlire stiff
sweFng
instac,
etraellular nmtr la with a high deomabe
ler, whie
i
gel
c
apical constriction
and cas
an
hav
mtal
specify which matea
measse in orde
deteri
conctionb
the
properte
to distngis
what drive
grnvt 92-20525.
anular
requlre
tness.
and cel
shp
t
Thes
th
matr
simublions
changes
to
between these mechanism, and to
pnmary gantration. Supported by NSF
Neural Development I (1348-1353). Monday
232a
1348
1349
NEK1, A MEMBER OF THE ELK CLASS OF RECEPTOR
TYROSN KINASES IS EXPRESSED IN HENSEN'S NODE. ((D.
Kenny, M. Bronner-Fraser, and C. Marcelle)) Developmental
Biology Center, University of Calffornia, Irvine, CA 92717
PLATELET-DERIVED GROWTH FACTOR RECEPTORS OF
MOUSE CENTRAL NERVOUS SYSTEM CELLS IN VITRO
To explore a possible role for receptor tyrosine kinases (RTK) in
early avian embryonic development, we examined the
distribution of two genes that belong to the ELK class of RTKs.
Expression of one of these genes, NEK1, was determined using in
situ hybridization. During early embryogenesis Nekl mRNA
expression was confined to cells of Hensen's node where it can be
seen from stage 4 until the completion of gastrulation.
Cells
expressing NEK1 are within Hensen's node as it regresses and
also in the newly laid down notochord. Later in development,
expression expands to cells of the presumptive neural plate and
floor plate. After several days of development NEK1 is expressed
in other tissues that include neural, placodal and mesenchymal
cells. Cells within the node differentiate into diverse cell types
that are critical for neurogenesis and somitogenesis. Presence of
NEK1 mRNA in cells of the node suggests a possible role incell
differentiation, and expression in the node and floor plate may
indicate a function in inductive interactions necessary for
Receptors for platelet-derived growth factor (PDGF) are likely to play a
key role in the differentiation of cells in the central nervous system. PDGF
patterning of the
((JiB.
The
limbic
e prtein
sysem-asociated memba
(LAMP) is a 64-68 kDa neuronl
surface
suboirtcal regions of theIhmbic systm. LAMP
byhbmophic bindi
and
me inked to
the
naeon
membrae
by
a
Rat-LAMP wu
oned fm
glycosyl-phospbatdylinosis (GPI)
cDNA
(Ckotec)usin degenera DNA prbes deduced from the LAMPN I
clond by PCR
mino acid sequence. Human-LAMP
of human cDNA
Both encode a 338
(Clontec) anchored by oligo prime from the at cDNA seqse
99%identity. Consevedpais ofcystines
amino acid polypepad withseque
a
was
sharn
idenify
the
off*
mai_rpas
three
stuccme
of the Ig-domains reveals
LI, FascicIn andother adhesion
OBCAM with
new
identy in the
homology
proteins. The
stafaunly.
with
speifi
g-fDs
on
Analsis of
TAG-i, N-CAM,
highst homology is between LAMP and
fim two domains.
Te
coem prusin
ha
eight pumtive N-
NorQth
sites.
bota nysis
linkedgycosyt dies and elev pose phoal
LAMP RNAprobesiniatd the pmsec of two mRNA transcripts in the aInt rat
ung
bra
whih arenot cqx sed in noel tiss. Twot scripts ofidaical wealso
detected inhum brain mRNA blot. In situ hy t analysis shows reticted
i
zed with LAMPimm
exrssion of LAMP tscripts,
parculy
The trecmbist prtein, expressd on the surface oftrsected
wihiDn limbic brin
is reonzed by at-LAMPmonooala
CHO-KI
ad
is
reased
body
by Pl-PLC
da indicates that LAMP is a new member of the immeoglobulin
trament.
col
men
with
hypot
that
by NIMHgl
GPt-linkage
LAP
ad
resated
ptcie in limbic
ntMH4SS07.
anatomical exprsso, conistent with
sysem
our
devdopmt and fencton. SuWported
is type 1 (NFM) is one of the most freuety
vonRecinghausens neuob
inherited autosomal dominant disorders in humans. Although the symptoms and
severity of the dies me variable, the mnjority oftissus affected inMFI me of neural
is 1 (aNFI) 432-bp cDNA which ve have
crest origin. The avian neuroo
cloned is 82% identical to the human NFI gene at the nucleic acid level and 93%
the
to
preicted
protidn
identica
sqce. We have used this probe to eblsh the
normal exprsion patern of aNFI in eary chick embryos Northe bbt anslysi
aNFI
traript expressed as ealy
Hamburger & Hailton
shows a large (12.6kb)
stage 11 (40-45 hours in oo) that pesis into adulthood. IA situ hybridization
aNFI
is
ubiquitously
in
the early embryo while
experiments show
exprssd
a
a
prtin, as delineated by antibody saining. is
neual crest cells t stages 13 (2 days inovo) nd
more highly expressed in
20 (3 days i ovo) dneua
suboetof
crest derivatives at stage 29 (6 days in ovo). NFI is a tumor suppressor gen which has
been shown to regule p2lras in vitro. Since p2lras ha been implicd in cell
differentiation, it is likely that NFI is also important to the process of
coll
neumn differntiaion. We me using
approache so
Fs, we have produced seveal
investigate the role of NFl in neuronal diff
crest
src-transfeomed quail newal crest cell lines by infecting primary quail
cultures with the PAIOI temprature sensitive mutant of the Rous Sarcoma
Second, we me genataig mouse neural ct cell lines from mmortomice
differentiation, spcfically
neuWal
virus.
mice
which am transgenic for an inducible-promoter conouct of SV40 re T angs. We
the
can manipulate tese sytems to differntia into nesrons in culte, d
changes in NFI levels and isoforms during in vitro differentiation. To pinpoint the.
ion, our third approch is to invesipte she
involvement of NFI in neuronal d
ES
cells.
diffrutsin capacity of cells defcet in NFI using NFI
nve"at
Medical
1351
HOMOPHILIC BINDING BETWEEN RECOMBINANT AND NATIVE
LIMBIC SYSTEM-ASSOCIATED MEMBRANE PROTEIN
SELECTIVELY REGULATES
NEURITE OUTGROWTH. ((P.Levitt,
V.A.Zhukareva, and A.F.Pimenta)). Dept. of Neuroscience and Cell
UMDNJ-Robert Wood Johnson Medical School, Piscataway
Biology,
08854.
NJ
We have recently shown that the limbic system associated membrane
protein (LAMP) can facilitate substrate adhesion, via homophilic binding,
and neurite outgrowth of neurons from
areas of rat embryonic brain.
rsults provide direct evidence that LAMP expression supports
The presentadhesiveness
of primary brain cells on LAMPincreased cells. CHO-Kiandcellsgrowth
were transfected with full-length LAMP
transfected
cDNA. Stable transfectants were obtained by calcium phosphate
and cloned by limiting dilution. Recombinant LAMP is
precipitation
on thesurface of CHO cells and susceptible to PI-PLCtreatment
expressed
has been shown previously that LAMP binds homophilically;rapid
fluorescent Covaspheres. Here,
aggregation occurs between LAMP-coatedLAMP
from the rat bran, bind to
Covaspheres, coatedwith
CHO cellstransfected with LAMP cDNA, forming aggregates of 3 5 beads
on the cell surface. Monolayers oftransfected cells were used as a culture
substrate for embryonic neurons from limbic (perirhinal cortex) and non
limbic (olfactory bulbs) areas of rat brain. LAMP-transfected cells showed
a markedly enhance ability to promote neurite outgrowth of perirhinal
neurons, known to express LAMP on the cell surface, compared with
olfactory bulb neurons that are LAMP-negative. These results support the
as cell adhesion molecule through
hypothesis that LAMPandfunctions
regulate neuronal outgrowth and
homophilic interaction, can
possiblytarget recognition. Supported by NDMH grant MH 45507.
specific
It
affimty-purified
-
selectively
1353
1352
THE ROLE OF NEUROFIBROMATOSIS-1 (NFl) IN THE DEVELOPI
NERVOUS SYSTEM ANDNEURALCREST. ((AL Kavka &K F. Barl)
Depwmnt ofAnatomy and Cdl Biology, University of Michiga Medical School.
Ann Arbor, MI 48109-0616.
neurofibromin
Mississippi
cells
and Neuobogy, Medical College ofPensylvaa, Philadelphia, PA 19129.
Inc., Proein Chenistry, S. Sm Fracsco, CA 94080.
molecu intect
of
.
tech,
glycopotein expressed in cordcal and
Anatomy, University
are found on central nervous system cells isolated from fetal or
newborn mice and maintained in titro The presence of PDGF receptors
has been shown with colloidal gold-labeled immunocytochemical markers
at the electron microscopic level. PDGF receptors are sparsely distributed
over the surface of type 1 astrocytes, apparent type 2 astrocytes, and neurons. Receptors appear to be preferentially associated with filopodia-like
extensions of the cell membrane. The existence of functional receptors
was confirmed using the impermeant, water-soluble affinity cross-lsnking
agent bis(sulfosuccinimidyl)suberate to covalently link radiolabeled PDGF
to its receptor. The PDGF/receptor complexes could also be immunoprecipitated with the same antibody as was used in immunocytochemical experiments. The improved resolution of these techniques allows definitive
identification of PDGF receptors on cultured mammalian central nervous
other than oligodendrocytes. These data expand the range of
system
possible roles of PDGF during nervous system development.
1350
MOLECULAR ANALYSIS OF THE LIMBIC SYSTEM-ASSOCIATED MEMBRANE
PROTEIN (LAMP): A NEW MEMBER OFTHE IMUN40GLOBULIN SUPERFAMILY
HIGHLY CONSERVED IN HUMAN AND RAT. ((A. F. Pimente, V. Zh
A.
skeva, B. S.
Fisher ad P. Levitt)). Depl. Neuocin
Reinoo. C. Grimey, W. Helzel
d Cell
Biology, UMDNJ-Robert Wood Jobhon Medical School Piscatawy, NJ 08854; Dept
Anatomy
of
receptors
system.
nervous
Hutchins)) Dept.
Center.
AND FUNCTION OF ANNEXIN II IN DEVELOPING RAT
LOCALIZATION NEURONS
IN VITRO. ((W.J. Zats and P.C. Bridgman))
SYMPATHETIC
and Anatomy and NeurobIology, WashIngton University
Medicine
Departmerls d
School d Medicine, St. Louis MO 63110.
Annexln II is a Ca2+-and
Is
protein whose
and PC-12 cel dllferentaton. To knvstIgate annexn
neuronl
durhg
enhanced
role nourke outgrowth, we have examned Ka ddatbion and ihbted
1ts plays
fnctIon devebping rat yIXthC nerons cuture. Immunoluorecence
revead that both annehdn II and ks p11 lght chain were datrbied
exression
I
a
in
in
in
microscopy
in
puntae pattem throgu them c1
at the
body staining
a
neurke and growth oone. In the cel
membra
for
coqswrd to
body,
annrodn
Their
p11
grater plama
Anneidn and p11 appeaed paslaly coocake growth cone.
thickened ceotral doain, but also extended
staining was greatest thedomain.
the neumns were m'croijed wth a
throuot
the perlheral1 artbody, When
both annexin 11 and p11 wer perthly
antiannexdn
poWcbnal
eded patches,
together with the
sequestered
bho plama
alffted,
antbody.
abl for at bast
the
and
th
remained
sen.
neurons
soma
wer
nwmplogyd
4.6 days clture. Folowng a 3 hr recovery perId, newIte outgrowth from
or
neurn
hrs.
was measured for up to 6
Alhough
antbody- coutrorneded
dNlferert for the firt 90 mh, arnbody-injed ceb
outgrowth was not statistcally
and by 280 min had grown only 28 % d the dItance d the
thrter,
grew poorly
The mean outgrowth veocilty 252 min for autuiodycornrol (P < 0.001).
wa -0.0012 t 0.024 snvrmin (n . 55)
0.147 t O.2 pWmmn (n .
ijected
oe
42) for control (P < 0.001). Foynne percent d the aw0body-irjcted coe we
this time 17 % d the controla (P < 0.001). Those corilnu to
ratracthg
grow had
grwth cone morphlogs. These data idcate that annexi
II.
In
to
in
jcted
Annesdn VI darbutlon
not
no
gro
nes
in
in
at
n
at
1
-.
or
p11
necessae
andior ocalzatlon may be
functon
in cultured
sympathetic
nerons.
for normal
outgrowth
Monday. Neural Development I (1354-1359)
1354
233a
1355
OFANTI-N-CADHERIN ANTIBODIES ONTH E
XEN OPUS RETINA
HISTOGENESIS AND IFFERENTIA ON OF THE
RV VIVO ((TD. Folsom, J. Leonad, and D.S. Sakaguchi)) Signal
Transduction Training Group, the Neurascience Program, and the Deptof
Zoology and Genetics, Iowa Stat University, Ames, IA.
In the preset study, we have investigaed the role of N-Cadhrin during the
dif
of the Xenopus retina. Functio
normal histgenesis and
blkxing antibodies directed aginst N-cadherin
injected into optic
vesicles of stage 23 embryos. Embryos were allowed to develop for 36 hours.
imm
unoist
using
development of the retia was thn examined
irected against the snptictrminal retd pteins sy
d
antibodies
(p65) and Rab 3A In control eyes, the anibodies labeled the outer andinner
plexiform layers and the optic fiber layerof the retimarveahing its distinct
laminatio. Compared to thecontrol eyes, those injed with the anti-Ncadhern antibodies appearedimnmaure, exhibiting unusual lamination patterns
or no lamination of the
In addition, the eyes often lacked a prominent
lens and other morphological abnormahites were obsered including unusual
normal eyes.
invaginations of the eye cup and small
retnotectal project
was also examined in antibodyinjected embryos at
24 hours following injections. Horseradish peroxidas was
approximately
use d to label the visua projecton fron the injected eyes. Many optic fibers
a relavely normal cous
from the antibody injected eyes followed
the optic
the antibody does
rot block axon outgrowth. However,
tectunm, rvealing
the projection from injected eyes tended be delayed their growth when
compared to nonimmune andbody injectedcontrols. We conclude that Ncadherin plays an essentia role durig the normal histogenesis and
of the vertebrate retina. Supported by grants from the NSF, the
differenttion
ISU Biotechnology Council and the Carvvr Tns
EFFECFS
wer
reti
to
I IDENTIFIC O
OF A NEURONAL COLLAGEN RECEPTOR: EXPRESSION
OF a2l WEGRIN IN THE DEVELOPIN
FUNCTIONAL ANALYSIS
AND
DiB. Gervin,
MdNsgy, and D.O. COegg))
BDBdow,U
Unvaity ofCalfornia SBaBatw, Santa
Rnate
CA9310. (Span.
Bradshaw.)
nrvons
the
all
exo
migon,; amo
uo to guide aniao ofcell
RETINA. ((AD.
NeosciencResech
Barba
K-&
by
In the doelophig
exe
of
mic
AD.
matrix
smm
md
en
cc
doc
edr
by
data-
Qt2 im
aY
highe
at the
n
day 1.
culs
d
The
the
npt
With
res
Reta pngo
ntlre
ioe ydeecne-i_
d
a
expressed
epsiOf02I
Leves atemiryomic day 4. As develepinesa roceeds,
litl orso
by postal
ideiid
neotic
1 colsgenreceptr
t2l
n2lxsein mid mRNA were
rn.
significast
Although cull
a
inea m edim
p
We have ideiedinigr
q2
developing avin
is
bavebeem
of colge
ewie onogw
samonderzkod.
tens~O
ofdevelopingemtryos.
prntigacvke
dfor endiyo nic retid cullS, eik
in di
povides apotentia
uoba
fainly Of
fao,
SW
O
oc gow
he Colla
synae
n ate exu
onesaprese
ehihemaric
comp
that
lite if
a2 is
inlogrin
Pr-culls. By employing function
exprasedcm unifeentd retinal n
amias a collagen reeptor,
blocking asalbdies we have foted thatA iesser
mediatin bath cull adhesio
andp nr cms outgrowth by embryonic retinal cull on
fioaction
bath collgen type I mid type IV.
of
-n
regulatd with
down
dhis' nsgrin
in
amociatd widh
thedifsetto
data
mid
wtol aWm appeas to be
activity of
p in p a
rtnald cdellsbose
sggesta
dk
die expession pater
w
forecaol in the whdy
role
events
avim retina
ovelpm ontof the
to
1356
INTEGRIN v41 AND VCAM-1 AREE(PRESSED IN THE DEVELOPING
P45D (PNS0AROM) IN TIHE PRIMARY CULTURES
ASTROC-YME BY PCR AND DAL,UNOCYTOCflsrY ((ainat I-L Zwam
Cheeg)
Poipaltoa Casicil. 1230 York Avasie, New York, NY 1002 1.
NSODAROM is the enzyme that catalysi the conversion of androgen to estrogen in the brain,
cesrgen is required to promote netwona growt asd regulate the sexual behavor
minimal.
Rcasen studie also sugges that estroge
agaLni Aluheimeors
may he invevedin the protectio
IDENTIFICATION OF AROMATASE
RETINA AND MEDIATE PROCESS OUTGROWTH
MOUSE
Bradshaw and
((G.M. Can, A.D.
Ceg.)) Neunroence ResearchInsttue, Utvray of
Barbar Ca. 93106.
OF RAT
D.O.
ClMorrisa Sa
mid C. Yasi
teractns are
o
hi the spelatn
DurIng rel devebpmet, ce
l
of relina
pwnpe,
aXonal paSthldrt and
and the
are
rlefsti of the
segregatnio of alemalt cea wd Me lyers
e
and homoplic eel
h
wIe p
of
ure
vertbrate r
adhesion nolecule are btly medae these ert ad delition of col
adheson nmlcules expre_d hI the deveopI reina dsod alow a better
Ial deveopnt In
o the moeculrm
anme Invovd In
undrstadI
n
inmt
inherant
edIated
VLA-4 (a41) and countr receptorthe vascterl adhesion
by
molculwe- (VCAM-1).Prouslywwreported theeprssIo of mRNA encoing
syqsogepnes,
diseas
cneste
aunil
at-
deopIn cick reI
hi
usng
are
nigran
We
tods ertaigt
sowble VCAM-1suppots pocess
reconant
hi cAr
odes spnet
the
protein,
acidic
resub sugos
betwee
arole
that this enzyme
Fl2ID ME (1: 1,
of
P450AROM
II
re-amplified
then
above to detec
in
astrcytes
the
2nd PCR reaction
a
using
P450AROM
also studied
was
dreurmining
by
yield
mRNA that
the
with the
action of
1358
oqp
IL-li5
conclusion:
dose-
a
estadiol
to
peared
he
spetiric since its action
astrcytes
sine
the brai
in
by
are
a new
to
cedh
role for
prdoduc
in prtctn
ee
was
blocked
IS
ALTERED
N-CADHERN
MOLECULAR
BY
EXPRESSION
DUWUNG
FUNCTION
AND
Fenre-Cornwell,
REINAL DEVELOPMENT ((iC.
Grnnwald)) D _atmnt of Antomy, Pathoogy,
Univrty, Philadel, PA 19107.
NEURAL
GENETIC
RJ.
and
Buoo
Jeknon
CeU Biola, Tboms
ad
G.B.
TIHE
eatAsto
mid expres
IN PROTEN, IS UP-REGULATED IN GLOSIS AND GUOMA.
((D.M. Jaworaid,
Yale
GlIt
N-cadberin
molcue
is
cadberin fmildy of
to mediae ceii-cu
d
been
N-cadherin
Optia
fincton depends
f the
a
shown
which
de
v ia
its
aic
methods
for
tunatd N-cnherin
domais
pr_tin
mnauker
i
(8-a).
promoter to
in
ly
e_enI
both
homophilic
the cytos icet
dsevlin
ina
embmyoni
seon
or
potion
desiwgn
encodi
retin
mRNA
of th
via
molecul
(2)
xracblular
e
hitochemially
Intt enbyonic day 7 chick
enxyo no"ul
pTBI,
which uilizes the
eprI-o of 8-gal, Nd pSG5NCAD(+) or (-),
_dsense N-cadherin mRNA, respetvely, dnven
Rwus
wbich
ct
by
exprem truncutd N-csdherin, or
SV4O promote of the pSG5 plasmd kxprm vector. Folowin thee days in oran
Whole mount emimbion reveaed ta
tui, ssa were fixed d staid for
wer
to be incorporftd
icaed by 8-gal expr
on
retn
cel
Whik blg noues could be detectd asmog cut ranelced with pTBlI Uad/or pSGS, no
neurites were obsrved amnwg markd culls
N-caherin
essing antsn
Aberrant nturile growth
observed along
marked cul
tncae
Niad peubation of N-cadherin retsin
These dat suggt that pa
cadhei
the constru
in
mRNK
wa
neurie
lOPM
expssn
amog
retinal
NRSAEY06470 to MC2FC and ROIEY06658
to
nuron
UIBO.
Supported
by
NIH
Hockfield))
contribute to the
cel
Section of Neurobiology,
Medicine, New Haven CT
06510.
vnmet of the CNS.
for
omyoliation
While
rqepre
sitocytes
a more
fucinlyheterogenonus populstion of cells. Severalextracellularmatrix
curimuponent areexprsussd by aitocytes
(HA) and the HA
incuding hyalur
Previosly, we repoted the cloning and
restricted tothe CNS (JCB
regulationof BEAB,a Abinding
with gl
125:495) that is tmpoally and spataly regulated in para
enesis.
drepto
fCYlEr alyze dBEHAB exre
hRA
hypCtopy
C.dconditions promoascyte
We
I
now
blot
analym of priay
tRocyten
or
with the
wh plsmid
drive
on
during
of
wegd respo sible
adesion
gowth
etinal deveopment by
mRNA
ideufy successlly transected
by
vir
directed at
hioduced
N-cadhri
ase
mima
and (3)
wer
murite
with
di teactio
of N-cdn
which coud (1) be
of eher
retin
smrcoma
the role
ng
veco
m
domain
e-peimea
Th
pasmid _ep
drive the exPri
dtect
o
extracd
its
cl
calcium
GJMZ Kelly and S.
University School
Prviu
sdies have
underping remodeling.
growth
is
adult rats
invaion.
mfpare
breast
danag
BEtAB
could
prmmy g
and
is
l
(l
cultu
and
hyperplasisY
BEHAB
Is
abundt
Northern
expressed by
Assues
wa
was
might play a role in glin
performed following a cortical
verified
.BEHAB
in the
vicinity
eHA
hasbeenrelported toplay
expession is
brain. hot is
to normal
colon
dt
HA
shown
s
Glioss
ignifcndy o-egultd
gliomawasexamined beca
indicates
wheher BEHAB
To
in
oro
stab wound
cutue
of the stab. BEHAB expression in
cpificandyup-reguled
ntmdetectbl
in
anileintu
in
non-neurkl
glioblastoma
tumors, inclxudng
resonse to CNS
lug Incrad BEHAB expreion
have sigios
implications for the role ofHA ad HA ind
nd
CNS
posifaintinsue remodeling dPiin.g
tmogednbysiat.(Suppored by EYo6511
and
injury and
EY06451.)
diring
factoirs
in
PiooAROM
supporting theunwons.
ad
EXPRESSION OF BEHAB, A CNS SPECIFIC HYALURONAN
BI
1
by anti-ILli
involved in synptic reorgaiization
xt iu id provding biological
otllutadd
am
1358
OF
5'-
mid
growth, the ability of astrocytes
vito suggeting
PERTURBATION
prinmer,
antzmsepMm derdcribed
idntci eas in estmadol production by
astrocytes. Intedeasidn-li (IL-18). a kownP50AR?oMA nKxduatorin gtatadt mid extragoninla
non-neurml cells, induced adoedependent isdbitionof estsdiol prdodction by astrocytes. The
cultures. Testosterone induced
fornerm
MORPHOLOGY
a
the
further
bp product. Aromaitization by
production by astroyte
168
herd of
was
was performed
(sense) and 5'This productwa
[y-32pJ-radiolabeled
a
a
llary
using
PCR
of
TrGGAGAGTTCATGAGAGTCTGA-3- (sme), ftoethe
hiegi
ffrs
mpicainstepa. The
greate
fri
localixd
masrcytes
in the
mechaiical
Was
study
was
5'-ATACCA0GTCC`rGGCTAC`rG-3'
IUI rAAATATGATGCC-3' (aniseose)
gScnnate a 272 bp product
specific primers
two
cal cnditio
altered
in two
unsocytochemical
Im
shown
presenc
hot-nested RT-PC3
onmirnied by
using
dirthshowtu man
owth fommouse and chick
ld by mownmonal
inhibitory
antibody.
nou
P450AROM
nasrcytes.
fthe
developing retnal cels.
NEURITE
detamined.
yet bans
in astroytes mid the
isolated from the cerebral
cels w
masker of astrocytes.
a
agnat rat
outrwth Is Wopct
dth and chik beta-I sburit Thes
and VCAM-1 hI meiadIag interactons
rkrtgrin
and that
mous
not
aittred in sera n-suppkanensed
camid
cortx,
pathoogicsl
Nixed
estrasici.
tats by
1-day-old
of
prodhuce
cells to
chan
growthcones
the cerebral
growth located
neurons degemesate
this
vlv)
reacton (Cann, GM. and Cego, D.041991)J. Ce# ib.
sud
we report the detection of VCAM-1
integhi ata4
eipessIon dneveping mouse reIVCAM-I eNesin is especaly stn
I the optc ber layer and opti
head, at Ines when retinal ganglon co
polynm
115:11Oa). In
where
mediui. for a day.
w
then sepwar
from oligodentAocyo- by
cottaud aiderserum-4reecconditions for 24
n
"mioig
Thpuity of masocytes
90%
stto allng using anasiibody aginst
judg
by
m
kgln
neuronal
areas:
forebrain. the
P450AROM
ability of
hiegn
the ick
moduates
hormone
basa
disorder. However, the soaurc e(s) of esuogen in crebralt cortex hat
Studies were petformed to investigat
the expresion of
to
bynqhoposisis,
since this
hippoesnpcms and
hara
Neural Development I (1360-1365). Monday
234a
1360
1361
BINDING BETWEEN THE NEURAL CELL ADHESION MOLECULES
AXONIN-1 AND NR-CAMIBRAVO IS INVOLVED IN NEURON GLIA
INTERACTION. ((D.M. Suter, G.E. Pollerberg, A. Buchstafler, and P.
Sonderegger))Institute of Biochemistry. University of Zurich, CH-8057
*Max-Panck-instItute for Developmental Biology,
Zurich, Switzeriard.
Department of Biochemistry, D-72076 TObngen,
EFFECTS OF L-SERINE ON THE GROWTH PATTERN OF NEURONS IN VITRO. ((1. Savoca, U. Ziegler, and P. Sonderegger)) Institute
of Biochemistry, University of Zurich, CH-8057 Zurich, Switzerland.
molecues comprising both immunoglobulinImmunoglobulin superfamily
type
and
IIke domains have been found to be Inoled In a
l te f5bronectin
and
heterophilk
of
ktracthant rv ovide the molcular
variety homophilic
basis for many different cell-cell and cell-matrix contacts during
system. Recently we have shown that the
morphogenesis of ternervous
mo
gii
nural cal adheslon
kmoles axornn-I and Ng-CAM (neuron
chicken
stituting the culture medium. We have examined the influence of non-essential
amino acids on embryonic chick dorsal root ganglia (DRG) neurons invitro.
DRG-neurons were grown for 16 h in a serum-free, chemically defined medium
containing minimal essential medium, nerve growth factor and N3 (insulin, triiodothyronine, corticosterone, transferrin, putrescine and Na-selenite). If
ine was supplemented at physiological concentrations (10-200 AM), the neurites
were up to 100 percent longer and developed a more complex branching pattern. The growth cones were smaller than in control cultures without serine.
Gernmny.
c
Interaction, which IsIrnolved
a
adhesion molue) undergo
hhterophilic
also interacts wih Nr-CAM
in neurite outgrowth. Here we report that
of
(Ng-CAM related cell adhesion molecule). Firs evidence for an
other
than
was
found when fluorescent
axonin-1 withmolecels
Ng-CAM
coupled axonin-1 atthe surface
beads (Covaspheres) covailely
carrying
(DRG) in an
bound to glial cells of cultured chicken dorsal root
Interactin that could not be perturbed wlth anti-Ng-CAM Fab fragments.
Preincubatlon of these cultures wih aritbodies against potentlal receptor
candidates revealed that only antibodles against Nr-CAM inhIbit this
that
I
these DRG
in
mmunostainIn andsi
interaction.
hybriaion revealed
glial cells indeed express Nr-CAM at their suriace. The bindIng between
axonin-I and Nr-CAM was confimred with Isolated molecules using a
cultured in the
Covasphere aggregation assay. dissociated DRG cell
ensheathment
presence of anti-axonin-I or anti-Nr-CAM Fab fragments,
of neuritas by the gIlal cab was markedly perturbed. We conclude that
the
membrane and Nr-CAM of the gial
binding between axonIn-I ofneurite
tth early phase of axon
cells may have a role during
cell
axonin-I
kiteractiDn
ganglna
were
tth
morphology of neuronal processes originating from
depend on the substratum as well as on a variety of components
The length and the
in culture
neurons
con-
ser-
The described effects of Lserine were concentration dependent, stereospecific
and found on several different substrata, such as laminin, Ng-CAM and axonin1. Simllar observations were made with neurons of the central nervous system
such as embryonic retinal explants. Addition of other non- essential amino acids
to DRG-neurons had no effect. We conclude that serine, althongh it belongs to
the group of non-essential amino acids and is not known as a neurotransmitter, is an important factor modulating neurite outgrowth and branching of the
central and peripheral nervous system neurons in vitro.
ensheathmenl.
1362
RAPID REGULATION OF NEURITE EXTENSION AND RETRACTIONBY ARACHIDONIC ACID METABOLITES. ((N.R. Smalheiser))
Dept. of Pediatrics, MC 5058, University of Chicago, Chicago,IL 60637.
Recent studies have indicated a role for arachidonic acid (AA) metabolites in regulating
F-actin levels in non-neural cell types. Since rapid-onset neurite extension on laminincoated substrata and acute lysophosphatidic acid-induced retraction in NG108-15 cells
both F-actin dependent responses, the possible regulatory roles of AA and its
cyclooxygenase (COX) and lipoxygenase (LOX)metabolites were examined in these
two assays (methods in DeveL Brain Res. 62: 81-89 and J. Neurochem. 61: 340343). Outgrowth measured at 1.5 hr post-plating was stimulated by AA (1 micro
M) and by the COX inhibitor indomethacin, but was inhibited by phospholipase A2
AA
inhibitors (BPB, ONO-RS-082, quincrine)and by the 5-LOX inhibitor
Acute
increased neurite lengths with little effect on probability of neurite
inhibitors,
A2
AA,
and
was
inhibited
by
PL
was
also
by
retraction
enhanced
neurite
by AA861, and by the FLAP inhibitor MK-886. The DAG lipase inhibitor RHC80267 had no effect in either assay. Cells exposed to high doses of PL A2 or LOX
inhibitors exhibited decreased contractility, in that many neurites failed to retract when
detached mechanically from the substratum. These results indicate that phospholipase
A2-derived formation of AA, and its metabolism via 5-LOX, are not only neceary for
acute neuritechanges in either direction, forward or backward, but that activity in this
pathway can actively regulate responses of neurites to other stimuli. It is possible that
some of the effects of AA and its metabolites on synaptic plasticity may be mediated
directly at the level of the neurite. Supported by NIH HD 09402, NS 26055 and the
Brain Research Foundation, Inc.
are
AA861.
initiation.
1363
CRITICAL PERIODS OF OPIOID SENSITIVITY IN A PHARMACOLOGICALLY
SUBPOPULATION OF ASTROGLIA DURING DEVELOPMENT.
Ryan and C.S. Turbek)) Department of Anatomy
((KF. Hauser, Gurwell,
KY 40536.
School of
&
University of
DISTINCT JA. S.E.
Medicine, Lexington,
Kentucky
Neurobiology,
glial
In free
Inltrellularcalium (KCC2li)
development, oplod-Irduced chanrge
messednIn prmary mixed glial cultures from 1-day-old mouse cerbra. Prior
To
explore the ontogeny of
esponsiveness
during CNS
to opbids
wer
opid
[Ca82]i in Irocytes,
can affect
growth. Opioid effects w reexprd
ch s [Ca2
immunoreacwe,
acidic protein
measured by using
days 1-10, 14 and 21 Invitro. Changes lC821i
ic1 opiold
analis of ratlometrlc dye fura-2. The
computer-sidedImage
caused signifIcant Ises InlCa21
U69,593 In100 nM
agonist,
in some astcytes. Equimolar nentrIonsof the opioid antagonist, naoxone,
or the selective
K antagonist, nor-binaltorphimine,bocked the effects of U69,593.
In
and
aonts can
vence
that
asocyt
(GFAP)
gIal flbury
flat
In
(tp
1) astrocytes
on
wer
the
entrtion,
and 5[D-Pen2,D3orpaD-Ala2,MePhe4, Gly(o051enkephalin)
agonistsfoploid
UW9,593
(Ca2+i.
t
ype
of
In
In
a
subpopulation
I
ncrases
[Ca&'li
signilIcant
Moreover, ICa2+i Increases were Induced by but not i and6,agonists. When
thep cntae of type astrocytes owing signIfIcant Incaes in [Ca24liwas
examined at dferet times in vltfO, the propordion of type astrocytes responding
to opioods changed dramatically. Fewerthan 20% of astrcytes responded before
few
responded.
day 4in culture. At days In vitr
in confluent cultures responded. This suggests that transient, develpmentally
regulated periods of oploid responsivenes occur In a pharmacologIcaly disnct
populatlon of astocytes. Critical pedods of opioid sensitivity during develpment
may be, part, defined by regionsl and temporal differences in the functional
expression ofK opiold receptors by astrogla. Supported by NIH grant DA 06204.
Selective
Pen5penkephalin)
receptor tvse
(100 nM)
did not effect
caused
I
ascytes.
xi,
I
I
44
60-90%
astrocytes
However,
In
1364
1365
DN VITRO EFFECT OF OKADAIC ACID ON NEURITE OUTGROWTH
IN RETINAL EXPLANTS. ((M.E. Vaxqucx and R. Chiesa)), Department
of Ophthalmology, Columbia University, New York, NY 10032.
NEURiTE OUTGROWTH OFPC12 CELLS IS INHlIBiED BY
rANUSAND BOTULUM-A, -B and -C NEUROTOXINS
((G. Bi, and R. A. Steinhardt)) Departent ofMolea and
Cell Biology, University of Califonia at Berkeley, 391 ISA,
Several reports indicate that okadaic acid (OA), a potent and specific
inhibitor of serine/thronine protein phosphatases 1 and 2A, can indwe
neuronal cytoskeleton disrption. This effect is due to changes in the
phosphorylation state of the cytoskleletal proteins, primarily, the
microtubule associated proteins MAP and Tau. Since the neuonal
cytoskeleton plays a pivotal roll in neuritogenesis and neurnal
degeneration, an study to delineate the effects of OA on neurite outgrowth
Rednal explants taken from six daysin refinal explants was uner
old chick donor embryos (white Leghorn), cultured on collagen gels,
covered with nutrient medium (BME eniched with optic lobe extract,
5OOg/ml) and incubated for three days. After 24 hr post explantation, the
cultures were treated with nanomolar concentations of OA (0.5-10 nM)
for 48 h. Neurite number (NN), length (NL) and growth index (NGI) were
determined at the end of the expeim in an inverted microscope coupled
to an image analyzer. We have found that exposure to OA (0.5 to 2.5 nM)
induce a dramatic dose dependent reduction (50-80 0%) of the rate of NN
and NL, with an overall decrease of NGI. Viability of the explants was
unaffected in this range of exposure, 10 nM induce a complete inhibition
of neuritogenesis. In conclusion, OA at concentations low as 0.5 nM,
ificantly inhibits the outgrowth and affect the morphology of neurtes
without impairment ofthe viability of the cultures.
as
Berkeley, CA94720-3200
To investogate the role ofexooytosis in neurite outgro , we
used the well-chaacterized bacterial neurotoxin targeting
sion complex to disrupt
vesicle do
proteins in synaptic
NGF
c
in PC12 rat pheoebromo
vesidle exocytosis
primed PC12 cells were plated on matrigel oated glass
with
coveraip for a few hours before being microisjected
control buffer or one of the four neurotoxins: tetanus toxin,
s.
(cleaving synaptobrevin), botulinum-B (cleaving and
synaptobrevin), botulinum-A (cleaving SNAP-25),length was
betulinum-CI toxin (cleaving syntaxin). Neurite
outgroth in all of
40 hours after injection. Neurite
measured
by
injected PC12 eafs
four
compared to that in control buffer injected cel These results
suggest that membrane addition by exocytosis mediated by
similar do
ion complex (snaptobrevin/SAPis partially responsible for neuite outgrowth
25/syntaxin)
cells.
PC12
the
toxins
is inhibited
30-50%
a
in
Monday. Neural Development I (1366)
235a
1366
ULTRASTRUCITURAL AND MORPHOMETRIC MANIFESTATIONS OF CHRONIC
ETHANOL TREATMENT ON RAT SUPRAOPTIC NEURONS. ((J. Joshua, T.
Thrower, A. Andrews and W. H. Woods)) Department of Biology, Philander
Smith College, Little Rock, AR 72202. (Spon. by B. Lyn-Cook)
The synthetic activity and/or release of posterior pituitary hormones
(oxytocin and vasopressin) which are produced in part by the hypothalamic
supraoptic nucleus (SON) are strongly inhibited by ethanol. The present
study was designed to determine the effect of chronic ethanol
administration on the ultrastructure of SON neurons.
One week old
Sprague-Dawley rats were given daily intragastric 3.0 g/kg doses of ethanol
until 4 wks, 3-4 mo or 6-7 mo old. Brains were removed and hypothalamic
regions containing the SON were processed using standard EM procedures.
Electron micrographs were studied for ultrastructural changes, and
cytoplasmic, nuclear, nucleolar cross-sectional areas were determined
from the micrographs using a Cal Comp digitizing pad connected to a
computerized image analysis system. Total counts of organelles which
appeared altered (mitochondria, neurosecretory granules (NSO) and
lysosomes/dense bodies) were made for each cell. The most significant
changes in soma, cytoplasmic and nuclear areas of SON neurons were the
smaller size of the 4 wk controls and experimentals as compared with the
older control and experimental groups (3-4 mo and 6-7 mo). Likewise,
there was a significantly smaller number of NSGs per 100 pm2 in 4 wk
controls and experimentals and between this and the 3-4 mo and 6-7 mo
control and experimental animals. Lysosomes and mitochondrial number
also increased per 100 pm2 in 3-4 mo and 6-7 mo control and experimental
groups over that of the 4 wk. However, the experimental groups in each
case showed a significant increase in the number of highly vacuolated and
generally enlarged mitochondria over that of controls.
Synaptogenesis and Synaptic Plasticity (1367-1370)
1367
KINETICS OF COMPARTMENTALIZATION AND DIFFERENTIATION
OF THE GOLGI APPARATUS IN MUSCLE FIBERS DURING
DEVELOPMENT OF THE MOUSE DIAPHRAGM. ((C. Antony1, B. J.
Jasmin2, M. Huchet3, J.P. Changeux3 and J.
Institut Jacques
Monod, Paris, Francel; University of Ottawa, Canada2; Institut Pasteur,
Paris, France3.
Cartaudl)).
Shortly after the onset of synaptogenesis (El3-14), compartmentalization of
the expression of the genes encoding the acetylcholine receptor subunits
occurs (Piette et al., Dev. BioL 157: 205-209, 1993), suggesting a regional
specializaion of the machinery responsible for the targeting of the receptor to
the postsynaptic membrane. Indeed, in adult chicken or rat muscles, the
Golgi apparatus (GA) appears both to be restricted to the subneural domain of
the endplate and to be biochemically differentiated (Jasmin et aL, PNAS, 86:
7218-7222, 1989; and in preparation, 1994). In the present work we
analyzed the kinetics of relocation of the GA in the developing diaphragm at
various times after innervation using a panel of antibodies mapping to
different subcompartments along the secretory pathway. We show that i)
compartnentalization of most of the GA markers occurs at E16, i. e. 2-3 days
after innervation, ii) some GA markers are no longer expressed even in the
subsynaptic Golgi, iii) by contrast, the rough endoplasmic reticulum or the
intermediate compartment, labelled with rabl, do not display
compartmentalized patterns of staining, iv) upon denervation, a burst of
reexpression of all Golgi markers is observed. Taken together, these data
show that in innervated muscle fibers only part of the secretory pathway, i e.
the GA, is compartnenslized and placed under nerve controL
1368
PRESYNAPTIC LOCAUZATION, MEMBRANE ASSOCIATION, AND
AXONAL TRANSPORT OF GUINEA PIG TYPE I BRAIN HEXOKINASE ((JA
Gamer, KD Unse, and RK Polk)) Cell and Neurobiology, USC School of
Med., Los Angeles, CA 90033; Beckman Instruments, Fullerton, CA 92634.
The identification of central nervous system presynaptic terminal proteins
has been of increasing recent interest Previously, we reported unique
characteristics of a 118 kD protein (pp118) axonally transported to the
presynaptic terminals of retinal ganglion cells as part of slow component b
(SCb), the proteins hought to compose the cytoplasmic matrix Afthough one
of many proteins in SCb, pp118 uniquely co-isolated with the synaptic
junctional complex upon biochemical hacionation of the radiolabeled
synaptic regions. Also, pp118 was found to exhibit a marked increase in
hydrophobicity, and thus potential relbtonship with membranes, after entry
into the presynaptic environment. Purification of pp118 from synapses and
amino acid sequencing of proteolytfc digest fragments revealed that the
protein was a guinea pig isoform of the glycolytic enzyme Type I (brain)
hexobnase. Further biochemical treatments support the identity of the
protein. Polyclonal antibodies made to pp118 reveal an expected restiction
of the protein to CNS neurons and an enrichment of the antigen in
presynaptic terminal areas of neurons. One question in the study of neuronal
hexokinase has been the fidelity of its known reversible association with
mitochondrial membranes. While it is clear that hexokinase does associate
with mitochondrial membranes, it is not known if that association is restricted
to mitochondria, or might also represent association with other membrane
systems. The preferential enrchment of putative hexokinase during synaptic
junctional complex puification was unexpected, and could resuft from its
sbtng association with membranes of the presynaptic portion of the synapse.
This enrichment was not mimicked by similar biochemical enrichment of two
intrinsic mitochondrial enzymes. Under the conditions used, few mitochondria
appear to be identifiable in the fractions highly enriched for ppl 18, though
synaptic 'junctional structures are visible (Matus and Taff-Jones, 1978).
1369
1370
PICCOLO,
EXPRESSION OF AGRIN ISOFORMS IN DEVELOPING RAT: AN IN
'Dept
SITU HYBRIDIZATION STUDY ((D.M.Stone and K.Nikolics)),
Department of Neuroscience, Genentech, Inc., South San Francisco, CA.
94080. (Spon. by J. Mather)
A 420 KDA SYNAPTIC JUNCTIONAL PROTEIN
LOCALIZED TO THE ACTIVE ZONES OF SYNAPSES IN THE RAT
CNS ((C. Cases-Langhoff, K. Takei*, P. De Camilli*, C. C. Garner))
of Pharmacology, Yale University, New Haven, CN 06536; NRC,
University of Alabama at Birmingham, Birmingham, AL 35294.
We have utilized a molecular and
immunological approach
to isolate and
characterize novel structural components of synaptic junctions from the CNS of
the rat. This has lead to the identification of cDNA clones encoding a 420 kDa
brain specific protein called Piccolo. Antibodies generated against short
segsnents
coding sequence
of
have been used to determine the
and
subcellular distribution of Piccolo in rat brain. At
level,
Piccolo show a similar
with the
vesicle
associated
the
of dendrites and cell
bodies. This
that Piccolo is associated with
pattern
spatial
light microscopic
punctate expression pattern
synaptic
protein synaptophysin, outlining
profiles
punctate
suggesting
synapses was confirmwd by EM analysis. Using the ABC method peroxidase
reaction product has been shown to be concentrated in the presynaptic nerve
terninals at the active zones of synapses. EM-immunogold labeling of cellular
fragments of cerebelum show gold particles decorating the cytoskeletal matrix
in between synaptic vesicles of synaptsones. These data suggest that Piccole is
part of the cytskeeal network ankoring synaptic vesides to the active zone of
these synaptic junctions. This hypothesis is supported by biochemical studies
revealing that Piccolo is tightly assoicated with synaptic junctional
preparation. Piccoio appears to be preforming a central role in the formation of
synaptic juctions since it appears in synapses, both in vivo and in vitro, as soon
as they are formed.
Agrin, a large multi-domain protein localized to the extracellular matrix at
the neuromuscular junction (NMJ) and derived from spinal cord (SC)
thought to play a key role in synapse formation at the
NMJ and possibly in the brain. Several different alternatively-spliced
isoforms of rat agrin have been identified, which differ by the inclusion or
exclusion of small inserts at three sites in the C-terminal half of the
molecule, and by nicotinic acetylcholine receptor (AChR)-aggregating
activity on skeletal muscle fibers (Ferns et al., Neuron 11, 491, 1993). We
used in situ hybridization histochemistry to examine differential isoform
expression in developing rat. Six 36-mer oligonucleotide probes were
designed to distinguish between known mRNA isoforms at both the Y site
(Exon 28: contains either a 0- or a 4-amino acid insert) and the Z site
(Exons 31-34: contains either a 0-, 8-, 11-, or 19-anino acid insert). From
developmental age E I S-PO, Y4ZO was the major isoform expressed in the
nervous system: in mitotic ventricular zones, dorsal root ganglia, and
diffusely throughout the brain. YOZO was not found in nervous tissue, but
motor neurons, is
specifically labelled microvessels within the brain and SC; expression was
not detected in peripheral blood vessels, however, suggesting a potential
role for YOZO in blood-brain barrer function. Y4Zl9 and Y4Z8, the forms
most active in AChR aggregation, were expressed specifically in SC
motoneurons; Z19 expression declined from E15-Pi, whereas Z8
expression increased slightly from E15-adult ZI1 could not be detected.
These data are consistent with results from PCR analysis (Hoch et. al.,
Neuron 11, 479, 1993), and suggest that different agrin isoforms may
serve different functional roles.
Synaptogenesis and Synaptic Plasticity (1371-1376). Monday
236a
1371
THE EFFECr OF ACRIN ISOlORMS ON CULTURED XENOPUS MYOTOMAL
MUSCLE CELLS. ((D.F.
Daggettt
CellBiol. and
NK
Haaelll
D. Stnel,
J.R.
and Cur. in NurobioL.,
Genentech, Inc., S. San
,
Anat
olicA
and IKB.
UNC, Chapel
Frandsco,
CA
Hill,
and
of Ophthalmolo, Univ. of Pittsburgh, Pittsburgh, PA 15233.
Deept.
Agrr n has long been imiplcted as a molecu involved in postsyaptic
of theneuromuscular juncin. As a atep towards furte
differentiati
which agrin induces the aggregatin of
have begun to characterize its activity in
Xenopus muscle cell 14 hr incubation with full-length souble recombnant chick
dusters on the ventral
agrin isoforms led to the formation of numerous small
aurface of cells with differing efficacea A.B.>A,B,,=A,B. with
being
dependent as
the
inffective. This activity appeared to be tyrone
aboished the effect
After
kinase inhibitor tyrphostin RG50864 (80 tM) completely
30 all agrin isofom could be labeled with an agnn
incubetion at 4-C for min
antibody in an evenly distributed, punctete manner on the muscle cell surface.
After a
22C
Untreatedcells ahowed negligilesating withthis
incubation, the activeisoforms induced ventral AChRclustering, and agrin was
to the edg of theaell and at the few dorsal
enriched at ventral dusters
showing agrin redistribution. However,
dusters, in agreement with previousstudies
in addition to the dustered agrin, with al isoforma there remained a punctste
distnrbution across thesurface of the cell, suggesting thatit was notall redistbuted
to sites ofcdustered AChRs Agrin bindingto the myotomal cell surface and
induction of AChRclustering were Ca'-dependent, inhibited by Ca'-freemedium
have
with 1 mM EGTA, as previously shown in other systems. Asp
been implicated in synaptognesis, the distnrbution of perlecan,the major myotomal
undersatnding the nehn
(
AChR
acetylcholine receptors
by
we
AChRR
A,,
,
antibody.
14hr,
coae
heparan sulfate
and
chondrodtin
sulfate
were
studied in reference to agrin binding sites. The even, punctateagr labing, the
punctate, fibrousperlecan labeling and the sparse, punctate CS labeling showed
distinct distributions, yetshared some areas of enrichment and others of oioal
overlap.
The relevance of
prtoglycan
to
agrin's
activity
is
being investigated.
(Supported by NIH, Muscular Dystrophy Association andGenentech, Inc.)
1373
SPONTANEOUS CLUSTERING OF ACETYLCHOLINE RECEPTORS IN C2
MUSCLE CELLS IS ASSOCIATED WITH CHONDROITIN SULFATE. ((I. Mook-Jung,
Colge of MedIIe, Universy of ArIzona,
and H. Gordon)) Departmnt of Anm
Tucson, AZ 85724.
Proteoglycans have beenImpllcated In thecustering of acetylcholine receptors
(AChRs) on cultured myobubes and at the neuronuscular junction. We have
prevbusly descrbed theIsolatIon of genetic variants in protoglyn bIosynthesis
from the C2 mouse musclecoell e andhave caacteedhI del the phenotype of
one of those variants, S27.S27 Is defectivein the biosynthesis of glycosaminoglycans (GA(is), fails to form spontaneous clusters of AChRs, does formnerveassociated AChR clusters, doe not bind laminin to the surface ofmyotbes, and
responds only weakly to agrln (J.Neurosci.,13Z5 595; Neuron,11:491-502). Two
othe oftheorihl variant,S1i and S26, formnmobes but fal to sponanously
cluster AChRs (Abstract 410, ASCB 1993). The three variat show dIferentboadand are especially deflclrthi thesynthesis of
spectrum defectshi GAGbI
chondroltn sulate (CS) chains thatelute at high salt hI ion-exchange chromatography.Heterokaryona formed in pair-wise fusbns of the variants sponnouwly
clustered AChRs and recovered syrnhesIs of GAGs, espedally of CSe tln at high
As lt is highly urlikely that defects hi AChR
salt Inbn-exchange chr
clustering would have arisen through a chance association with three different
defecs hI GAGbosynihesis, we conclude that there is a requirement for poper GAG
bbsynthesishi AChR clustering. We were also able to manipulate GAG biosynthesis
In wild type C2 cells wlth severalp adim and to examIe the effects on AChR
clustering. Chiorate was found to hbi both GAG synthesis and the clustefing of
AChRshi a dose-dependent manner. When extracellular calcium was raised from 1.8
to 6.8 mM in cultures ofwild ype C2myotubes, both the frequency of spontanos
AChRclusters and the level of cl layer-assolated CS were Increased. Culture of
wNld type C2nmoyubes in the presence of chonasklte ABC eliminated cel layerassocated CS and prevented the ornation of AChR clusters. Cell layer hoparan
suNate was unaffected. Treatment wlth chondroktinase ABC only prevented AChR
clustern I begun prior to the fomaton of spontneou clusters. This Uggests that
CSis required In the initiation but not themairtenance of AChRcusters.
1375
DEVELOPMENTAL REGULATION OF A PROTEIN KINASE C ISOFORM LOCALIZED IN THE
NEUROMUSCULAR JUNCTION. ((L Hllgenberg and K. Miles)) Department of Anatomy and
Ceil Biology, SUNY H"eth Science Center at Brooklyn, Brooklyn, NY 11203.
-
Protein kinase C (PKC) has been suggested to play a role in synapse formation and
signal transduction at the neuromuscular junction. The specifi PKC lsotorm(s) Involved
In these various proesses has not been Identfled.
Skeletal muscle has been shown to express high levels of mRNA encoding for cPKCa
and nPKCe. To examine the protein distribution of nPKCe we raised a
antisera against nPKCe. The affinity purified antisra specifically rcognIzed an 80 kDa
protein highly enriched In skeletal muscle compared to other tissues and long term
phorbol ester treatment of myotube cultures resulted in the down-regulation of this 80
kDa protein. A comparison of the subcellular distribution of nPKCe and cPKCa In rat
skeletal musce reveaisd that nPKCe was enriched In the membrane traction whereas
cPKCa was more abundant In the cytosol fmraction. Furthermore, nPKCe was found to
be postnatally developmentally regulited wth a 4-told increase in expression occurring
during days 4 to 21. Expresson of nPKCe In rat spleen, another tissue expresing
detectable levels of this isoform, was not developmentally reguiated during this time.
Only a moderate Increase In cPKCa expression In skeletal musce was obeved during
postnatal days 4 to 21. Immunocytochemical staining for nPKCe hI skeletal muscis was
associated wlth the sarcoismma and appeared to be predominantly located at the
nouromuscular junction. This lmmunoreactivtty persisted after denervation suggesting
that it was aiso localized at
that the stain Is posaynaptic. Staining for cPKCa
motor endplate but the moat lntehra staining patterns were extralunctlonal.
that
nPKCO
is highly and spciftically
The results of our experiments demonstrat
expreaed In the membrane fraction of skelel muacle. The developmental increas In
nPKC5 exprmoon during the time of synapse maturation and the distribution of nPKCe
in nouromuscular junctions support the hypothesis that the nPKCe isoform pisys an
Imporant role In the neuromuecular synapse.
peptidh-spcilftc
revzesd
1372
POSTSYNAPTIC CLUSTERING OF GLUTAMATE AND GABA RECEPTORS
IN CULTURED HIPPOCAMPAL NEURONS. ((A.M. Craigt and G. Banker2))
'Department of Cell and Structural Biology, University of Illinois, Urbana, IL
61801 and 2Department of Neuroscience, University of Virginia School of
Medicine, Charlottesville, VA 22908.
Several immunocytochemical and physiological studies have demonstrated
of
receptors at postsynaptic sites on neurons,
a concentration
but
neurotransmitter
has not been clear whether receptor clusters are selectively localized
opposite terminals that release the corresponding neurotransmltter. Using
antibodies against the excitatory AMPA-selective glutamatereceptor subunit
and the inhibitory GABAA receptor 12/3 subunits, we found that these
different receptor types cluster at distinct
sites on cultured rat
subunits clustered on cell bodies
hippocampal neurons. The
and dendritc shafts opposite GABAergic terminals, while GluR1
mainly on
spines and was associated with glutamatergic synapses.
The metabotropic glutamate receptor mGluRl a, which is not endogenously
at
detectable
levels in cuitured pyramidalcells, was expressed in
expressed
the neurons from a defective herpesvirus vector. The expressed mGluRl a,
like
was restricted to the
domain and formed postsynaptic clusters on
spines. Furthermore, mGluRl a and
formed clusters at many of the same postsynaptic sites, suggesting that
similar mechanisms may regulate their localization. Chronic blockade of
evoked
release did not block receptor clustering at
sites. These resuits suggest that complex mechanisms invoMng nerve
GluRl
GABAARI2/3
postsynapfic
dustered
dendritic
GluRi,
dendritfc
somatodendrltc
GluR1
postsynaptfc
transmitter
terminal-specific signals are required to generate such a postsynaptfc receptor
mosaic.
Supported by NIH NS17112, NS09248
and
NS33184.
1374
CHICK ARIA IS CONCENTRATED AT EMBRYONIC NEUROMUSCULAR
Sandrock A.,Yee A.& Flschbach G.))
JUNCTIONS
Neurobiology Dept., Harvard Medical School, 220 Longwood Ave, Boston
MA 02115
((A.Goodearl,
Chick ARIA
(acetylcholine receptor inducing activity)
was
purified and
cloned bymeasuring increased AChR expression on the surface of cultured
primary muscle cells (Falls et al. 1993 Cell 22 801). It is a member of a
diverse family of growth factors including GGF, NDF and theheregulins,
encoded by asingle gene that generates many isoforms through alternative
splicing. As motor neurons express ARIAmRNA during embryonic
development, we propose that ARIA acts in vivo as a trophic factor secreted
bymotor neurons that regulates focal neurotransmitter receptor expression
(and probably other synaptic specialization) on its target muscle. We have
developed a panel of polyclonal antibodies raised against sequences from
in order to investigate this hypothesis. An antiserum
proARIAl, aP1 isoform,
(HM22) specific for the N-terminus of proARIAl gives strong staining of
neuromuscular junctions (nmjs) in slow (anterior latissimus dorsi, ALD) and
fast (posterior latissimus dorsi, PLD) muscles in late (E18) embryonic chicks.
Although no ARIA staining is seen in motor neuron cell bodies, nerves
immediately proximal to the nmjs are lightly stained, in a pattern that appears
to be extra-axonal.
Thbus ARIA may be rapidly transported from the spinal
cord and accumulate at nerve terminals. Preliminary studies using
electron microscopy suggest that ARIA at nmjs is concentrated between nerve
and muscle cells in the synaptic cleft. HM22
is first
observed at E16, some days after initial synaptic contact, suggesting that this
isoform may play a role in thematuration and maintainance of synaptic
may play in the
receptor levels. The role that other
initial formation of synaptic AChR clusters is
under investigation
using other antisera.
immuno-
imnmnoractivity
currentlyisofonrns
neurotransmitter
1376
ARE THE NEUROTOXIC EFFECTS OF METHYL MERCURY
(MeHg) MEDIATED THROUGH THE ACTIVITIES OF PROTEIN
KINASE A (PKA), PROTEIN KINASE C AND/OR
ACETYLCHOLINESTERASE? ((M. Davis, M. Darville and 0.
Turner)) Department of Biology, Morgan State University, Baltimore,
Maryland. 21239. (Spon. by T. J. Robinson)
In our attempt to clarify the neurotoxic basis of MeHg at the enzyme
level in synaptosomes from the brain of rats, we investigated the effects of
PKC and
activity in the bmin of
MeHg on the
working hypothesis was that the effects of
Sprague Dawley rats.areOur
mediated through the phosphorylation of brain
MeHg
as well as by the inactivation of the
proteins by PKC and
enzyme. In this study, the animals were treated with
for
5
from the forebrain,
(3
MeHg mg/kg, i.p.)
days. The
midbrain, and hindbrain of these animals were prepared according to
standard biochemical techniques. The PKA and PKC activities were
determined using the
Protein Kinase Assay System
Reaction Kits (Sigma
(Upstate
Biotechnology, Inc.) and the
Chemical Co., St. Louis, Missouri) was used to assay for
activity. The results of our investigatiDons showed that
a 1.43, 9.4, and 1.1 fold increases in PKA
MeHg treatment producedand
1.6 fold. increases in PKC activity for
5.5,
activity; and, 1.92,
isolated
from
the forebrain, midbrain and hindbrain,
synaptosomes
PKA,
acetylcholinesterase
neurotoxicity PKA
acetylcholinesterase
synaptosomes
Non-Radioisotopic Ellman
acetylcholinesterase
gespectively.
Additionally,
an
inhibition of
acetylcholinestemwa
activity
observed in synaptosones of all of the brain regions following MeHg
These increases in PIKA and P3KC actiivity observed, suggest
treatment.
that MeHg produces its neurotoxic effects by intercting synergistically
with both the cAMP and the inositol pathway and by inhibiting
acetylcholinestemse.
(Supported by Grant #SRC-7 (20) 2 T34 OMO
7977-11 from NIH).
was
237a
Monday. Synaptogenesis and Synaptic Plasticity (1377)
1377
F1/GAP-43 LEVELS ARE ALTERED AFTER THE INDUCTION OF
HIPPOCAMPAL MOSSY FIBER LONG-TERM POTENWIATION
((D.T. Rivera, M.L Escobar, E Barea-Rodriguez, B.E Derrick and J.L
Martinez, Jr.)) Departments of Psychology and Cell and Molecular
Biology, University of California, Berkeley, CA 94720.
Hippocampal long-term potentiation (LTP) is a long lasting process of
synaptic plasticity that currently is considered the best model of memosy
storage in the mammalian brain. Thus far, two forms of hippocampal
LTP have been characterized: NMDA receptor-dependent and opioid
receptor-dependent mossy fiber (MF) LTP, which is associated with
bilateral synaptogenesis even in the absence of seizures (Escobar et al.,
Soc. Neurosci. AbsL in press). To explore further our previous findings,
we measured the levels of F1/GAP-43 after the induction of MF LTP.
Fl/GAP-43 is a neuronal, PKC substrate that has been associated with
synaptic plasticity. Using Western blot analysis, we measured the
protein levels of F1/GAP-43 four and seven days after the induction of
LTP at the MF-CA3 synapse of an anesthetized rat Both sham and
seizure control animals were used. We observed that F1/GAP-43 levels
are decrased in the side where the stimulus was applied, but increased
in the contralateral hippocampus at four days. At seven days following
LTP induction, F1/GAP-43 levels were reduced in both hippocampi.
Since F1/GAP-43 is associated with growth, its increase in expression in
the contralateral side after 4 days suggests a possible regulatory role in
the development of new synapses following MF LTP. Supported by
DA01495 (JLM), NSF-DIR 9101951 (DTR), CONACYT MEX (MLE).
Endoplasmic Reticulum (1378-1381)
1378
A mRNA SEQUENCE RESEMBLING A -1 TRANSLATION FRAMESHIFT
INVOLVED IN SRP RECEPTOR MEMBRANE ASSEMBLY. ((J.C. Young
and D.W. Andrews)) Department of Biochemistry, McMaster University,
Hamilton, Ontario, Canada.
The site of signal recognition particle (SRP) mediated membrane targeting of
nascent secretory proteins is the SRP receptor. Using deletion mutagenesis and
a cell free assay system, we have now mapped the region of SRa required for
membrane assembly. Examination of the translational properties of mRNA
encoding SRa revealed a sequence necessary and sufficient to cause ribosomes
to pause during translation. RNase protection and primer extension assays (toeprinting) demonstrated that the pause sequence is positioned within the SRat
mRNA such that the SRa membrane assembly domain exits from the ribosome
just prior to pausing. The primary sequence and predicted secondary structure
of the mRNA at the ribosome pause site closely resembles a -1 frameshifling
sequence. Moreover, it was possible to demonstrate a small amount of -1
frameshifting at this site in vilm. By separating membrane bound polysomes
from cytoplasmic ones it was also possible to demonstrate that membrane
assembly correlates with ribosome pausing. Thus, ribosomes translating SRa
mRNA sequences 5' of the pause site are either cytoplasmic or tethered to ER
membranes by the 3' end of a previously targeted mRNA. Ribosomes translating
sequences beyond the pause site are membrane associated. Experiments with
puromycin confirmed this result and indicated that at steady state translation,
the 3' end of the SRa mRNA is attached to the ER membrane via the
interactions of the nascent SRca polypeptide.
1380
RECONSTITUTION INTO PROTEOLIPOSOMES AND PARTIAL
PURIFCATION OF TIE ENDOPLASMIC RETICULUM ATP
TRANSPORTER. ((E. Guillen, and C.B. Hirschberg)) Department of
Biochemistry and Molecular Biology, University of Massachusetts Medical
Center, Worcester, MA 01655.
Recent studies in vitro demonstrated an ATP transpr in the membrane of
the endoplasmic reticulum (ER) of mammals and yeast This transport
allows ATP, which is synthesized primarily in mitochondria, to be available
in the lumen of the ER for reactions requirng energy durng folding and
polymerization of proteins as well as phosphorylation. Purification and
charcteion of this tansporter is important to (a) determine whether it is
regulated and thereby affects the amount of ATP available in the lumen of the
ER for the above descsibed reactions, (b) understand the mechanism of ATP
translocation and (c) detemine the transporter arrangement within the ER
mmbrane and its reltionship with other cellular ATP transps. Towards
bne proteins were solubilized with detergent and
this goaL ER
inc
ated into unillar phosphatdylcholhne liposomes. The resulting
proteollposomes transported ATP in a satrable manner with an apparent Km
of 0.5 pM, a value very similar to that of intact ER-derived vesicles.
Transport of AT? into proteoliposomes was inhibited by DIDS and
atractyloside in a manner similar to intact ER-derived vesicles. The above
proteoliposomes were highly specific towards solute transport: nucleotide
sugars and nucleoside phosphates which do not enter the lumen of the ER
were not transport
Supported by NIH grant GM34396.
1379
RER-65, a memnbrane protein of the Rough Endoplasmic Reticulum is
related to p63, a protein of an ER-Golgi intermediate compartment.
By Y-J., Chen, A.Stieber,N.K.Gonatas,University of Pennsylvania School of
Medicine, Phiadelphia, PA 19104, and W.S.Lane, The Biological
Laboratories, Harvard University, Boston, MA 02138.
RER-65, identified by monoclonal antibody 2H1, is a protein of the rough
endoplasmic reticulum (RER) of several rat cells including neurons,
hepatocytes and pheochromocytoma (PC1 2), (Chen et al.
J.Histochem.Cytochem. 1991, 39,635).We report here that RER-65 and
p63, a protein of an ER-GoIgi intermediate compartment of primates, are
closely related (Schweizer et al. J.Cell Sci. 1993,104,685).
By immunoelectron microscopy, RER-65 is localized in the RER and
nuclear envelope of brain neurons; by Westem blotting, mAb2H1 reacts
with a 65 kDa band of liver rough microsomes. Partitioning in Triton X114, extractions in carbonate, and tryptic digests of membrane fractions
from PC1 2 cells, show that RER-65 is an intrinsic membrane protein
with a 10-15 kDa cytoplasmic segment. RER-65 does not display
intrachain disulfide bonds but it may form interchain disulfide bridges and
homomultimers. Metabolic babeling of PC1 2 cells in the presence of
tunicamycin, digestions with 0-glycanase and chromatography of PC1 2
membrane fractions on a concanavalin-A column show that
RER-65 is not glysosylated.
Amino acid sequences of nine peptides, 132 residues, derived from tryptic
digests of RER-65 purified from PC12 cells, reveal 75% to 100%
identities with p63, while two peptides,35 residues, of RER-65 show
no identities with p63. The biochenmical and sequence data strongly
suggest that p63 and RER-65 are cosely related.
Supported by N IH grant NS 05572.
1381
CHARACTISATION
IN THE
OF YEAST MUTANTS
DOLIC55DL-LIliXKD
PATHWAY. ((J. Roos, N. Dean, J. Eu, and W. J.
Lennarz)) Department of Biochemistry and Cell Biology, State
University of New York at stony Brook, Stony Brook, NY 11794,
OLIOOS CCIARIDE
N-linked glycoproteina are the product of two bioaynthetic
processes in the endoplassic reticulumi the assembly of the
and
and
the
translation
oligosaccharide
lipid-linked
In the yeast
glycosylation of nascent polypeptide chains.
Baccharoayces corevisaae, the enzyme that transfers the
oligosaccharide chain from the lipid carrier to nascent
We
have
polypeptide chains is still poorly understood.
simple, yet highly snsitive assay to screen for
devloped
This
in
defective
assay
peptide glycosylation.
yeast mutants
utilizes endogenous oligosaccharylpyrophosphoryldolichol, the
end product ?A, the dolichol phosphate synthetic pathway, and an
for
N-linked
substrate
I-labeled
peptide
exogenous
glycosylation. Characterization of mutants identified by this
screen allowed us to group the mutants into three classesi those
defective ln (1) dollchol phosphate synthesis, (2) lLpid-linked
ynthesis, or (3) the transfer of the
oligosaccharide
A putative temperatureoligosaccharide chain to proteln.
sensitive Class 3 mutant (JRY163) has been identLfied and two
genes that affect glycosylation in thl mutant have ben cloned.
MNG is an essentLal gene that suppresses the temperature
sensitivity of JRY163, but not the peptide glycosylation dfefct.
NUGi
effects the oligosaccharyl transferase mutant, wbpl-1, ln
The socond gene, O083, rescues both the
the same fashion.
growth and peptide glycosylation defect of JRY1Y63, yet has no
a
effect
efforts
when
are
introduced
into
focused
the
on
the
f
wbpl-1
fect
of
mutant
8I50
on
strain.
other
Current
mutants
in
the glycosylatLon pathway and the subcellular locatLon of Neglp.
In addition, the role of 0873 in N-linked glycosylation is boing
studied. (Supported by NIB grants GK33184 and 01133185 to 13L).
238a
Endoplasmic Reticulum (1382-1387). Monday
1382
1383
ISOLATION OF MUTANTS DEFECTIVE IN KARMELLAE
MEMBRANE BIOGENESIS IN S. CEREVISIAE ((A. Koning, S.
Nygaard, K. Johnsen, G. Hedden, and R. Wright)) Dept. of Zoology,
University of Washington, Seattle, WA 98195
GENETIC CHARACTERIZATION OF S. CEREVISIE MUTANTS
DEFICEENT IN ENDOPLASMIC RETICULUM-ASSOCIATED
DEGRADATION (ERAD). ((.E.Ernaga, E.D.Werner, I.V.Karpichev,
and A.A.McCracken)) Department of Biology, University of Nevada,
Reno, Reno, NV 89557
Proliferation of a specific type of ER membranes called karmellac can be
induced by a ten-fold increase in the levels of the integral ER protein, HMGCoA reductase (HMGR). This response allows us to investigate the
mechanisms that underlie cellular membrane biogenesis. Using a vital
membrane dye, DiOC6, we have screened mutagenized yeast cells for
defects in karmellae biogenesis and identified several non-conditional
mutants and many temperature-sensitive mutants that still have high levels
of HMGR. Several of the non-conditional mutants, AK1, 2, and 4, do not
generate karmellae with elevated levels of HMGR from a multicopy HMGR
plasmid, but do generate karmellae with higher levels of HMGR from a
galactose-inducible HMGR plasmid. In contrast, AK3 generates low
amounts of karmellae with both the multicopy and galactose-inducible
HMG-R constructs. A temperature-sensitive mutant, AK5, does not
generate karmellae at the permissive growth temperature, but does at the
restrictive growth temperature. One mutant,SN6, has been identified that
does not produce karmellae at any level of HMGR in the cell. Interestingly,
karmellae deficiency inSN6 co-segregates with improved growth on media
containing galactose and raffinose as the carbon sources, and reduced
growth on media containing only galactose. This phenotype has been used
to identify genomic clones that complemnent the reduced growth phenotype
on galactose, and that allow the mutant to generate karmellae. A 12 kb
genomic clone that complementsSN6 is currently being analyzed.
1384
ER-ASSOCIATED PROTEIN DEGRADATION IN YEAST.
((E.D.Wernerand A.A.McCracken)) Departent of Biology, University of
Nevada, Reno, Reno, NV 89557
Secretory proteins that are incorrectly processed and assembled acclate
in the ER and are selectively degraded by a quality control mechanism
associated with the ER (ERAD). This proteolytic process is thought to occur
of the ER utilizing novel proteases that are not
within an earlyco
associated with previously reported intracellular protein degradation systems
(McCrackenand Kruse, 1993, Mol.Biol.Cell 4:729). To study this process,
we monitored the degradation of the mutant form of human .1-anti-trypsin
(AlPiZ) expressed in various yeast strains. Our results showed that this
ERAD substate was not stabilized ina strain deficient in vacuolar protease
ng enzyme UBC6.
activity, nor ina strain deficient in the ubiquitinco
However, when the reporter substrat was monitored by ELISA and pulsechase protein radiolabelling in strains with mutations which alter the
"chymotrypsin-like' activity of the yeast proteosome complex, the rapid
degradation of AlPiZ was restricted. For example, the half-life of A1PiZ
in the double mutant Prel-1 Pre2-2 (Heinmyer, et al., 1991, EMBO 10:555)
was increased to 80 mintes compared to the half-life in the parent strain of
52 minutes. These results suggest that ERAD may depend, at least partially,
on a proteolytic activity of the yeast proteosome complex. In another set of
experiments, we studied the effects of ALLN and PMSF, two membrane
permeable protease inhibitors often used to characterize proteolytic activity.
In the presence of either inhibitor, strains expssg AlPiZ show a 4-5 fold
increase in substrate. Onq possible inpretation of these results is that
multiple proteolytic activities are involved in ERAD. (Supported by grants
to A.M. from the American Cancer Society -MV551 and the Cystic Fibrosis
Foundation #G789).
Yeast mutants have been identified that lack the ability to degrade certain
ERAD reporter proteins, including an aberrant form of al-anti-trypsin
(A1PiZ). These mutants have been shown to stabilize this protein ina preGolgicompament, most likely the ER. Pulse-chase protein radiolabelling
experiments showed that the half-life of AlPiZin various mutant strains
ranged from 50-120 minutes compared to 30-38 mimutesin the wild type
strains. The genetic analysis of these mutants was carried out tofacilitate the
identification of the gene(s) involved in ERAD. Tetrad analysis ofthese
mutants was performed to determine the number of genes involved in the
mutant phenotype. About 40% of the mutants characterized conformed to
simple Mendelian genetics, i.e. 2:2 segregation of phenotype. Further
genetic testing revealed that the phenotypes of all but one of the mutants was
expressed in a manner recessive to the wild type gene. Also, the phenotype
of at least one of the mutants was determined to be controlled by two genes.
Complementation analysis of these mutants identified at least seven
complementaion groups. Experiments examining the stability of the mutant
phenotypes were conducted to identify mutants competent for molecular
cloning by complementation of the gene(s) responsible for ERAD.
(Supported by grants to A.M. from the American Cancer Society -MV551
and the Cystic Fibrosis Foundation #G789).
1385
DEGRADATION OFAN ER-RETAINED FORM OF VSV GLYCOPROTEIN LEADS TO
CLASS II PRESENTATION ((S. M. Bartido, S. Diment, and C. S. Reiss))
Departments of Biology and Pathology and the Kaplan Comprehensive Cancer
Center, Naw York University, New York NY
10003-6888.
A20 lymphoma clls infected by a vaccinia recombinant incorporating the 'poison
tail' construct of the vesicular stomatitis virus glycoprotein IVSV GI
and
Bergmann, Cell 34: 513) express a mutated form of the protein that is retained
in theendoplasmic reticulum (ER). Pulse-chase studies show degradation of the
protein (TT -4h). Immunofluoresence studies as wel as ENDO
sensitivities
show the protein to be limited to the ER. This degradation was found unaffected
by monensin or by leupeptin or NH4CI. These results also indicate that autophagy
is an unlikely mechanism of protein degradation. A neutral, non-reducing
environment, which is characteristic of the ER, is required. Pulse-chase
experiments utilizing the proton ionophore CCCP, which equilibrates the pH in the
intracellular compartments with that of the outside milieu, indicate optimal
conditions occuring at pH 6-7 and not at pHI 8. The use of diamide, a thioloxidizing reagent, resulted in inhibition of protein degradation suggesting the
possible involvement of serine or cysteine proteases. An inhibitor of cysteine
protesses such as E-64 did not block the degradation. We have found that
presentation of epitopes derived from VSV G in this system is affected by NH4C,
emetine, and Brefeldin A (Reiss et al Cal Immunol. 139: 229;
Together
our resuits indicate that proteolytic fragments
of the protein generated in the ER
o
lumen may traffic with the nascent class 11
complex to the compartment of
peptide acquisition. Subcellular fractionation studies are
being carried
out to identify the site of peptide binding.
Supported by NIH Grant All 8083 to CSR.
(Rose
D/H
1992).
presently
1386
138T
DEGRADATION OF P-SUBUNITS OF GONADOTROPINS RETAINED
IN THE ENDOPLASMIC RETICULUM OF HYPERSTIMULATED
GONADOTROPHS. ((P. Rosa, M. Bassetti, and D. Lodi Pasini)) CNR
IN VIVO PHOSPHORYLATION OF CALNEXIN OCCURS ON THE
CYTOPLASMIC DOMAIN A REGION REQUIRED FOR
COPRECIPITATION
WITM CLASS MHC MOLECULES((G.
G.
Capps, S. L Moseley, and M. C.
Department of B
University of Califomia, Santa Cruz, A
Calnexin is a major calium-binding protein of the endoplasmic
retiulum (ER) which is implicated in the biosynthesis of class Major
Histocompatibiliy Compblx (MHC) molecules and other cell-surfaceif
glycoproteins. Calnexin is phosphorlated in vitro, but it is unclear
vWo phospho ation of calnexin oocurs on lumenal or on
cytopasmic resides. This issue is of particular importance for
understanding calnexin's chaperone
functions because previous
studies in this laboratory showed that not al class I MHC molecules
with phosphorylated calnexin and that association with
calnexin
Cytopharmacology, Department of Medical Pharmacology,
University of Milan, 1-20129 Milan, Italy. (Spon. by P. Rosa.)
Center of
the fate of the gonadoopin -subunits, accumulated in the
endoplasmic reticulum (ER) of gonadotrophs after gonadectomy, using
markers for the ER, the lysoms and the sectory granules on antenor pituitay ultra-thin fozen sections. After castration, the intacllular evels of the
subunits were found to increase more than that of the a-subunit. Both
gonadotropin subunits co-localized in secretoy granules with secretogrnnin I,
a protein present in secretory granules of many specis. Conversely, the psubunits, but not the a-subunit and secrtogranin II, were localized in the
dilated cisternae of the ER and the ransidonal elements as well as in
irregularly-shaped vacuoles with one thick membrane. Using makers for the
ER (PDI), the prelysosomal compartet and lysosomes (cathepsin D and
lgpl20), we found that these vacuoles correspond to a degrdative
ates, which may represent different
compartment with two types of in
stages in the pathway of gonadotropin -subunits degradation. These vacuoles
do not appear to derive from autophagy, since vesicles
rounded by a double
or multilamellar membrane containing profiles of dstnae of the ER together
with small amounts of the cytoplasm were never detected Moreover, they do
not correspond to cnnophagic bodies, since the lakmr contained psubunits as
well as a-subunit and SgI. Taken together these results show that
rpha are degraded by a
snits reined in the ER of gon
gonadoin
diffent from the clsscal auophagy andis not dmilarto the
non-autophagic pathway for the diversion of the inracisternal ganules
accumulated in the ER to lysosomes, recently described in hyperstimulated
thyrotoph (Noda andFarquhar, J. Cell BioL 119, 85, 1992).
We invesdgated
Zuhga))
logy,
in
rylated
correlates with slower acquisition of Endo H
We now show thatpoorylated calnexin is
phosphorylated exclusively on its cytop
domain in vivo.
Proteinase K treatment of vesicbs rom cells labeled with [5S]
methionine and [s1P104
th
H2Ld and from calnexin. Surprisingly, this tr
alo resuits in the
loss of coprecipitated calnexin in immunopreipitates of H-2Ld and in
the oss of coprecipitated H-2Ld in immunopcipiates
of calnexin.
is required
cytop
Thus, the cainexin
for its
nc domain
coprecipitationofwith class I MHC
molecules. This is acalnexin
surpsing
resuit in Eght the observation that phosphorylated
efficlently coprecipitates with H-2Ld molecules which lck a
domain. Thus, is unlikely thatdomain
domain
the cytopasmic
cvtopbasmkc
of the cla
ozf calnexin associates
I
with the cytopbsmb
that
the
of
MHC molecule. These
findings
sugest
assoclation
calnexin with class I MHC molecules is influenced in Dart by the
conformation of the cainexin molecule. (Supported by NSF grants
DCB-9196051 and DCB-9096241)
removes
rmoves
Monday. Endoplasmic Reiiculum (1388-1391)
239a
1388
1389
RETINOL-MODULATED PROTEIN DISULFIDE ISOMERASE ACTIVITY
OF RAT LIVER TRANSMONAL ENDOPLASMIC RETICULUM. ((E.
Jacobs, R. de Cabo, D. J. Morrd and D. M. Morr6)) Department of Foods and
Nutrition, and Department of Medicinal Chemistry, Purdue Univesity, W.
Lafayette, IN 47907.
ALL-TRANS RETINOL INHBITS BINDING OF GTP-y-S TO A 55 KD
PROTEIN OF TRANSITIONAL ENDOPLASMIC RETICULUM OF RAT
LIVER. ((J. Stevenson and D. M. Morrd)) Department of Foods and Nutrition,
Purdue University, W. Lafayette, IN 47907
The formation and fusion of transition vesicles (TV) from transitional
endoplasmic reticulum (TER) to cis Golgi apparatus (GA) was reconstituted in
our laboratory in a cell-free system from rat liver (Nowack et al., BBA,
1051:250, 1990). At an optimum conc. of 1 gg/ml, the rate and amount of
transfer was approx. doubled over 1-2 h incubation and was found to be the
result of an effect on TV formation. A protein disulfide isomerase (PDI)
activity of TER, stimulated by retinol with reduced and denatured RNase as
substrate correlates with retinol-stimulated budding with TER incubated under
the complete conditions required to induced vesicle budding in a cell-free
transfer assay (ATP plus ATP-regenerating system and cytosol). Oxidized
glutathione or a mixture of reduced and oxidized glutathione also supported the
retinol-stimulated activity. With oxidized and denatured RNase, retinol, as
well as NADH, stimulated reactiviation in the complete system. However, in
the presence of NADH, retinol at all concentrafions tested, inhibited, rather
than stimulated. The results are interpreted as evidence for a and retinolstimulated PDI activity of rat liver TER that may play a role in vesicle budding
or membrane trafficking. Supported by USDA 9301253 and Phi Beta Psi
Sorority.
Nowack et al. (Biochim. Biophys. Acta, 1051: 250-8, 1990) previously
described a role for retinol in stimulation of formation of transition vesicles
from transitional endoplasmic reticulum (TER) of rat liver and suggested that
this stimulation was due to retinol sparing cytosolic GTP. Zhao et al.
(Biochim. Biophys. Acta, 1055:230, 1990) described a retinol-inhibited
GTPase activity of rat liver TER. This activity was monitored and isolated by
solubilization of isolated TER membranes with C12E8 detergent and by DEAE
cellulose and gel filtration chromatography. Active fractions with retinolinhibited GTPase activities also had retinol-inhibited ADPase activity.
Western blots of active fractions incubated with 5'-p-fluorosulfonyl
benzoyladenosine (FSBA) (an adenosine analog) with detection using antiFSBA sera (gift of R. Geahlen, Purdue Univesity) with and without GTP or
ADP revealed a band at 55 kD that was competed by the nucleotide. Blotted
fractions incubated with [35S]GTP-y-S revealed a radiolabeled band at 55 kD
with intensity that correlated with the retinol-inhibited GTPase activity.
Binding of GTP-y- S to TER was greatly reduced with the addition of retinol.
The findings suggest that the retinol-inhibited GTPase has a monomer Mr of
55 kD on denaturing gels. Supported in part by USDA 9301253 and Phi Beta
Psi Sorority.
1390
ANALYSIS OF THE ENDOPLASMIC RETICULUM PROTEIN TRAFFIC
RESPONSE ELEMENT (ERPTRE) OF ERP72, ALUMINAL ER STRESS
PROTEIN ((N. Marcus and M. Green)) Department of Molecular
Microbiology and Immunology, Saint Louis University School of
Medicine, St. Louis, MO 63104
ERp72, a resident protein of the endoplasmic reticulum (ER) is both a
stress protein and a member of the protein disulfide isomerase family
of proteins. The promoter region of the murine ERp72 gene contains
several potential transcriptional control elements, including multiple
CCAAT elements and Sp1 and AP-2 consensus sequences. Functional
analysis of the ERp72 promoter identified an 82 bp segment that is
sufficient to mediate the response of the ERp72 gene to such stresses
as exposure to the Ca"+ ionophore A23187 or accumulation of
incompletely assembled immunoglobulin p heavy chain in the ER. This
82 bp sequence contains two CCAAT elements and a putative AP-2
site and has some limited additional homology to protein traffic
responsive sequences of other members of the ER stress protein family.
Mobility shift analysis using the 82 bp fragment showed no significant
difference in the pattern of bands produced by extracts derived from
control and stressed cells. Competition experiments using a set of
overlapping synthetic oligonucleotides spanning the 82 bp segment
demonstrated that a 30-mer containing one of the CCAAT sites could
abolish the binding to the 82 bp sequence. This CCAAT site is a good
candidate for interaction with CP-1. These studies should allow us to
elucidate the target of the intracellular signaling pathway initiated by
protein traffic in the ER.
Supported by: NSF Grant MCB-9317238.
1391
CROSS-LINKING OF BETA-LACTAMASE TO A HIGH MOLECULAR
WEIGHT PROTEIN COMPLEX IN THE LUMEN OF THE ROUGH
ENDOPLASMIC RETICULUM. ((H. Pestana, R. MooreBatchelder, S. Alcala, P. Tamirisa, and J.B. Denny))
Division of Life Sciences, University of Texas at
San Antonio, San Antonio, TX 78249.
We have added the membrane-permeable cross-linking
reagent BSOCOES to material sedimented from cellfree translation mixtures synthesizing beta-lactamase.
The sedimented material included rough microsomes. Cross-linked material containing processed
beta-lactamase appeared at the top of a 4% and a
12% polyacrylamide gel, and was still observed if
RM were treated with proteinase K following crosslinking, indicating that the material was located
in the RM lumen. The high molecular weight
complexes were readily apparent in sedimented RM,
but could not be immunoprecipitated using antibodies
to beta-lactamase, even though uncross-linked betalactamase was immunoprecipitated. We have shown
that the epitopes recognized by the antibodies are
not destroyed by cross-linking and that free betalactamase that has reacted with one end of BSOCOES
The results therecan still be immunoprecipitated.
fore indicate that the lumenal RER proteins have
bound to beta-lactamase in a way that prevents
Beta-lactamase may
access of antibody molecules.
thus enter a complex of RER lumenal proteins.
Golgi Complex (1392-1393)
1392
EMP47P. A YEAST HOMOLOGUE OF THE VIP36/ERGIC53-FAMILY OF
POTENTIAL INTRACELLULAR LECTINS, REQUIRES ITS C-TERMINAL
KXKXX,MOTIF FOR LOCAUSATION TO A POST-ER COMPARTMENT.
((S. Schr6der, F. Sc&hmmOler, B. SingerOger# and H. RJezman)) Blozertmrum,
University of Basel, Switzerland; #present address: EMBL, Heldeberg,
We have determined the primary sdtucture
of
the Integral
1393
membrane protein
Emp47p of Sacehwomyces cerevisiae (orignaly termed p44; Singer-KrOger et
at., 1993, JBC 268, 14376-14386). The mature type I transmbrane protein
has a moecuar weight of 47-103K. Emp47p has a weak but satistically
sgnificant hology o ERGIC53 (Sehindler et at, 1993, Eur. J. Cel Biol 61, 19). The homobgy extends Into the short cytopassc tail with an ER-Iocalsatlon
signal of the KKXX-(ERGIC 53) and KXKXX-(EMP 47) type, respectvely. The
homology to Vlp36 Is restricted to the N-terminal -280 residues, which
constitute a domain odginayekntfed In VipW as being homologous to plnt
lecdns (Fiedler and Slmons, 1994, Cell 77, 625-626). Deletion disruption of the
EMP47-locus does not elcit any readily observable growth detect. By
Immunofiuorescence Emp47p is locaized to punctate structures with no
apparent spatal affinity br ER, vacuoles or plsma membrane, reminiscent of
Golgi-staining. In fact Enp47p colocalizes in immunofluorescence wfth the
presumed Golgi-protein Pmr1p (Antebi and FirK 1992, MBC 3, 633454) and
fractionates on sucrose gradents ike several Golgi-markers. In addition,
Ermp47p with an engineered N-inked glcosyLon site, displaying a wild type
lmnwnoflurescence pattem, receives a1.6. and a1.3-mannose-modificaions.
We show that dhngfg a sgle lysine in the K)XKXX-otif leads to the escape of
the mutant protein to the vacuole. Enmp47p may be the 1irst exmpe of a protein
with a fucton KXiOKX-mO and a G"oi steady state dibuln
WITEIDRAWN
240a
Golgi Complex (1394-1399). Monday
1394
1395
PROTEIN RETENTION IN THE GOLGI APPARATUS ((T.
Nilsson and G. Warren)) Cell Biology Laboratory, Imperial
Cancer Research Fund, 44 Lincoln's Inn Field, London WC2A
3PX, England
IN VTRO REASSEMBLY OF A POLARISED GOLGI
STACK FROM M[TOTIC GOLGI FRAGMENTS ((C.
Rabouille and G. Warren)) Cell Biology Laboratory,
Imperial Cancer Research Fund, 44 Lincoln's Inn Field,
London WC2A 3PX, UK
We and others recendy demonstrated the importance of the
membrane spanning domain in retention of resident Golgi
cnzymcL To explain etntion, we have posulated that Golgi
enzymes ptcipae in the formaion of heteo-oligoms. Thcse
oligomers would conslat of tousand or more molecules of
related ("kin") nature which would be large enough to ensure
exclusion from budding transot vesicles. We have shown that
medial Golgi enzymes have the capacity to form hetero
oligomers and are prsendy invesdgating their role in organclle
architecture. The pesence of resident medial enzymes is a prerequisite for maintaining the cisternal structure and subte
changes in their primary structure result in drstic alterations of
the Golgi sucue. We propose dtat the role of hetero oligomers
is to maintain the flattened apecarance of the Golgi cisterna and
to prvent fencstration of the membrae.
a
On the onset of mitosis, the Golgi apparatus undergoes
dramatic morphological changes: The stack disassbles
into small fragments which form the Golgi clusters
(Lucocq and Warren, 1989, JCB,109:463). Exiting mitosis (at
telophase), the Golgi reassemble into the characteristic
polarised stack as the transport resumes (Souter et al, 1993,
JCB,122:533). To monitor the reassembly of these mitotic
Golgi fragments, we have developped an in vitro assay
using similar approaches than Misteli and Warren (1993,
JCB, 125:269) for the disassbly of the stack. Preliminary
studies showed that the Golgi can reassemble into a
polarised stack after it has been fragmented under mitotic
conditions.
1396
1397
IDENTIFICATION OF FACTORS REQUIRED FOR THE
DISASSEMBLY OF THE GOLGI COMPLE DURING MiTOlS.
((PA. Barr, T. Mistelli, and G. Warren)) Cell Biology Laboratory,
Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London
WC2A 3PX. (Spon. by G. Warren.)
GOLGI ANKYRIN: ASSOCIATION OF AN
WITH THE GOLGI COMPLEX ((K. A. Beck,
Nelson)) Dept. Molecular and Cellular
University School of Medkice, Stamford, CA
complex disassbles into clusters of
throughout the cytoplasm. After cell
reassemble to gve a functional copy of
the Golgi complex in each of the daughter cells.
Immunodepletion of coatomer from mitotic cytosol prevents this
disassembly process, indicating that ARF and coatomer
dependant vesicle formation is required for mitotic Golgi
breakdown. Further investigation indicates that Golgi
disassembly is a two stage process: the first step requiring only
ARF and coatomer and giving rise to Golgi stacks with shorter
cisternae (core-Golgi stack), and the second step bein the
unstacking and fragmentation of these core-Golgi stacks m an
ARF and catomer indeendant fashion. We are now isolating
components from mitotic cytosol which are required for the
second step in Golgi disassembly, the unstacking and
fragmentation process.
During
mitosis
Golgi
small vesicles dispersed
division these vesicles
1398
CELL-CYCLE DEPENDENT PHOSPHORYLATION OF GLNTIN,
A CYIOSKELETAL-LIKE GOLGI MEMBRANE PROTEIN.
((A.D. Linstedt and H.-P. Hauri)) Dept. of Pharmacology, Biocenter,
Basel, Switzerland CH-4056.
The Golgi complex vesicularizes at mitosis and then reassembles in
each daughter cell as a centrally localized, subcompartmentalized, and
stacked membrane network. We have identified a protein, giantin, that
may be involved in regulating Golgi structure. Subcellular fractionation and EM studies indicated that giantin is localized to cis and medial
Golgi cisternae. Sequence analysis and expression of truncation
mutants showed that a C-terminal membrane anchor and adjacent
amino acids are required for proper localization of giantin. The cytoplasmic domain of giantin, comprising over 3000 amino acids, is largely
composed of heptad repeats. Giantin's migration on velocity gradients
and gel filtration columns suggested that the cytoplasmic domain
forms a -250 nm coiled-coil rod. Glantin from non-synchronized cell
cultures was resistant to extraction with non-ionic detergents, and
bound to microtubules in an in vitro assay. Thus giantin is unique in
being a cytoskeletal-like integral membrane protein. Two dramatic
changes were observed in cells blocked at mitosis: giantin became
readily extractable,.and the incorporation of phosphate into its cytoplasmic domain was increased -20 fold. Giantin was also phosphorylated in vitro by p34cdc2. These findings suggest that phosphorylation
of giantin may regulate cell-cycle dependent changes in Golgi
structure, and that giantin may link the Golgi to the cytoskeleton.
ANKYRIN ISOFORM
J. Buchama and W. J.
Physiology, Stamford
94305-5424
The Golgi complex has a sructual organization composed of sequentially
armnged comprments. While much is Inown about how this structural
organization relates to Golgi function, little is known about how it is
maintaine We have previously demonstrated that an isoform of spectin
immunologically related to m i ey oyte spectrin (£1) resides
at the level of the Golgi apparatus (Beck et al., (1994) J. Cell Biol. in
press), suggesting that a spectrin-based membrane skeleton plays a role in
Golgi
organization and function. To
the
examine
nature
of the int
ion
of this spectrin isoform with the Golgi complex we sought to identify an
isoform of ankyrin that associates with this organelle. Two lines of
evidence in support of a Golgi associated ankyrin are presented. First, by
indirect immunofluorescence we show that an antibody raised against
purified canine erythrocyte ankyrin stains
the
Golgi complex
in
a variety
of cell types including MDCK, NRK and 293 cells. In addition, we have
prepared an HA-epitope tagged, recombinant human erythrid ankynn
cDNA composed of an 384 amino acid portion of the 90-kDa band 3
binding domain. We find that upon transient expression in human 293
cells, this construct localizes to the Golgi apparatus; plasma membrane
staining is also observed. Te presence of spectrin and ankyrin isoforms
at the Golgi appars suggests that these two proteins exist in the form of
a macromolecular complex analogous to the spectrin membrane
cytoskeleton associated with the plasma membrane. We are currently
examining the association of these proteins with the Golgi complex at the
ultrastcal lewVl by immuno-electron microscopy.
1399
EFFECTS OF WINIBTORS OF SPHINGOLIPID SYNTHESIS ON
LOCALIZATION OF GOLGI PROTEINS. ((MCW.Maceyka and C
Machamer)) Deparmet of Cell Biology and Anatomy, The Johns Hopkins
Universty School of Medicine, Balime, Maryland 21205
has boen shown that ftansmembrane domains contain information
for the correct locaization of many Golgi memnane proteins.
Because diffnin Lipid conios'on through the sectoy pathway are
to
thought exst, proin locaization may be drectly related to membrane
co
Son. M glycpra from the avian
v
infcdous
It
neessary
co
(IBV)is targeted to the cis Golgi although indirect
evidence suggests that its localiration may be maintained by retrieval from
later
ments
her
by
tight retention.
first encountaphn
Be
potins
in the
in the cim Golgi, we asked
wheter ongoin phingolid synthess is necessary for correct llin
of IBV M pro The inhibitor l-phenyl-2-(decanoylaino)-3secret
pway
p
morpollno-l-propanol (PDMP) blo
ucosyceamide and
Treatment of BHR-21 cels with PDM1P: 1)
the steady-stat ditibuon of IBV M frm the Golgi to the
ERas assessed by i2di)c
_
. V dows the movement of
seps the secretoy patway as assed by pulseitnemtproteinp atll
chase experiment with VSV-0, and 3) does not chage aning pattern of
sphingoyelinsytei
appearsto shift
IL, which is thought
to be
stably retained
rathr
than
aypiments, p-chloroalanine, an
to roEdihibute the IBV M protein to
the ineneateco
ent, while there is no effect on mannosi iI
locaizaionL Tese data suggest tha when aphingolipid synthesis is
blocked, the change in steady state disribution oFIBVMmay be due to a
decrease in anterograde taffc and/or an increae in retrograde traffic.
dyna
lly retrieved. In
inhibitor of ceramide synthesis, appears
Monday. Golgi Complex (1400-1405)
241a
1401
1400
ANALYSIS OF THE GOLGI COMPLEX. ((E.B. Cluett and C.E.
Machamer)) Department of Cell Biology and Anatomy, The Johns Hopkins
University School of Medicine, Baltimore, MD 21205.
LIPID
lipids in the structure and function of the Golgi complex is
unknown. Evidence indicates that compartmentalization of lipids may occur
in the Golgi complex. Both sphingomyelin and cholesterol are preferentially
concentrated in the trans region of the organelle, but the significance of this is
not understood In the case of cholesterol this is particularly intriguing, since
the pathways and mechanism by which cholesterol is transported within the
cell are unclear. The transport of newly-synthesized cholesterol appears to be
unaffected by BFA treatmet or 2
a 0°C temperature block, suggesting a Golgiindependent transport route. Cholesterol is also presumed to influence the
flotation of intracellular membanes during subcellular fractionation. As a first
step in investigating the role of lipids in the structure andfunction of the Golgi
complex, we have isolated a highly-enriched population of intact Golgi stacks
from rat liver. Lipids were extracted and analyzed byHPTLC and scanning
densitometry with results comparable to previous studies. Furthermore, two
populations of Golgi complexes could be isolated which were virtually
identicalin protein profile, enzymatic marker activity, andlipid composition,
including cholesterol. Only the amount of cholesterol esters differed in each
fraction. Interestingly, a heavier fraction, which appeared to be smooth
ER/Golgi-like membrane, was devoid of galactosyl transferase activity but
contained an unexpectedly significant amount of cholesterol. These studies
are currently being extended toMDCK and WIF B cultured celllines.
The role of
TWO RELATED GOLGI-RESIDENT
CHIMIRIC PROTEINS ARE
RETAINED BY DIFFERENT MECHANISMS. ((O.A. Weisz, C.S.
Sevier, and C.E. Machamer)) Dept. ofCell Biology and Anatomy,
Johns Hopkins University School of Medicine, Baltimore, MD 21205.
We have been investigatng the mechanisms by which membrane proteins
are retained in the Golgi complex. Previously, we showed that the first
membrane spanning domain (ml) of the M glycoproten of infectious
bronchitis virus (formerly caUled IBV El) contains cis Golgi retention
information. When the membrne spanning domain of a protein that is
delivered to the plasma membrane (VSV G) is replaced with ml,
efficiently
the resulting chimera (Gml) is retained in the Golgi. We recently
demonstrated that Gml forms large oligom ers which are insoluble in Triton
X-100 and SDS (Weisz, 0. A., A. M. Swift and C. E. Machamer (1993) J.
Cell
1185-1196). Point mutants of Gml which disrupt the Golgi
The
Biol.22,
spanning domain do not form
that the
oligomers. Experiments using truncated Gml constructsofsuggest
tail
a
in
the
role
plays
tight Golgi retention Gml. When the
cytoplasmic
Gml tail is replaced with theIBV M tail, the resulting chimera (GmlM) is
retentioninformationin the membrane
retained in the Golgi, although at high expression levels, some of the
protein reaches the plasma membrane. Surprisingly, internal deletions of
the GmlMI tail result in rapid delivery of the chimeras to the cell surface.
This suggests that Gml and GmlMK are retained in the Golgi complex by
different mechanisms. We are currendy investigating whether these
mechanisms involve the dirt retention of ml-conaining proteins or the
retrieval of these proteins from later compartments.
1402
1403
CHOLESTIEROL-NDEPENDENT TARGETING OF GOLGI
MEMBRANE PROTEINS. ((M.M. Rolls*, M.T. Marquardt+, M.
Kielian+, C. E. Machame_o)) *Departsent of Cell Biology and Anatomy,
IDENTIFICATION OF 4 NOVEL PROTEINS OF THE GOLGI COMPLEX USING
HUMAN AUTOANTIBODY PROBES. ((Marvin J. Fritzler, Kevin J. Griffith, Edward
Chan)) University of Calgry, Calgary, Alberta, Canada T2N 4NI and 'T Scripps
K.L.
Research Institute, La Jolla, CA 92037.
Bronx, NY 10461.
Autoantibodies directed agist the Golgi complex have been reportedi the sea of
patients with certain autoimmme, infectious and neuro-degenative diseases. The
ofdiffcrent cellularmembranes may provide
physical basis for specifictargeting of some membrane proteins. In
that cholesterol could play an essential
been
it
has
suggested
particular,
role intranamembrane domain-mediated loclization of proteins to the
Golgi complex (Bretscher, M.S. and S. Munro,1993, Science 261: 1280-
Human auanb directed agaist the Golgi complex wrae used to screen
complex.
cDNA exprsson lbraes. Four groups ofcDNA clones, designated golgin 95, golgm 160,
study. These cDNAs wer ch_id
CDF20, ACJF 5.21 were selected for fuher
reacvities of the recombinant
by resrictbon mapping and DNA seq
analysis
antibodies were eamid by Westeni nobti using
proteins with
Johns Hopkins University School of Medicine, Baltimore,
and+Department of Cell Biology, Albert Einstein College
MD 21205,
of Medicine,
Distinct lipid compositions
a
1281). To determine whether cholesterol is required for thetargeing of
Golgimembrane proteins, we exanined the localzation of several Golgi
resident proteins in cholesterol-deplted cultured cells.Insect cells
cannot synthesize cholesterol and canbe depleted of sterol by growthin
delipidated senum We used the C6/36 cell line derived from Aedes
albopicts (mosquito), which after four passgesin delipidatsd serum
containedundetectable levels of cholesteol (Marquardt,MIT. et al,
1993, J. Cell Biol. 123:57-65). Because no probes are available for
mosquito Golgi proteins, we expressed two well-cha ized
mam malian Golgi enymes using a Semliki Forest virus-based vetor.
In non-depleted C6/36 cells, both p1,4-galactosyltransferase andawere localized to the Golgi complex by indirect
mannosidaseI
immunofluoresence microscopy. When the same proteins were
expressed in cholesterol-depleted C6/36 cells the staining pattern was
indistinguishable from that soen in non-depleted cells. Thus, at leastin
insect cells, cholestrol does not appear to be required for targetng of
several proteins to the Golgi complex.
objective of our research is
to understand the nature
of
argets
in the Golgi
and
human
recombinant protein produced in E. coi and by
pcipitation using in vitro
translation products. Antibodies affinity purified by adsorpn to the recombinant phage
The binding specificity
Golgi staining HEp-2
Golgi
complex was confirmed byd ting that the reactivity cokocalized to the same
subcellular organelle bound by wheatgeSm agglutiin and antibodies to the coatomer
protein,PCOP. InWvtro translation prodcts usingthese cDNA templates produced 40
5.21), 65 (golgin 160) and -95 (golgin 95, CDF20) kDa proteins Sequence analysis
(ACJF
of all four cDNAs indicated that they encode unique proteins, 3 of which (golgin 95, golgin
160, CDF20) had 20-2Woh sequence identity to the heavy chain of myosin and kineis.
Analysis for secondary stmcturedemonstated that golgin 95 had coiled-coil domain
(golgin 95) and CDF20 has a predicted alpha helical structurethroughout the majority of
protein reprodued
on
to the
cells.
a
the
protein.
sequences.
None of the sequenc
had
potential
HEp-2 cells, the autoantgma
tranamembranc domains
After befeldin A treuent of
or
wer
sign
redistrbuted
5 min, absent at 10-15 min and then reconstituted 120 minutes after eptnishing with
fresh media. Insummary, we have cloned and identifed 4 novel components of the
but stuctul analysis suggests that
complex. The function ofthese proteins is not
they are part of myosin-like family of proteins assoced with the Goigi complx.
after
Golgi
known
a
1404
1405
PERMEABILITY CHANGES IN LIPID BILAYERS INDUCED BY
ILIMAQUINONE, A GOLGI DISRUPTIVE METABOLITE. ((IS. Fisher,
S. Sohraby, F.G. Grillo)) Dept. Nephrology, WRAMI, Washington, DC
20307; Dept. Physiopathologie, Univ. Libre Bruxelles, Brussels, Belgium.
DIARYLSULFONYLUREA LY181984 ((S. Moya-Camarena,
ANTITUMOR
D. M. Morrd, M.
W.
L.-Y. Wu and D.J. Morrd))
Recently, Takizawa et aL (Cell 73:1079, 1993) demonsrated that 25 pM
ilimaquinone (IQ), a metabolite from sea sponges, reversibly disrupts Golgi
membranes in several cell culture lines. Its mechanism of action is unknown.
We eamined the effects of IQ on the properties of lipid bilayers. Bilayers
were "painted" on a 250 p diameter aperture and consisted of 15 mg/ml 1,2Diphytanoyl-sn-Clcero-3-Phosphocholine in decane. Membrane ca ce
be
(60 Hz triangle wave) was used to estimate bilayer area. Specific
conductance (G) averaged .04 pS/cm2 for untreated bilayers (150 NaCl, 10
MES, pH=7; AV= ±100 mV, DC). Addition of 1.0 pM IQ (trans) to the
ce were
bilayer inased G about 2-fold. Bilayer noise and ca
unchanged by IQ. Addition of 10 pM IQ inreased conductance 4-fold within
2 min. G continued to increase over the next 40 min to values 14-fold greater
than untreated bilayers. I-V plots were linear. The response was independent
of pH (6 to 9) and was not selective for Na+ or Cl-. After IQ (2 to 14 p, a
step change of bilayer voltage caused an additional exponential component in
the decay of the current response with a time constant of about 10 s. We
conclude that similar permeability changes of Golgi cisternae after IQ may
occur which could be important in undrstanding the mechanisn(s) leading to
membrane breakdown and loss of Golgi function.
We thank Dr. Vv;ek Malhotra, Dept. Biol. UCSD, for the kind gift of IQ.
A RESPONSE OF HELA CELL GOLGI APPARATUS TO THE
MacKellar,
Paulik,
Department of Foods and Nutrition and Department of Medicinal Chemistry,
Purdue
University, W. Lafayette,
Indianapolis,
IN and Eli
Lilly
Research Laboratories,
IN
Growth of HeLa cells is markedly inhibited by the antitumor
N-(4-methylphenyisulfonyl)-N'-(4-chlorophenyl)urea
diarylsulfonylurea
In
(LY181984). parallel studies, the drug was shown to block an ameliorideinsensitive proton export by HeLa cells (E. Sun et al., Purdue University,
Results Unpublished). Evidence was sought for a similar inhibition of proton
transport at the level of the Golgi apparatus. If cells were pretated 1 to 3 h
subsequently challanged with monensin, the resultant
with drug and the
monensin-induced vacuoles were smaller and less abundant The total volume
of the monensin-sensitive trans Golgi apparatus compartment was reduced by
more than
treatment with LY181984. The swelling response was
unaffected by sulfonylurea in
cells (e.g. MDCK or
PC-12 cells).
A stuctully similar but inactive analog N4methylphenyisulfonyl)-N-(4-phenyl)urea (LY181985) was without effect on
Golgi apparatus morphology following monensin treatment. The results
active antitumor
suggest a response of the trans Golgi apparatus to the
trans Golgi
cells
70%/o by
sulfonylurea that resulted
cssternae.
Experiments
sulfonylurea-unresponsive
in reduced acidification of the
to
identify
an
antitumor
apparats
sulfonylurea-responsive
enzymatic activity as a marker to guide isolation of Golgi
membranes from HeLa homogenates are underway.
appartus
Golgi Complex (1406-1410). Monday
242a
140?
1406
EFFECT OF TEMPERATURE BLOCKADE ON THE PROCESSING OF
PROTRH. ((E.A.
Nilini, Perez de la Cruz and D. Jackson)) Division of
Endocrinology, Brown University, Rhode Island Hospital, Providence, RI
I.M
02903.
AtT20 cells with a cDNA encoding preproTRH we have
Using transfected
previously reported that the processing of proTRH (26 kDa) begins in a
subcompartment of Golgi Complex (GC) with proteolytic cleavage at two
15 kDa
positions in the center of the molecule, generating an
intrmediate peptide and a carboxyl-terminal 16.5 kDa intermediate peptide. In
this study, we investigated the effect oftempeature blockade on the initial and
AtT2O cell line, cells were pulsed
subsequent processing of proTRH. In the
with 3H-leucine for 4 hours at 37 OC followed by a 90, 120 and 180 minutes
chase at 20 OC, in order to accumulate peptides in the trans-Golgi network, and
compared to a chase at 370C as control. The release of peptides was further
measured by increasing the discharge in response to PMA (phorbol 12myristate ester). Peptides were immunoprecipitated followed by analysis on
SDS gel elotoihoresis.
Endoproteolytic cleavage of proTRH to generate the 15 kDa and 16.5 kDa
intermediates was not blocked by incubating the cells at 20 OC; however,
generation of smaller intcmediates and cryptic peptides was seen only at 37 OC.
After 120 minutes chase, the 15 kDa peptide at 20 OC accumulated five fold
over the level at 37 OC. In contrast, the 16.5 kDa intermediate did not
accumulate and was further processed with the level only 50% of the amount at
amino-trmminal
kDa
in the GC while that of the 15 kDa intrmediate occurs in the
This data
370C.
suggests
that further processing of the 16.5
to
smaller
forms occurs
secretory granules (SG) after leaving the GC. We conclude that processing of
proTRH begins prior to packing into SG and that N- and C-terminal
intermediates are subjected to differential processing.
Caifomria,
hIssitute,
A novel method was developed to introduce fluorescent probes selectively into the
compartment of trans-Golgi in living cells. Normal human skin fibroblasts
microinjected with a suspension of uniform-sized liposomes (70 ± 1 nm
101
diameter) containing fluid-phase fluorescent indicators, such as
(SR). Liposomes fused with trana-Golgi and delivered their aqueous-phase contents
with a half-time of min
15
at 37 'C. mfocal microscopy showed Dolocazation of SR
Effcient liposome
with the lipid-phase trans-Golgi-specific
fusion in vito required 37 'C temperature, ATP, and precise liposome size. Fusion
was also sensitive to NEM but not to GTP-y-S. To measure pH in the trans-Golgi
aqueous
were
sulforhodamine
mlarker NBD-ceramidde.
compartment, two membrane-impermeant
fluorophores
were
encapsulated in
liposomes: fluorescein-sulfonate (pH-sensitive, pKa=6.3) and sulforhodamine 101
(pH-insensitive). These compounds were chosen for their self-quenching when
present at high concentrations in liposomes, bright fluorescence when diluted in
After
trans-Golgi, non-overlapping spectra, low membrane permeability, and
fusion of microinjected liposomes, confocal cellimages were collected by a cooled
(oil
x100,
N.A.
1.4).
Fluorescein-to-sulforhodamine
signal
CCD-camera
objective:
ratios (F/SR) werecalculated from background-subtracted pixel intensities integrated
over the area of the trans-Golgi. In normul human skin fibroblasts, F/SR was .230
±
0.009 (SE,n=89). The relationship between F/SR andtrans-Golgi pH was calibrated
in vivo using solutions titrated to various pH containing bafilomycin, the ionophores
monensin and CCCP, and highK. Simiar pKa values (-63) were determined in
vitro and in
vivo. Trns-Golgi pH was caklculated from F/SR and the calibration curve
to be 6.20 + 0.05. pH was sensitive to bafilomycin, weak bases, solution anion
pKa.
content, and cAMP stimulation. These results provide the first direct masurment of
trans-Golgi pH in living cells and introduce an effective methodto deliver fluid-phase
indicators into the secretorycom
t.
1410
Acw=RMcnzOP',EBNWtKShUCR REGMOANLAX
APPARAVS (GA) WIDIINIDOPIISOFIHCAMALSNBUROM IN CULTUR.L ((E. Tonr,
0. Seward)). DepL of Nuauckence. Univbsity of Visginia
(2IarIousville, VA 22908
an
ervecells,
N
mogouly
RNAsla
mRNAs
eemd eua
dkl dihlbgeof
vh
eaode
som
so beuuu
usglyosyled, orguseles
IDdieRE td ieGAmetbePotip
evadasd de; pme
of
at low
aIC.
musdg
g
or m
dug
dads Thepsn
op
mere
es
dh E tGA comapex in enluned hi;wocundlemrnsby
syeiiwd
wy
_me0 or3H-plecomewhm
by ham
betwen
wee-scir-.eat-wi
aesiadwid BfA.due
r
amk
gbpnkA"p1 labdidg ih3Hn
slrm onnsthent
w*bvocked
BDrid
A (BfA).When
orb
am
syndheized
xm meaoftcmdy
heimcelbarmeaainisied at 2DIC.exit
Of
fas ike GA is bbcke& ds way.
spouable so dede tde saim ofa
Il
e
eGA).
cen (14 days in
ad
_o
gadwor(m di
(s deER)
glyooproisd
horntheER
vitro) won pruiacu
d
(S
ta l)
heGA lb loked
so
W
wilh BEA (5 pghal, orat 15 ,20 3PC (cotl
3H eannoe
Uced
pilac
(200 p3eCAa for h ud chaad in medisontining naulbeed
30 asi
or
s
N21 mdaenah
we
glon.
or3
lkllaetwawefinedsdjIorpouAdfor
ag= for
me
waepnlxed atW
won ep
do GA hd
bee
bloked
ees-ceoflocalg
laim_ca
only
_o ee
W
hsropod
do thttias
_ea
dmd 6e,lbe
t
s
uesenoof
W
dandritc labeing
neimos warepuls4abel
gwujxeuep ovearcafbodies nd
do bkbm of
iD
yd
of ldin wit pbk ispm
no nomism&a tdeIR
GA
dotno
Wadatg
Alp mle atvties
widhIn
forlacsoe
Mr Xo
_t-ofreceidy 1yntyhuiaSigly
avail
dhe
t
edwh
fom
dyonpaot
d_oft
wads
norq. Inomc
ddcoe
When
forMAP-2Imiddfy
3,,eR
bodibesoawnndeimswereheavily lddee When cells
20l
at at2-P
DB A. bedingwinreuicidtocei bodies and
Ilbue.ofxo.1bbe sdku Xtdseeofrexetly syndtzed
dseIb
dno-i.
at l
Fuseliaa,
Santarone*,
Girolamo.+, Corda+,
We have previously shown that brefeldin A (BFA), stimulates the mono-ADPribosylation of two cytosolic proteins of 38 and 50 KDa (De Matteis et al., PNAS
91:1114). The reaction is blocked by known inhibitors of mono ADP-ribosylation such
as nicotinamide, aminobenzamide and novobiocin. We now show that, in addition to
a coumarinic ring, such as dicumarol and
novobiocin, other compounds
coumermycin A1 can inhibit the BFA-stimulatnd ADP-ribosyltaraferase. Dicumarol in
known to block the NAD binding site of DT dinphorase and of the mitochondrial NADH
oxidase complex
quinone binding site sugesting that
1. Both these enzymes contain amight
bind quinones. The effects of
also the BFA-dependent
several natural and synthetic quinones were thus examined. They were all found to inhibit
the reaction with varying degrees of potency. In order to corrlate these in vitro results
the
complex
and
with in vivo events, the effects of
agents on Gogig
were studied in rat basophilic leukemia (RBL) cells by immunofluorescence and electron
with the
(labelled
microscopy. In the presence of these compounds the Golgi complex
antibody) lost
II
FITC-conjugated lectn from Helix pomatia and anti-mannosidase
sensitivity to BFA and appeared as spot-like seacture located in the perinuclear area At
the ultrastnuctural level this spot was seen to corispond to a tightly packed ring of Golgi
the above mentoned inhibitors suppressed
cinternae around the centrosome. Finally,
constittive glycosaminoglycan
port from the cis-Colgi trto the ans Golgi network
an enzyme
(TON) but not from TON to the plasma membrane. Our results suggest
conuaining NAD and quinone binding sites in involved in the regulation of constitutive
secretion and in the mechanism of action of BFA.
work was partialy supported by the Agenzia per la Promoznine
Acknowledgments.
Sud).
Sviluppo del Mezzogiomo (PR-2 and PR-3) and by CNR (Convenzione CNR-Mario Negri
containing
ADP-ribosyltransferase
coumarsnic quinone
tara
This
e
lo
1409
1406
TRANS-GOLGI pH MEASURED IN FIBROBLASTS BY MICROINJECTION OF
LIPOSOME-ENCAPSULATED FLUOROPHORES ((0. Seksek, J. Biwersi, and A.S.
University of
San Francisco
Verkman)) Cardiovascular Research
In
CELLULAR EFFECTS OF INHIBITORS OF BFA-STIMULATED ADPRIBOSYLTRANSFERASE. ((0. Santini', M.G. Sciulli', A. Colanzi', A. Mironov
M.A De
D.
S.
M. Di
G Innamorati', A.
Matteis.* and A. Luini)) *Unit of Physiopatology of Secretion, +Laboratory of
Molecular and Callular Endocrinology and 'Laboratory of Molecular Neurobiology,
di Ricerche Farmacologiche Mario Negri Snd, 66030 Maria Imbaro, Chieti,
Istituto
Italy (Spon. by A. Luini)
sume was
so
e
demndriesocaee indiamndlud milie elyestiesuanihevechxis
ofdh ER-A co.mpbm Sppmwd by MS1233 to OS.
orgrni
tionc
berac erisfic
115 IS A GENERAL VESICULARTRANSPORT FACTOR RELATED TO THE YEAST
ER-GOLGI TRANSPORT FACTOR USOlp ((S.K. Sapperstein, D.M. Walter, A.R.
Grosvenor,
J.E. Heuser, and M.G. Waters)) Department of Molecular Biology,
Princeton University, Princeton, WJ 08544.
p
vesictuar
A recently discovered
transport factor, termed p11, is required
along with NSF and SNAPs for in vitro Golgi transport 15 is a peripheral
membrane protein found predominantly on the Golgi Biochemical and
electronmicroscopic analyses indicate that p1 15 is an elongated homo-dimer
with to globular "heads" and an exlended "tail" reminiscent of myosin N.
We have clonedandsequenced cDNAsfor bovine and rat 1 The predicted
translation products are 90% identical, and each can be divided into three
bovine protein consists of an aminodomains. The predicted 108
terminal 73 kDo globular domain follwod by a 29 kDa coied-coil
acdic domain of
dimerization domain, aminker segment of 4 kDa and ahighly ER
3 kDa. p1 5 is
p,a protein requedfor toGolgi
p has a
Usol
vesicular transport in the yeast Saccharomyces cerevisiae.
15 and
similar"head-coll-acid" domain structure. The head domains of pland
728aa,
Usol p, which are about 25% identical, are simiar in size (651
respectively), and they possess two highly homiogous regionsThe first is
62%identical and 74% similar over 34 residues and the second is 60%
identical and 77% similar over 53 residues. There is a third region of
homology (50%identity over 28 residues) between the coEded-cil and acidic
domains. The acidic domains at the extreme C-termini of p1 and Usol p do
Since pl15 is
not appear to be conserved at the primary sequence
required for intra Golgi transport, and Usol p is required for ER to Golgi
transport, we propose that pl15 may be a general transport factor.
pl
pl 5.
kDa
homologoustoUsol
level.
15
Monday. Blood VesselsI (1411-1416)
243a
1411
LDL INCREASES ENDOTHEUAL CELL VULNERABIUTY TO SHEAR
STRESS. ((M.S.F. Clake, KA Pitda Jr., KS. Mdowand P.L
MCNs1))
Bly
bw dansiy lpopictsi
Elead plma
nical
-tharoad
as
paru lion
of
lechan lciy-In
ro s.
ucd EC
repot he
expour
t
of
factor-
for th
rela
0.005) th
reveabd
of
LDL-4reatd
toxs
andlor mor
nu
at
of
*vu.
badsc
fbrolast
o
f
(sudfered
lees
Flw
uravvd
EC
cytofluorometly
she
had
Sheared
membrane
ous
We
The
wa
ureated EC.
LDL-tread EC releasd 24old mor (p< 0.02) bFGF tn sheard
conrol EC. LDL treamt of EC resubAd in an
0% incae above
km em
dcol rol, and an binrase in
evekls oftl
ontrol
cholesterol
phospholpid rao from 0.6 to 1.3.
Stedys"ta
-
otrop
threscence
mebane
th
reveaed
fluktpy (PLF)
LDLWreabd EC
sigdflctiy lower hn that of ctol EC. When
PMF of LDWItreatd EC ws raisd usin the membane
fluldzimg
shear
alty
agen AaC, we obsved a sgnd cut reducion In EC
th
whereas
reducion In PMF, usion
sti
ste
ant
PFP8, had th opposte affw Our dat
LWI-induced
hincea in ECvwu abilitytovmechani s*essmaypromotesendothellal
th
do o.sceosI.
t
ijry i
of
wa
a
poo
,
inlllation
1413
tunmor
a
celUs
from the circulatoy
seconday tissue loca
the
mignte
monolayer of
or
lymphatc systems,
endodelil
cells
(EC) lining
fiboneci mroorouscel culture is and E-STIM Endothelial Cel Culture Medium
promotes the rapid formation of confluent EC monolayers withbarrier fncto i vuiro.
These EC monolayers were used to examine the migrato of human prmasytumors and
in wwo measatic potentials, including meanomas, sacomas,
tumor cell lines of differing
carcinomas
of
various
origins. For each
used
tumor type,cor
ng
normal cells
or
and
less
controls. Cells
placedintoiserts contmag EC
and allowed to bind to and invade the endothelium for up to 24hours, then fixed
invasive cell lines
monolayers
were
as
we
and processed for microscopy (phase or SEM) or for quantitation of invasion (by microscopy
or y scintillation counting of ICr-labeled tumor cells). In each case, tumor cells that were
highly invasive inWio showed rapid invasion of the endothelium inWitro, migratingthrough
to the underside
of the insert.
In
hse ara
of
invasion, complete scattering of the EC
monolayer was observed, while adjacentregions of the monolayer which were free of tumor
cells remaiedintact. Control cells weremuchkss invasive, with most cells remaining only
wealdy atached to the apical surface of the monolayer. Invasion of the endotheliumby highly
invasive cells couldbe decreased or inhibitedcither by pro-treatment of EC with antibodies
against certain CAMs (e.g., P-sdectin), or by treatment of the tumor cels with anti-tumor
compounds such as taxol, vincristin, or vinblstine. These results demonstra th t the
BioCoat Transendothea Tumor Migration Environmentserves as physiologically relvant
a
insitro model oftumor cell extravasation during metastasis, which can used to study the
extravastion process, to assess the metastatc poential of varioustumor cells, and potentially
to examine the efficacy of candidate anti-tumor
be
dtrapmstics.
1414
WING ENDOTHELIAL
PERMSELECTIVITY. ((R.N. Feinberg and
E. Cafasso)) Department of Anatomy, Cell Biology and Injury
Sciences, New Jersey Medical School, Newark, NJ 07103
CHICK
Normal vertebrate limb development requires
the formation of
characteristic vasculature. Vascular patterns of embryonic
but little is known
limbs
have been extensively studied,
about the permeability characteristics of the
developing
The microcirculation of the chick wing was
circulation.
examined by in vivo confocal microscopy following the systemic
injection of a graded series of fluorescein isothiocyanate
(FITC) labeled dextrans. Chick embryos at stages 21-23 were
injected with FITC-DX-150, 70, 40 in order to establish how
selective the endothelial lining of microvessels are to
macromolecules of various molecular weights. A confocal image
of the perfused wing bud was obtained by image
analysis
software. Videodensitometry, over a gray scale range of 0255, was used to quantitate the amount of tracer found within
the interstitium. Images were acquired at 2, 7, and 12 min
postinjection. FITC-DX-150 and 70 did not extravasate from
the endothelium of limb microvessels for all three stages.
the surrounding
FITCDI--40 was found to extravasate into
Thus, differences in
all three stages.
interstitium for
microvascular extravasation were attributed to the size of the
molecular tracer rather than the stage of the embryo.
These
data indicate that embryonic wing microvessels
demonstrate
efflux
across
to
macromolecular
the
permselectivity
provide a
basis
endothelium. The present results
for
additional permeability studies and challenge our
concepts
about the role of diffusible morphogens in limb development.
a
1415
A NEUROPHIL SECRETORY PRODUCT INCREASES ENDOTHELAL
CEI CALCIUM AND MONOLAYER PERMEABILITY. ((AJ. Huag, K.R.
Haner, T.T. Chen, and S.C. Siverstin))
epmmeasts of Medicine
an
Physiolg, Cohsmbia Universy, Colege of Physics & Sugo, New York,
NY 10032.
P
ucs
barrier
by
reguates
(PMN) migrate
betwe
crawl
PMN
tanst
120:1371-1380).
woes
EC. EC
acrn
an
EC
molcule
by
mandodelbal ceU (EC)
coolic
fre
acrs
com
eteoftUa is
ty
(Ca
calcium
at al. 1993.
monolayr (Hu
PMN bindnto and mirtion
by tie ligation of EC arhe
pmti
J.GCeZ BIoL
acmnied
on Ie
PMN
Wteraction of PMN sereoy
wth EC. To determine
whoher PMN-indced increases in EC 1Ca++]. and EC monolyer pemabiliy,
ectrical rance, e inited by tie liaon
a meassred by transendot
of EC urfac
PMN
product,
examined
effict
of cross lining EC srface m bcules and of cell fee a e
derived from
to
evets.
PMN
Cell frec
t
IMLPlsmulated PMN increased EC [Ca++], ad EC monolayer pernebility to sons in
a na _riodependent fahkon MAb 60.3, dircted againstP2 istgrn onthe
PMN srface, inbibed 85 4% PMN adhesion to and migration acws EC
but did not prevent PMN-inucd bxnr
EC [CaL in response
to fMLP. PMN tplasts, which lack grals and respiraory burs acivity,
and
by
t
products
molels or
a
tie
on
migrt
wit
tstatic
the lumenal surfaces of the vessels. To model this prcess oftumor cell extravasaton, we
nwtro eanvironmnst, the BioCoate Transendothel
hawv contcted
Tumor Migrtion
Environment. Previous workfiom our laboartories has shown that a combinaton of BioCoat
an
ECto 240 mgdchoLestsrUdL
vess
coitrol
pm t of
mle* cCl nmogen
smoot
ilasVW severity d shear-d*ed injry h
IL-realsd EC Vth sured mechnIcal
smherng
20%; p
vsl
injry ooaun
fbr te
m
vascular
a
Duringinvasion into
cdM (EC)
andothala
(WI)
rsk
Is men
alheaogmnesis In m*o ad a*
groAh facto (bFGF), poter-
la
Colg
Mdical
Anaomy,
&
Augus, GA 30912. (proo R. Cadwel)
Gaorge,
meha
Caul
ment
1412
CONSTRUCTON OF AN ENVIRONMENTFOR MODELLING TRANSENDOTHELIAL
M IGRATION OF TUMOR CELLS. ((BJ. Del Buono)) Becton
Dickinson Collabotive
Biomedical Products, Bedford, MA 01730.
om
and increase
tat
PMN.
molecues- with
provide
evio
Crs
muibodie
pernme- bility of EC
ICAM
I
a
as
com
vral
oier
EC
EC [Ca-1.
bctinbfaled tD
hmatrctastbnulatd PMN
ad
product which snc,reases BC [Ca4L and EC monolayer per-me akility.
OVEREXPRESSION OF A MUTANT MYOSIN UGHT CHAIN (MLC) IN
ENDOTHEUAL CELLS ATTENUATES THE INCREASE
IN
ENDOTHELIAL
PERMEABILITY ((P.G. Nagpala, H. Lum, G. Nikevics, G. Nowak,
S. Ono, D. Lodme', P. de LaneoW, and A.B. Malk)) Rush Medick
Univeriy lEnois School Medcie Chicago, IL60612.
of
Colege, and
of
enkhl_
Contra_ion of
cal (EC) in resporne to infammaty mecatDr resut
in incread EC permeabit. We hypothesed tt ph
MLC
and
by MLC kins aclivng the cadin-mnyosin motor
endothelalo
coracon. The role of MLC phoephorlallon on EC contrcion we inveigated by
stdablbrodon of inmortefied human umb5csi vein EC kw EAhyb 928 wIt
vector confinin
mutant rat aoric smooth muscebMLC cDNA (muMLC). The
muMLC wa gerae by conve
thr18 and serB aleS and
The
muMLC DNA ws paced under the contol of the promoter region in th retrovira 5'
lng bnrini repet and wa teggd at the end with an 11 amino saod myc
epipe for idekaln. The transfedad cle showed no appernt moo
difrence fom th control cel. Co-immunopeii
wth
confirmed the
presencedof ogenous muMLC in the
cel. The muMLC trarnfectante
showed decreasd MLC phohoya
Both the
and muMLC
b
showd imlr basal taneendotellal pneabit to
values 0.340.0B and 0.41±0.07 pifin,
Human
repecval-y).
(1 laM) caused 26-fod increase over basene in
_
b
po..
of
whereas muMLC transfetente
w1rg
O
memat
an
a
a
al1a9.
to
myoin
_trasfeted
_ansfeate
responded with 1.7-fod increase
a
MLCK- dependet
irease
'ni-albunin
ceabnceof '5i_.
m-hronbin
'1tunmin;
(trarew'd
endothelyl
a
beselne. These findngs indicate that
MLC ser19 andfor thrl8 in pert mndate fe
over
hophomlon of
pennabit in response
to
pro-inllmmatoiry
meditore.
Since
mulSon of mer9
thri8 did not inhibit
resporns, ph
MLC by protein ins C may alo conribte to the response. (Supported by
HL45638, HL27016, and Amnecn Heart Aawo ation of Chicago).
the
erir
of the
1416
CHEMOATTRACTANT-STIMULATED NEUTROPIILS SIGNAL PHOSPHORYLATION OF MYOSIN REGULATORY UGHT CHAINS IN ENDOTHEUAL
CELLS. (tEA. Hiuxebeghl, Z.M. Goeckaler2, A.J. HUan3, R.B.
WyaoImersl4, ad S.C. Silverstein'l) Depts of Physiology' end Medicine3,
Colunbia Uniesity, New York, NY 10032, and Depts of
and
Anesthesiology4, Saint Louis University, Saint Louis, MO 63104.
Pathdogy24
Chemotaxing neutrophils
ondothelial cel (EC) monolayers by
crawling between neighboring ECs. ECs iceas their intracelllar C2 in
response to chemo
attractant-st umdted neutrophils. Agent that block this
C
jnctions and transendothelial
rise inhibit opening of
neutrophil migration (L Cal Bio. 135:355 119931). Inrease in EC Ca2+
promote phoephoryltion of reulatory myosin light chains and EC retraction
(PNAS 87:16 119901). To determin whether neutrophils
promote myosin
light chain phosphorylation, ECs were prelabebd with 32P4 or u5methionine, incubated with neutrophils in the presence or absence of
chemoattractant, and myosin was immunoprecipitated and subjected to
one- or two-dimensional gel analysis. Using both methods, phosphorylation
of EC myosin regulatory light chains increased 130-240% within 1-2
minutes afteraddition of chemoatractant-stimuated, but not unstimulated
neutrophils. However, in interleukin-l-treated EC monolayers, addition of
unstimulated neutrophils signaled a 1 10150% increase in phosphorylation
of EC myosin regulatory light chains. Two-dimensional gelanalysis showed
stimulated nurophils icreased both mono- and di-phosphorylation of EC
tave
intor-EC
a
lght
urfce
myosin regulatory
sudies
19 and threonine 18
activity. Thes
chai,
and
tryptic peptide mapping revealed serine
phshrltion,
results
suggest that
indicatin
myosin light chain kinas
phortion
of
the EC myosin
regulatory light chains plays a role in neutrophil migration across
endothelia.
Blood VesselsI
244a
1417
TRANSENDOTHELIAL MIGRATION OF HUMAN MONOCYTES IS
ATATENUATED BY THE STABLE PROSTACYCLIN ANALOGUE CICAPROST ((A. Griitzkau, S. Weliershoff and H. Graf)) Schering Research
Laboratories, 13342 Berlin, Germany
prostacyclin
(PGI2) and
Recent data indicated additional properties of
exceeding the well known antiaggregatory activity on platelets,
its analogues
such as enh
of endothelial barrier function, inhibition of neutrophil
adherence to vascular endothelium and inhibitory effects on leukocyte chemotaxis. The aim of the present study was to investigate PGI2-dependent
mechanisms responsible for the regulation of monocyte extravasation during
inflammatory conditions. In this respect the influence of the stable PGI2analogue Cicaprost (ZK 96480) on the tansendothelial migration of human
monocytes in response to various chemotcic compounds was investigated
in an in-vitro model for the vascular endothelium. For this purpose endothelial cells isolated from bovine pulmonary artery were cultured on porous
poycarbonate-membranes separating a two compartment system. The upper
compartment contained monocytes prepared from fresh human blood by
of 2h mocounterflow centrifugation elutriation. During an incubation
nocytes migrated across the endotihealized membranes into the lower
decreased
migration
in
a
ttrnsendothelial
dose-depenpartment. Cicaprost
dent manner mainly on the level of the endothelium. Rolipram (ZK 62711),
IV specific phosphodiesterase, potentiated the inhibian ihibitor of the
tion evoked by Cicaprost, whereas the cAMP analogue dibutyryl cAMP only
caused an additive effect These studies suggest that prostacyclin and its analogues are capable of modulating the extravasation of monocytes during inflammatory processes via a cAMP-dependent mechanism.
ent
tme
co-
(1417-1422). Monday
1418
EXPRESSION OF THE GROWTH FACTOR-INDUCIBLE GENE PS4 IN
BALLOON INJURED RAT ARTERIES AND IN HUMAN ATHEREC-
TOMY SPECIMENS. ((R Mora, C. Haudenschild5, and G. Liau)) Department of
Molecular, Biology, and Department of Experimental, Pathology*, Holland LAboratory,
American Red Cross,
Rockville, Maryland 20855.
We recently identified a novel serum and growth factor- inducible gene called PS4 in
rabbit vascular smooth muscle cells
Sequence analysis of a full-length PS4
cDNA revealed an open reading frame encoding a polypeptide of 276 amino acids.
The deduced protein sequence exhibits a 94% identity with human TSG-6; a specific
TNF and IL-1 inducible hyaluronan binding protein synthesized by human fibroblasts.
However, unlike human fibroblasts, expression of PS4/TSG-6 in vascular SMCs is regulated by both cytokines (IL-1) and growth fsctors (FGF-1). To determine if PS4
(SMCs).
has a role in the development of vascular lesions, we examined the expression of this
protein in a rat vascular injury model as wel as in human atheretomy specimens
An intense immunostaining for PS4 was detected in the rat
whereas in the
medial layer and in non-ballooned carotid arteries
was not expressed. This confinement to the neointima suggests that PS4 may be involved in vascular remodeling
neointima
PS4
also highly expressed in human atherectomy specimens. The highest level of
PS4 immunolocalized in
was found in the
PS4 was
PS4 immunoetaining
macrophage-rich areas.
the macrophage cytoplasm as well as diffusey in the matrix immediately surrounding
the lipid-laden macrophages. In regions which were macrophags-negative and rich in
SMCs, PS4 expression, when observed, was predominantly intracelluar. We conclude
PS4 expression
that the
in
localization of
is consistent with its regulation by growth factors
vvoi
and cytokines in vitro and increased
of PS4 is a marker of phenotypically
and
associated
with
activated
macrophages. (Supported by NIH Grant
SMCs is a
of
a
Research
Career
Development Award HL-02449).
recipient
HH-37510,G.L.
altered
1419
1420
ADHESIONMOLECULE
ENDOTHEUN-1 DOES NOT STIMMLATE CELL
EXPRESSION IN HUMAN UMBILCAL VEIN ENDOTHELIAL CELLS. ((R.
Zaragoza,G.P. Budzik and J.R. Wu-Wong)). (Spon.G. P. Budzik) Abbott
Laboratories, Pharmaceutical Products Division, Abbott Park, IL 60064
STABLE TRANSFER OF GENES INTO ENDOTHELUAL CELLS USING UPOSOME
MEDIATED TRANSFECTION AND RETROVIRAL TRANSDUCTION.((P.G. Nagpolet H.
Coag/ Rush
Lum, and A.B. Melik)) Departnent of Pharmacology, Rush
Presbytorin-St Luo 's Medical Canter, Chicago, IL60 12. ISpon. by EA. Everitt)
Endothelin (ET-1),
a
21 amino acid peptide, is
potent endothelium-
a
derived vasoconstrictor. Previously it has been reported that ET-1
stimulates the expression of intercellular adhesion molecule-1 (ICAM-
1), vascular cell adhesion molecule-1 (VCAM-1), and
endothelial cell
adhesion molecule (ELAM) in human brain microvascular endothelial cells
(McCarron et al., 1993). In this study we investigated theeffectB of ET-1
on CAM expression in human umbilical veinendothelial cells (HUVEC).
ET-1 up to1 LsM had no effecton the expression ofthese CAMs in HUVEC,
although TNFa at 10 nglml stimulated the expression ELAM,ICAM and
VCAM by 5.2, 4.0 and 8.3 fold, respectively. Bindingstudies show that
HUVEC exhibits few ET binding sites (0.41 ±0.06 fmolmg). We further
treated HUVEC with phosphoramidon to up-regulate the number of ET
receptors and then examined the effect of ET-1 on CAM expression. After
phosphoramidon treatment at 0, 0.1, 1, 2 and 6 mM for 48 h, the binding
of 1251 ET-1 to HUVEC increased from a basal level of 0.41±0.06 to
1.8±0.2, 4.1±0.2, 6.2±0.6 and 11.9±0.9 fmoVmg protein, respectively.
In HUVEC pretreated with phosphoramidon (2 mM) for 48 h, TNFa (10
nglml, 4 h incubation) was again shown to induce the expression of ELAM,
FiM)
still failed to exert any
ICAM, and VCAM. However, ET-1 (0.01-1
effecton the expression of these CAMs. These data suggest that ET-1
in
HUVEC
does
not
stimulate
the expression of
receptor
to
its
binding
ICAM-1, VCAM-1 and ELAM.
Medkcl
EndotIhat coe (EC)wae mown to be refractoryto transtectionusingcalcum phosphate,
DEAE dextrn, and electoporaon. Problems in obtainingoffcient gene traer and
stable eprsionlimitthe appctn oftr nfection in EC. We compard two methods:
(RMT) and lipofectemine- medatedtrao n (LMT)
retrovirsl-mdiabd
protocolsthat resultd in persitt eox on ofvarouscDNAs in EC. Wedoned cDNAs
into retrovirel vectora pLNCXand pLXSN which containing theimmedate ealy promoter of
ofth
human cytomeglovu and the retrovirsl tong terminl repeatforthe tran
tle marker,the voctos Wo hadthe
insertedcDNA, (NPT) As aThe" constucte
used troduce
w
genes io two EC
gne.
phoephotwasferese
vein EC drivtve) nd HMEC(human dermal
lins: EA hyb92t(a human
pariles we generated by
microvscur EC). For RMT, replication incompetent
plsrrid conabucts into amphotopic retovrs packaging colls,
itoducing
EC. In LMT,
pA317. The viruscon rng cndioned medium was then used to
tbanfeci
noomycin
redpectively.
umbllcl
rerovlra
EC
were
tOnuc
inubated with
a
suspension of
lipofcteineo and
the
DNA.
The bfton
Tho DNA
efficincy wa about 1004-od grear in RMT than LMTfor both cei
f1hgment with NPT and theinsorted cDNAintgratinginto the EC chromosome was
lines
RMT. However, ths was notthec
definod, andthus both genes wero wacoexpresed
was necesy to etash
intdegration of random, and hence
for LMT in wtich DNA
donl
survMng coels in LMT made lecion ofcwhich
clonal populations. The low denit ater
in
populaions possible immediately
screning HMEC,but not in EA hyb92t
Both methods oftransfection yielded stabletwb
exrssingthe
eventuallyanddiod. inrted
gene for at least up to 2 monts poet traection confirmed
the
mar
by Wetewn bkL. Theme resut show that stable long term expression of exogenous DNA
in EC is poible using RMT and LMT,but th RMT is the prefrred metod itgive
of the marker andtheinsrted
higher transfectlon effency and cornient andcontegraon Heart
Association of
In
It
th
ectnts
gene. (Supported by HL45638, HL27016,
Americm
Chicago.)
1421
1422
IL-1 INDUCES PHENOTYPIC TRANSDIFFERENTIATION OF SKIN
MICROVASCULAR ENDOTHELIAL CELLS INTO SPINDLE-SHAPED CELLS:
RELEVANCE TO KAPOSI'S SARCOMA. ((LI. Romero, D.N. Zhang, and
M.A. Karasek)) Department of Dermatology, Stanford University
School of Medicine, Stanford, CA 94305.
DEVELOPMENT OF THE AORTIC VESSEL WALL AS DEFINED BY VASCULAR
MUSCLE AND EXTRACELLULAR MATRIX MARKERS
W.S.Argraves C.D.L)) *
('JEHungerford,
Cha,otesvile, VA 22908. +Biochemisty Labt,A
UniVersity VI
Red Cross, Rockvle, MD 20655. Cardivaseular Developmental Bbgy Center,
Kaposi's
sarcoma is a
neoplasm of vascular origen, histologically
characterized by an abnormal proliferation and perivascular
infiltration of spindle-shaped cells. The purpose of this
investigation was to deter-mine the effect of IL-1 and bFGF, two
cytokines known to be secreted and to stimulate growth of Kaposi's
sarcoma cells in itro, on transdifferentiation of normal
microvascular endothelial cells. IL-1 A and IL-1ba induced
morphologic transdifferentiation of normal epithelioid endothelial
cells into spindle-shaped cells in 24 to 72 hrs. Changes in
morphology
of factor VIII
were
accompanied by
immunostaining. IL-
a
decrease
effects
or
were
complete absence
reversible after 3
days of stimulation and became irreversible after 7-10 days.
Pretreatment of endothelial cells with cycloheximide (5gg/ml) did
not inhibit transdifferentiation. The effect of IL-1 was more
marked in the presence of bFGF (10 U/ml). In the absence of IL-1,
bFGF slightly stimulated growth without changes in cell
morphology; in the presence of IL-i, bFGF potentiated spindle cells
growth dramatically. These data indicate that IL-1 can induce
permanent transdifferentiation of endothelial cells into
mesenchymal like cells and that these cells become more
susceptible to stimulation by bFGF. These observations may begin to
explain the inital steps in the pathogenesis of
Kaposi's
sarcoma.
SMOOTH
Medical Universit
of South Carolna, Charlsto, SC 29425.
The bul"d ot vessel wallfrom s cellularand ltraelllarmatrix(ECM)
and maturaon of th cardovascular
S aacuical event in tde
events ta occur aftere iniil
system. However, very Ut es known abo
d
vasmur nstwot anasent ndolu, esabshed. Usiqn
quail
the
VWdlls X
roe ntand
as amodel (VSMC)
thoracc aorta
mot
o
devep
gveselwall has
edby
usig
St 12-22
nr luor qun
been _ oed
and
indces tatth de
monoconl enlbodes M bs) VSMC
differetan ofdebryonic VSMCS
vntral to
.
The
are
on
the
vental
sufe
ote
fusigW
cab
first
dosal p a ent t thee ndo lium, one er side dt midine. In orr to
aortae,
dorsal
addresse
rtd ttissue speicwy markers to VSMCs in
em n tissue, weNhave gnad MAbs om e n sselwa anIens.
1E12, which iske muscle
sourMAb,
vs
study
Criticaltoths
MAbs tob wnVSMC markers (suchs smoo
muscle
is
b
Imm
seen wi h a SMA
h
t osa
ms
msst, patemd
vascuar
muscle caD
of
b
bsto
foud
now
and
problms
n
td
lwaldel
alha adh) ianoo
pattn #aWs
tD Mrdiawnd vernrJ uno heel
cl'ril
spcrnin
1E12hlg
withth
we show t kmuolxuor
MAb, 8-12 hourspoynonal
labrin d
abo ob 1 showsa m d on siarto
he
beigwkiha
th of the VSMCmars Fin- lbngsufounii the VSMCe before estin
pecurson are prsntwit the d vesor wVaLSWeMtwCo.bdevelbui-I
expesslon to bean
early marker for
dIW
MVMs
Monday. Blood Vessels I (1423-1427)
245a
1423
PAF-INDUCED ALTERATION OF MORPHOLOGY, CYTOSKELETAL
ORGANIZATION AND lCAM-1 RECEPTOR EXPRESSION IN MICROVASCULAR
ENDOTHELIAL CELL MONOLAYERS. S.S. Kantak C.L. Jones, B. Singh, D.E.
Bossung and J.M. Onoda, Department Radiation Oncology, Wayne State
University Schoold Medicine, Detroit, Ml 48201.
We characterized the effects of short-term exposure of nanomolar
concentrations of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) on
those endothera cell reaponses (to PAF) which characterize PAF-kiduced kig
pathologies (e.g., edema). We examined PAF effects on edothelial cel
morphlogy using phae cornrast
mncocopy and
mirofiamerit
nt
and
luminal surface expression of adhesion receptors (i.e., ICAM-1) using
immunofluorescence microscopy. Exposure of confluent, contact-inhibited
munrne pulmonary mnicrwasculature endothelial cell monolayers to 10 nM PAF
results in significant celular retraction ocring < 30 minutes post PAF exposue.
in
Inasnuch as F-acin micfaments have been shown to play a significant
the regulation of endothelal mophology, we examined PAF effects on F-actin
organization and observed PAF-induced F-actin depolynmrization conomitant
with ceHular retraction. We observed that PAF-induced endothelial cell retmction
is a reversible event. Reorganization of F-actin fibers occurs > 8 hours post PAF
exposure, concomitant with restoration of monolayer morphology. Our results
indicate a direct causal relationhip between PAF exposure and the induction of
endtelial cell retraction and F-actin organization which are determinants in the
pathology of inflammation. The induced expression of endothelial adhesion
receptors is also causal for the inflammatory response. Therefore, we also
characterized the effects of PAF on apical surface expression of ICAM-1, an
adhesion receptor which is at east partialy responsible for maintenance and
amplification of the intlammatory response. We observed that 10 nM PAF
induces significant lCAM-1 expresion. These studies were supported in part by
CAS0465 and Gers on Radiation Oncology Center, Detroit, Ml 48201
rle
1424
APPROACHING THE ROLE OF CD34 IN VASCULAR DEVELOPMENT AND
HEMATOPOIESIS: LOCALIZATION, HEMATOPOIETIC POTENTIAL, AND
PROTEIN-PROTEIN INTERACTIONS. ((P.E. Young, C. Fennie, H. Avraham, J.
Groopman, & LA. Lasky)) Dept. Immunology, Genentech, Inc., South San
Francisco, CA 94080.
CD34 is
a
cell-surface protein expressed
on
vascular endothelial cells and
hematopoietic progenitor cells. Our lab demonstrated that CD34 presents
carbohydrate ligands to L-selectin in the high endothelial venules of the
lymph node and potentially throughout the vasculature. However, an
understanding of the function of CD34 in hematopdiesis and of its relative
importance to cell survival and/or differentiation at vascular locations
remains elusive. Towards this end, we completed an immunofluorescent
confocal analysis of the distribution of CD34 in developing murine embryos
throughout development. Our studies localized CD34 throughout the
vasculature, to endothelial processes and filopodia associated with
angiogenesis, and to hematopoletic progenitor cells within the embryonic yolk
fetal liver, and other intraembryonic sites of hematopoiesis. Our
results identified CD34 as the first known useful marker for the analysis of
mammalian vascular development. In addition, we used anti-CD34 affinitypurified antibodies to isolate hematopoietic progenitor cells from embryonic
sources. These studies demonstrated that CD34+ progenitors can give rise to
a variety of hematopoletic lineages In vitro, when cultured with appropriate
cytokines and/or growth factors. Finally, we isolated seven distinct proteins
via the yeast two-hybrid system that interact with the intracellular domain of
CD34 and may therefore be involved in signalling and/or CD34 function. Most
proteins isolated thus far show no significant homology with the database.
Interestinly, two proteins are 7096 identical to each other, and may represent
a novel family of unknown function.
sac, the
1425
1426
ENDOTHELIAL CELL CD36 EXPRESSION CORRELATES WITH PARENCHYMAL
TISSUE FATTY ACID UTILIZATION. (D. E. Greenwalt*, S. Scheck§,
and T. Rhinehart-Jones*)*Holland Laboratory, American Red
Cross, Rockville, MD, U.S.A. and §Loyola Narymount University,
DISIRIBUBlON OF PAF RECEPOR IN THE VASCUIATURE ((D.
Los Angeles, CA, U.S.A.
CD36 has been described as a receptor/binding protein for
thrombospondin, collagen, oxidized low density lipoproteins,
and long chain fatty acids. We have exaained CD36 tissue
distribution and expression in a mouse model. Nurine capillary
endothelial cell (CEC) CD36 expression was most prominent in
adipose and maary epithelial tissues which take up fatty
acids and store and/or secrete triacylglycerol and highly
oxidative tissues such as skeletal and cardiac muscle which
use fatty acids for energy production. CEC of the brain, which
does not utilize fatty acids as an energy source, did not
express CD36. We then hypothesized that increased levels of
plasma triglyceride (PT) would lead to increased expression of
CEC CD36. Both diabetic mice and mice fed diets high in fat
have elevated PT levels and were examined for cardiac muscle
CEC expression. NOD (nonobese diabetic) mice had PT levels 3fold higher than normal mice and expressed cardiac muscle CD36
at a level 7-fold higher than normal mice as quantified by
densitometry of immunoblots. Female mice fed ad l1bltua a diet
containing 40% fat (% caloric intake) for 5 weeks, expressed
CD36 at a level 4-fold greater than mice fed normal diets (9%
fat). In both diabetic and high fat diet mice, brain CEC
remained CD36-negative. These data suggest cardiac muscle CEC
can regulate CD36 expression in response to increases in PT.
142
DEVELOPMENT OF A HUMAN BRAIN MICROVASCULAR
ENDOIIHELIAL CELL LINE WITH BLOOD BRAIN BARRIER
CHARACTERISTICS: TRANSFECInON WnI SV40-LARGE T.
((M.F.Stins, P.V. Neman.i, KS.Kim)). Div. Infectious Diseases, Chlldrens
Hospital LA, 4650 Sunset Blvd, Boc 51, Los Angeles, CA, 90027.
Studieswith human brain microvascular endothelial cells (HBEC) have
been limited because these cells are difficult to obtain and culture in
vitro. Normal HBMEC usually showed signs of senescence at early
passage. We trnsfcted HBMEC with a pBR322 based plasmid
containing simian virus 40 large T antigen. The transfected endothelal
cells showed a somewhat spindly morphology, were positive for fictor
VIm carbonic anhydrase IV, took up fluorescently labeled acetylated low
density lipoprotein, and expessed gamma glutamyl transpeptidase,
demonstrating both their endothelial and brain origin. We have cultured
these cells past passage 15. The HBMEC were used as an in vitro model
of the blood brain barrier in
minmg the pathogenesis of E.gl
meningitis Le. the role of S-Fimbriae and OmpA in binding and invasion
of HBMEC. Binding to and invasion of HBMEC was significantly greater
for S-fimbriated and OmpA+ E..cli. These differenceswere not observed
with human systemic arterial or venous endothelial cells. In additon,
atvation of HBMEC by TNF reslted in a time dependent increase in
VCAM expre. In conclusion, our tanfecd HBMEC's are the first
reported human brain endothelial cells that retain morphologic,
phenotypic and functional chracteristics of normal HBMEC for an
extended period of time.
Predescu, S. Predescu, L Ihida and G.E Palade)) Division of Cellular
and Molecular Medicine, School of Medicine, University of Califomia,
San Diego, CA 92093.
Although the platelet activating factor (PAF) is one of the most active
inflammatory mediators known to date, little is known about the cellular
and subcellular distribution of its receptor(s). The latter has already been
identified as a membrane protein of 39 kD. In order to better
understand the signaling mechanisms responsible for PAFs unusually
high potency, the first question that we decided to answer concerned the
tissue distribution of the PAF receptor(s) (PAF-R). In our laboratory, we
raised monoclonal antibodies agnat synthetic peptides reproducing sbort
segments (12 and 14 as) at the N and C terminal parts of PAF-R. With
the anti N terminal monoclonal antibody, we localized PAF-R by
immunofluorescence on semithin frozen sections of lung, heart,
diaphragm, kidney and brain specimens. With the exception of brain, the
signal was restricted in all cases, primarily, to the vascular endothelium.
Using a preembedding Immunogold procedure, we localized the PAF-R In
small clusters on endothelial surfaces, without preferential localization to
any differentiated microdomain. A morphometric analysis revealed,
however, a greater signal density at the level of venoles than at the level
of any other segment of the microvasculature. With the same antibody,
we immunoprecipitated PAF-R from whole homogenates of the same
tissues, and with an ELISA we obtained quantitative data. The purified
antibody detected PAF-R at a dilution of 1:10,000 in the lung and at a
dilution of 1:150 in the case of brain, which correspond to calculated
values of 322 pmol/mg tissue protein and 26,8 pmol/mg tissue protein,
respectively. Supported by NIH Grant HL 17080 to GEP and by Bugher
Fellowship 001 to DP.
-
246a
Platelets (1428-1432). Monday
I
1428
REDISTRIBUTION OF PLATELET-BOUND FIBRINOGEN MODULATES
PLATELET AGGREGATION. ((J.D. Wencel-Drake)) Department of Medical
Laboratory Sciences, University of Illinois, Chicago, IL 60612.
ammsis converted fron *inactive'
agonist-stimulated platelets, the integrin
'active fibrinosen (FBG) receptor, thereby mediating platelet aggregation.
In
to an
an
FBG
an,sthat
agP
With time after agonist addition, at least two events occur,
beocmes
irreversibly bound to the platelet, and in the absence of stirring, platelets lose the
is
ability to aggregate. Since we previously identified a mobile pool of
actively internalized in platelets, we explored the possibility that both of these
events might result from the internalization of FBG bound to 'active'
As
Under conditions of irreversible FBG binding, fluorescence microscopy revealed
that bound FBG is rpidly redistributed by activated platelets to a surface
inaccesible, intracellular pool. To investigate the relationship between the rapid
internalization of bound FBG and the loss of aggregation response, unstirred,
washed platelets augmented with FITC-FBG were stimulated with thrombin
receptor peptide agonist (TRP) for a various times, and the samples were rapidly
brought to 4'C. FACS analyses revealed a time-dependent increase in the binding
of FITC-FBG to unstirred, TRP activated platelets. Rabbit anti-fluorescein was
subsequently added to quench surface-associated fluorescence and the samples
were re-analyzed. A plot of the percent surface-associated FITC-FBG revealed a
paaraleled a loss of aggregation
loss of FITC-FBG from the platelet surface that
response. Thus, the removal of FBG from the activated platelet surface via
internalization appears to contribute not only to the irreversible phase of FBG
binding, but also to the downregulation of platelet adhesiveness. To examine the
effect of inhibition of irreversible FBG binding on the loss of aggreption response
and the cellular localization of bound FBG, studies were repeated in the presence
of ZnCl2. Incubation of platelets with ZnCl5 resulted in the retention of
aggregation response in TRP desensitized platelets and inhibited internalization of
biotinylated FBG previously observed by immunofluorescence microscopy. Taken
colectively, these data sugest that internalization bound FBG represents a
fundamental regulatory mechanism that modulates platelet function.
HL-60 cells, a human promyelocytic stem cell line, have been used as
model leukocytes in studies investigating platelet-leukocyte binding.
Platelets activated bya-thrombin and related agonists bind to neutrophils
and monocytes. Several groups have strong evidence that P-Selectin
receptors on platelets and an unidentified ligand on leukocytes mediate this
binding. It has also been reported that GPIIbEIa receptors are involved in
platelet-leukocyte binding.
Our laboratory has been interested in the
functional interaction of these cells. We, and others, have demonstrted
that coincident with binding, activated platelets cause an enhanced
production of superoxide anion by neutrophils. Recently, we have also
shown that activated platelets can modulate superoxide anionin DMS0differentiated HL-60 cells in a similar manner. The purpose of the present
study was to investigate the relative roles of P-Selectin and GPIIbIIa
receptors in the functional inteacion of these cells. This was
accomplished by the use of monoclonal blocking antibodies against the
respective receptors and RGDS peptides in platelet:HL-60
while measuring superoxide anion. (HL-40615)
coincubations
1432
PHOSPHORYLATION OF CYTOSOLIC PHOSPHOLIPASE A2 IN
HUMAN PLATELETS. ((Y. P. CWte and M. B. Feinstein)) Department of
Pharmacology, University of Connecticut Health Center, Farmington, CT
06030-6125.
Phospholipase A2 (PLA2) catalyscs the hydrolysis of the sn-2 fatty acyl
phospholipids to liberate free fatty acids and lysophospholipids.
Recently a Ca2-dependent high molecular weight cytosolic PLA2
(cPLA2) that is specific for arachidonic acid (AA) has been described.
cloned and sequenced This enzyme has been identified and catezed
in a variety of cells and has been prposed to be involved in the release of
AA.
More recently its activation by phosphorylation has been
bond of
demonstrated in human
platelets and it has been proposed that it plays a
mobilization of AA in human platelets. The goal of this
determine the signal transductions involved in the
phosphylation of cPLA2 in human pltelets. More specifically we have
studied the phosphoylation of cPLA2 in human platelets stimulated with
a-throbn, throbin receptor pbptide, A23187, tasiggin and phorbol12-myritate-I3-acetate (PMA). We have shown that (1) Although the
release of the intracellular Ca2+ pool by thapsigagin or A23187, or the
activation of PKC by PMA, rsult in increed by phosphorylaton of
key
IL-2 is required, but not sufficient, for the conal proiration of T lymphoThe activation of ymphocytes ruires concurrent occupancy of the T
cellantigen receptor. IL-2 is known to induce the phosphorylation and
activation of PI-3-K in lymphocytes, but does not affect intracellular calcium
levels. In the patelet system, IL-2 presented alone does not result in pldelet
aggregation, however, it does enhance the platelet
response to
suboptimal levels of collagen or thrombin. This study focused on the effect of
EL-2 on patelet
Platelets exposed to IL-2 were
lyses and immunoPI-3-K.
a
precipitated withPI-3-K
specific antibody. The immunoprecipitates were
subjected to Western blot analysis with an antibody specific for phosphoof
tyrosine contaning proteins. The phosphorylationPI-3-K
(85 kDa subunit)
peaked at 5-10 seconds. This is much more rapid than that noted in the
the
PI-3-K
In
lymphocyte system which peaks at 15 minutaes. adddtion,
cytes.
activity was directly meaured. Plateets incubated with IL-2 for varying times
lysed and imunnoprecipitated with anti-PI-3-K. The incorporation of
was
from y`P-ATP into phosphatidylinositol substrate PI-3-K
by
y`2pd;2
determined by thin layer chromatograhy. The kinase activity was maximal
within 1a0 second incubation of platelets with IL-2 These studies indicate
that IL-2 modulatessignal transduconin platelets and may havesignificant
effects on platelet function in conditions whereIL-2 levels are elevated.
were
1431
1430
PLATELE;T MODUIALION OP E0O CELL PRODUCTION OF
SUPEROXIDEANION:INHIBIONOOFP-SEECINANDGPIbMia
RECEPTORS. ((D.G. Moon, P.S. Chin, T.B. Nguyen, S.M. Branley,
S.R. Braemer, J.W. Fenton,II and G.A. Wetmore )) Departments of
Biology and Pharmacy Prkcice, Albany College of Pharmacy, Albany,
New York 12208
the
was to
role in
work
1429
INTERLEUKIN-2 (IL-2) INDUCES PHOSPHORYLATION OF
PHOSPHATIDYLINOSITOL-34KNASE (PI-3-K) IN HUMAN
PLATELETS.((M.L. Aiken)) Univ. ofTexas Health Center at Tyler, Dept. of
Biochem Tyler, TX 75710.
cPLA2, (2)
phosphorylation
of cPLA2 induced
by
a-thrombin is
extracellular Ca2+, is inhibited by prostacyclin, not
GDPpS (in permesbii
and not affected by GTPS
by
platelets). These results demonstrate that thrombin can stimulate the
mchanism
a
phosphorylaton of cPLA2 by
independent of a Gem protein
and its downstramsignals, and may therefore be mediated by Py subunits.
independent of
mediated
PKC
or
PIATELET ACIVAnON BY SODIUM
INHIBMON OF HUMAN
N
AS VIEWED BY VIDEO-ENHANCED DIC
IROPRUSSIDE
Loftus, M.L Harlow and SJ Smith))
Department of Molecular and Cellular Physiology and
Division of Hematology. Stanford University School of
Medicine, Stanford, CA 94305.
MICROSCOPY. ((DJ.
We have used the technique of video-enhanced
differential
contrast (DIC) microscpy to examine the effect
interference
of a nitric oxide donor, sodium nitroprusside (SNP), on the
morphological transformation that takes place when
platelets become activated. In the absence of inhibitors,
activating agents
platelets atreatedW vth thrombin or other
undergo dramatic changein shape with extension of
numerousiflopodia. Plateletfllopodia exhibit extension,
retraction, waving, bending and other complex movements.
Near completeinhibition offllopodial formation and
movement is seen in the presence of 100 gM SNP. Platelet
adhesion and spreading on glass surfaces, however, which
occursin the absence of activating agents, appears
unaffected by SNP. These results indicate that nitric oxide
specifically inhibits the platelet cytoskeletal transformation
that is associated with platelet activation.
Monday. Imaging Technology (1433-1438)
1434
1433*
3-D AND 4-D
IMAGING OF LIVING CELLS WITH DIC AND POLARIZATION
Inoue)) Marine Biological Lab., Woods
MICROSCOPY.
((S. lnou6 and T.D.
Hole, MA 02543, and Universal Imaging Corp., West Chester, PA 19380.
We
report here
247a
a
practical method for rapidly acquiring and generating
high-resolution 3-D and 4-D images of live cells and developing embryos
non-confocal, DIC and polarization (pol) microscopy. Current biological
confocal microscopes do not allow confocal imaging in DIC orpol, and
low-noise confocal fluorescence images require several seconds to
acquire. Some confocal microscopes do permit video-rate image
acquisition, but their utility for the current application is limited by photon
statistics. Our method entails: Al acquire background-subtracted, lownoise, through-focal, high-numerical-aperture DIC orpoI video images (the
z-stack at rates of 30 optical sections per second, repeatable every few
seconds(Inoue et al., 1991, Biol. Bull. 181: 336-337); B) remove haze
from 16-bit gray scale images using filtered nearest-neighbor optical
sections; C) reconstruct volumetric 3-D projections with gray values of
intermediate planes interpolated as required; D) sharpen and adjust
contrast of the projections; E) play back as stereo movies from computer
memory or from a video-rate laser disk recorder. The high-rate image
acquisition (A), stack processing(B-D), and 3-D and 4-D playback (E) are
controlled by a personal computer equipped with newly developed
programs. 3-D and 4-D examples displayed at the ASCB Poster Session
will include: birefringent chromosomal spindle fibers in mitosis with
enhanced visibility; dynamic positioning of mitotic spindle in cell division;
through-focal, high-resolution 3-D images of developing sea urchin plutei.
(Supported in parts by NIH grant R-37 GM31617 and NSF grant MCB890816 awarded to S.I.)
in
1435
Theory, Application And Comparison Of Real-time, Fluorescence
Polarization Image Microscopy Using Polarized And Natural-Light
Excitation. ((Yuling Yan-Marriott & Gerard Marriott), Max Planck Inst. for
Biochemistry, 82152, Martinsried, Germany)
polarization techniques are widely used in the measurement of
orientational distributions, rotational diffusion of molecules in solution or in
interactions, and membrane fluidity in artificial
molecular
suspension,
liposomes and cells.The microscope measurement enables an analogous
study on microvolumes of cells, especially for samples of highlyordered
orientations, such as fluorophores aligned along membrane phospholipids.
These probes are easily imaged and the orientations canbe calculated from
the polarization values on a pixel by pixel base. In this study, we present a
derivation of fluorescence polarization using natural and polarized
excitations in the microscope, followed by some biological applications. A
multi-view fluorescence image microscope is employed for simultaneous
capture of two polarized emission images is employed in the microscope
measurements. In the microscope measurement, the numerical aperture
effect on the limiting polarization value of samples is well known. However,
so far, only the depolarization factor from emission cone effect has been
theoretically established. Here, we consider the depolarization from both
excitation and emission cone effects and as an example, a derivation for the
limitng polarization value for samples with 2-D isotropical frozen dipoles is
given. We also study the fluorescence polarization image microscopy with
natural-light excitation to observe the orientation and dynamics of ordered
molecules or artificial fluorescent lipids and fluorescent proteins
encapsulated within liposomes, and phalloidin labeled actin molecules in Factin intecting with myosin. We show that with natural-light excitation, the
polarization of randomly oriented dipoles is theoretically zero, which makes
it convenient to distinguish ordered dipoles from randomly oriented ones.
Fluorescence
4-Dimensional Digital Imaging and Display System for Dynamic Processes.
((C.F.Thomas, P.J.DeVries, V.E.Centonze, J.D.Hardin* and J.G.White))
Integrated Microscopy Resource and *Deept Zoology, Univ. of Wisconsin,
Madison, WI 53706.
We have developed a 4D digital imaging system to study intracellular and
multicellular dynamics. Images are
with a Sierra Scientific video
camera mounted on a Nikon Diaphot equipped with high extinction DIC
is used to preprocess the video signal
optics. A DSP-2000 image
before digitization by the Scion LG-3 framegrabber card. Images are stored
to
the
hard
drive.
The operating software is based on
digitally
computer
NIH-Image, a public domain Macintosh-based image processing package
run on a Quadra 950. Using the extensive macro language, the frameMac-1000 stage drive were integrated into a
grabber card and the
mouse-driven 4D acquisition operating system. We have incorporated the
following features for flexible data acquisition: user-definable region of
interest, choice of acquisition modes (time-lapse of single focal plane or zsetup, and variable
series), image annotation, graphic menu for
delay between time points. The data are subsequently organized into one
large Apple QuickTime movie where images from each focal plane are
appended together and compressed using the QuickTime JPEG compressor.
The dataset is then made into a navigable movie using Video Navigator
software from Radiant Interactive. This utility creates links between the
timeframes at different focal planes. The resulting dataset is viewed using
our custom-designed 4D Navigator program. This program allows the user
to move up and down in focus and forward and backward in time, go to
specific frames, set bookmarks etc. It also displays the current frame
coordinates. This 4D data display package can be applied to any optical
confocal, and 2sectioningmicroscopy technique such as
Photon excitation. (Supported by NIH Grant RR-00570)
collected
processor
Ludl
z-series
Nomarski-DIC,
1436
THE AUTOMATED INTERACTIVE MICROSCOPE: MEASUREMENT AND
OF CHEMICAL AND MOLECULAR DYNAMICS IN LIVING CELLS,
F. Lanni, D.L. Farkas, S.
D.W. Deerfield and
((A.H. Gough, L.D.
Taylor)) Center. for Light Microscope Imaging and Biotechnology, Camegie
D.L.
Mellon University, Pittsburgh, PA 15213
MANIPULATION Harris,
Fahiman,
goal of this project is to develop an Automated Interactive Light Microscope
(AIM) thatwill be capable of real-time acquisition, processing, analysis and display of
N-dimensional data sets from living cells, tissues and organisms. An advanced
will be possible when researchers can
generation of biological
measure and manipulate biological processes during the time of the events.
information fromliving
Todays workstations can obtain complex
event such
samples minutes to days after a linear experiment, when the
as
been completed. The development and use of the
division, hasWorkstation
of
has
demonstrated
value
the
hamessing
(MMLMW)
Ught Microscope
from different modes of
the distinct chemical and structural informatlon
into
a
machine-vision
when
semi-automated,
system.
light microscopy
integrated
Together with the growing list of specific reagents designed to measure and
the
allowed
has
manipulate cell chemical and molecular dynamics, the MMLMW
of cell and developmental functions. However, there
exploration of
are fundamental limitations of the present generation workstations that must be
the maximum information. The major limitations include:
overcome in order to
of focusing devices; (3)
(1) performance of computers; (2) speed and
and
of
speed
flexibility wavelength modulation for multispectral imaging; (4)
resolution and read out rate of solid-state cameras; (5) axial resolution of
motion
fluorescence microscopy; (6) intelligence and power of feature extraction,
and (7) integration and speed of switching
and event detection
tracking
between key modes of microscopy. Solutions to these limitations are leading to the
design and implementation of AIM. Recent advances in these areas will be
presented with examples of biological applications.
This project is supported by NSF grants: BIR-8920118 & BIR-9217091
The
experiments
temporal-spatial biological
Multimode
cell
available
mechanisms
gain
precision
algorithms
1437
1438
NEAR-FEELE SCANING OPTICAL MICROSCOPY AS A PROBE OF
FLTORSCENTF-LABEIED M 0BRANES. ((JeeseongHwng*,Eic Bezig,
Robert CQiirster# and Michael Edidin*)) *Dqprten of Biology, The Johns
OBSERVATION OF
FLAGELLA BY CONTACT
MODE AFM AND TAPPING MODE AFM(Kyoko Yoshioka,
Hideo Komizu and Hiroshi Miyamoto, National Inst. Biosci. &
Human-Technol. Tsukuba Ibaraki 305 JAPAN)
Isopkirs Univesity, Balimore, MD 21218 USA, #AT&T Bell Laboraiores,
07974 USA.
Mkmay Hill,
While convetional otical miacscopy has a diffraction limited resolution of
at best (where A is the wavelength of the light source), the Ner-Field
Scaing Optical Nfiscopy (NSOf etinely gaenrates imagesw ith resolution
as good as SOnm InourNSOMa fluorescently-labeled sample is illsuinated by
a taered fiber oic probe with an aperture diamter of 80nm and the probe is
raster-scaned over the sample at a distance of lOnm from the srface. The 2-D
imwity map of fleence light from those smal areas constructs an image
0.5
hly srlius of the probe apeture. We used
api
pobe the distribution of lipids and proteins in plasma nnbranes of
fibrlasts. Tei distnbution of fluoresctt lipid analogs in model
sy
was also imvestigoted and consared with the cell dta. Cell
ya
whose resolution is
e
NSOM to
hianm skin
n
mnmbranes we labeled with the flhorescerst lipid analog, BODIPY-PC
well as with uI_ted anti-HLA IgG. Patchy
distribution of the fluorescest lipid analogs idicative of lipid domains was sem
in all cells. These patches w"e not spatially correlated with patches of labeled
HLA molecules. In conhast, the distribution of fluorescence was uniform, not
plasma
as
oatd into single phase, DPPC, model
patched, when BCODIPY-PC was in
nI
n.ce, the patches of BODiPY-PC seen in cell nmemranes are
, domains, in thse nmemranes.
dmo
likely to reflect
BACTERLAL
Bacterial flagellum is an assembled structure of its protein subunit,
flagellin. By using contact mode AFM and tapping mode AFM, we
observed flagella of E. coli, S. typhimurium and its mutant SJW46
whose flagellin has deletion at central portion in amino acid
sequence. Each flagellum is mounted on the surface of glass
substrate and observed under ambient atmosphere. Flagella of wild
type E. coli and S. typhimurium showed normal helical wave
length of 2.7 u m, however that of mutant strain showed little
deformed wave form. Since flagella of mutant SJW46 showed quite
nornal structure in water from dark field light microscope
observation, deformation after drying process suggests that
flagellum of mutant SJW46 is fragile compare to that of wild type.
This is also ascertained from height measurement of each flagellum
filament. According to height of each filament, these flagella are
deformed during AFM observation more or less and order of
deformation is
*Author(s) and/or presenter(s) have noted
that
there is
potential
conflict
of
SJW46>SJWllO03(wild
interest.
type)>E.coli(wild type).
248a
Imaging Technology (1439-1444). Monday
1440
REQUIREMENTS FOR AND LIMITATIONS OF CONFOCAL IMAGING WITH
RED AND FAR-RED LIGHT. ((Christopher Cullander)) Departments of Pharmacy
and Pharmaceutical Chemistry, School of Pharmacy, University of Califoria, San
Francisco, 94143
1439
SEQUENTIAL FORCE IMAGING OF CELLS USING THE ATOMIC
FORCE MICROSCOPE (AFM). (S. R Marchese-Ragona and M.
Wortge) TopoMetrix, 5403. Betsy Ross Drive, Santa Clara, CA.
95054. (408)-982-9700.
tight will penetrate further (and with less scattering) into a specimen
excitationwavelengths
will, and the emitted fluorescent photons will exit the
are
*f long-wavelength fluorophores
specimen in a similar fashion. Consequently,
within a
used, ls possible to obtain high quality confocal images from deeper
thick specimen. While resolution is somewhat decreased
longer wavelengths,
with
biological material is much less autofluorescent when excited with red tight. The
acquisition of confocal images with red and far-red light thus has both advantages
and limitations, and an understanding of the latter is necessary both to acquire
high quality images and to avoid the misinterpretation of experimental data. A dual
set was designed for the Bio-Rad MRC-600 for imaging the fluorescent
tiiter
probes cyanine 3.18 (CY3) and cyanine 5.18 (CY5). Visualization of CY5 with this
Red
than shorter
instrument
measuring
Mkroscope is an extremely senisiive force
The Atomic Force
whichis routinefy used to measure the force exerted on the AFM tip by the sample,
with a routine force resolution inpiconwton
the
to nanorewton range. By plotting
to
tp, one can
tth tip-sample separation(positive or negative) vs. force
produce what is traditionally known as a force distance' carve. Analysis of the
dis tance curve allows one to extract information on micro elastict and
force
as
magnitude of any tip-sample binding that
plasticity of cell surfaces as welltth
al.,
may occur. Recently several groups (e.g. Lee e1 1994,
langmuir, 10, 354-357)
the
force-distance
curve to investigate the binding (unbinding) strength of
have used
utility
of the force-distance curve
the streptavidin-blotin interaction. Toincrease the
for cell blological studies we have develoed a sottware-dnven data acquisition
program that will perform a force-distancecurve for every pixel of a topographic
image. For a 200 x 200pixel image, 40,000 force-distance curves are generated.
In order to analyze this vast quantity of information in a convenient and meaningful
image isviewed in sequence, and can beviewed as 'movie',
a
way, each force
eachframe being animage taken at a particular force. Asthe force on the sample
increases (thetip pressing onthe sample) hard incompressible material appears
bright, while soft compressible matenal appears dark. We have used this technique
to imagefibroblasts in PBS solution. By analyzing the force movie one can determine(1) the optimum force for image contrasting, and (2) differences in compressibility of the cell surfaces. This technique represents a new form of microscopy,
where differencs in compressability are used as the contrast mechanism for the
image.
app6edtth
CY5
fiter set is neariy ideal, since
is excited almost stits absorption maximum by
the 647 nm laser line, and the collection fiher can be centered on its emisslon
In
the
568
nm
laser line happens to be at the emission
spectrum. contrast,
maximum of CY3, which thus limits the collectable CY3 fluorescence. The poor
quantum efficiency of photomultiplier tubes in the red region mandates a high
optical transfer efficiency for the system, and light throughput was maximized by
the use of special high transmission optical fitters with sharp bandpass cutoffs.
The transmittance of objective
decreases gradually as wavelength
increases, and the axial and lateral chromatic aberration of a given lens are also
since
far-red
not
focus in the same plane as lower
important,
photons may
confocal lightpath mirror efficiency rapidly degrades above
wavelengths.
700 nm, the loss of this portion of the CY5 emission spectrum was acceptable
since (a) the fluorophore is very bright and (b) these very long wavelengths will
probably introduce aberration. Imaging with long-wavelength fluorophores thus
requires the use of high quality optical components, bright dyes, and attention to
lenses
While
the selection of the objective lens.
1441
CONFOCALMICROSCOPY AND CALCIUM IMAGES IN HUMAN
EPIDERMAL KERATINOCYTES EXPOSED TO SULFUR MUSTARD
((RJ. Werrlein, J. Madren-Wsalley, S.D. Kirby and M.AE. Mol))
Pharmacology Division, United States Army Medical ResearchInstitute of
Chemical Defense, Aberdeen Proving Ground, MD 21010-5425.
Human epidermal keratinocytes (HEK) were grown on glass coverslips in a
mixture ofHam's F-12 and Dulbecco's MEM. From day 7, culture medium
contained 0.15 mM calcium. Experiments were performed on days 10-14.
Cultures, preloaded with Indo-lAM, were excited with a 355 nm laser-line and
subjected to image analyses using a Meridian ACAS-570 confocal laser
cytometer. From control populations, the emissions at 485nm (Ca2e-free
Indo-1) and 405nm (Ca2"-bound Indo-1) produced evidence of selective cell
loading. Epidermal cells at the periphery of seed colonies were relatively large,
morphologically distinct and brightly fluorescent. Smaller cells at each colony
core were not fluorescent. Five-minute exposures to 400
sulfur mustard in
buffer containing 0.15 mM calcium did not disrupt calcium images. They did
produce significant (p<.05) increases in ratios of Ca2e-bound Indo-l
emissions. Populations acutely
Indo-l, without increasing Ca2+-bound
exposed to 400 (SMsulfur mustard and to 1.02 mM calcium showed an early
post-exposure efflux of Indo-l, and a corresponding loss of calcium image/cell
definition. Control populations acutely exposed to 1.02 mM calcium, without
mustard, showed no efflux of Indo-l. Results indicate that acute, sinmltaneous
exposure tosulfur mustard and to increased calcium concentrations may effect
changes in the plasma membrane and selective permeability of basal-type HEK.
were
IuM
:Ca2+-free
Indo-l
1443
(Tllll),
a
T111.
side
a
a
a
of Guinea Pig amnion and chorion.
1444
KINETICAL STUDY OF DNA SYNTHESIS AND MORPHOLOGICAL
ESTABLISHMENT OF APPROACH
IN LIVING CULTURE CELL
((L. Lehong)) China Academy of Traditional Chinese
Medicine, Beijing, China
CHANGES
In this document, an unique approach has been developed by
which time lapse in vivo observation of dynamic behavior
of DNA synthesis were successively observed during 12 hours
and the paralleled morphological changes in the same cell
Living culture cells (N1H3T3)
were efficiently obtained.
were stained by Hoechst33342 at the possible limited
concentration and DNA changes was measured by cytofluorometry.
Phase-contrast photomicrographs were recorded in the same
cells in which DNA was examined. Morphologic features
(cell areas, nuclear areas and shape constant) and the
paralleled DNA dynamic synthesis were processed through
image analyzer controlled by computer. In order to evaluate
the method, the DNA synthesis time obtained by this approach
was compared to the one come from AH-thymidine ('H-tdR)
autoradiography. In addition, by using the established
method, some tumor cell lines such as A431 and HL-60 cell
were also observed and the effect of some anti-tumor drugs
derived from Traditional Chinese Medicinal herbs were
examined.
It
1442
CELL VIABILITY AND EPITHELIAL IMPERMIABILITY CAN
BE TESTED SIMULTANEOUSLY WITH THE STYRYL DYE
RH414
WHEN VIEWED WITH A CONFOCAL MICROSCOPE. ((DAVID CARTER)) Department of Zoology and Robarts Research
Institute, University of Western Ontario, London, ON, Canada. N6A 5B7.
The styryl dye RH414 (Molecular Probes Tllll) has significant advantages
over Propidium Iodide and Fluorescein Diacetate for distinguishing living from
dead cells. At the same time, it provides information about plasma membrane
distribution and the permiability of epithelial layers, when viewed by confocal
microscopy. This water-soluble dye becomes fluorescent when it interchalates
into the exposed face of lipid bilayer, but is not otherwise fluorescent. It
has excellent photostability, low toxicity, adn can be washed out of living cells,
once their viability has been established. Although designed as an indicator
of membrane potentials, signal differences are too low (<5%) to interfere with
its use as a vital stain. Living cells are revealed as a bright outline when their
If dye solution is presented to one side
plasma membranes accumulate
of an impermiable epithelium, only the exposed
of each cell is labelled, the
dye being blocked at the level of the tight junctions. In fully permiable cell
layers, both sides of the cell are labelled. When a cell ruptures, the membranes
of all its organelles are exposed, resulting in highly fluorescent cytoplasm
surrounding characteristically unstained nucleus. T111 has successfully been
used to demonstrate cell viability and plasma membrane distribution in range
of insect tissues. It has been used to demonstrate the absence of intracellular
movement of solutes across insect epidermis and to show the membrane integrity
was
concluded that the established approach may
effectively provide reliable dynamic information for vital
cell and can be applied to the research on the mechanisms
of anti-tumor drugs.
IMPROVED PHOTOELECTRON IMAGING OF HUMAN BREAST
CARCINOMA CELLS USING MULTIPLE WAVELENGTH EXCITATION.
((D.L. Habliston, K.K. Hedberg, G.B. Birrell and O.H. Griffith)) Institute of
Molecular Biology and Department of Chemistry, University of Oregon, Eugene,
OR 97403
Photoelectron microscopy (PEM) provides a sensitive low-damage method
imaging cell surfaces. The specimen is exposed to UV light, and the
photoejected electrons are accelerated and imaged. PEM serves as the electron
optical analogue of fluorescence microscopy, and provides both a topographical
map of unlabeled cell surfaces and the distribution of gold-labeled surface
antigens. A UV light source of 254 nm is used to generate maximal
photoemission (image brightness). However, the use of this excitation wavelength
can lead to charging in thicker specimens, and therefore PEM of cultured cells has
been generally used for only thin, well-spread cell types. We now find that
combining the short wavelength UV excitation with a longer wavelength UV
source (310-325 nm) greatly extends the range of cell thickness which can be
imaged. While the shorter UV illumination is primarily responsible for the
photoelectron emission from the biological surface, the longer wavelength
of
evidently serves to increase photoconductivity and reduce charging. The use of
excitation sources has the additional advantage of allowing the immunogold
label brightness to be varied relative to cell surface brightness, generating the
unique capability of tunable label contrast. The effects of varying the excitation
wavelength is illustrated here with PEM images of cultured MCF-7 human breast
carcinoma cells, both alone and in co-culture with thinner, normal cell types.
two
Supported by
PBS grant
#
CA 11695 from the National Cancer Institute.
Monday. Imaging Technology (1445-1448)
1445
NOVEL APPLICATIONS OF TOTAL INTERNAL REFLECTION MICROFLUORIMETRY (fiRM) IN UVING CELLS; MEASUREMENT OF CELL VOLUME
AND CHARACTERIZATION OF MEMBRANE-ADJACENT CYTOPLASM. ((S.E.
Bicknese, J. Farinas, HIP. Kao and AS. Verkman)) CVRI, U.C.S.F.
TIRM pemrmts the selective excitation of cell-entrapped fluorophores that are very
(within -100-250 nrn) the plasma numbrane. The effective excitation 'depth" is
determined by illumnination angle and refractive index. A TIRM microscope was
constructed to image cells nounted in a perfusion chamber and illuminated by Ar
near
and/or He-Cd laser beams directed at specified incident angles. To permit rapid
selection of incident beam angles, the microscope contained a plane mirror mounted
on a software-controlled microstepper motor, an off-axis ellipsoidal mirror, and a
hemicylindrical sapphire prism. Fluorescence was detected by a cooled CCD
camera or photomultiplier. Several biological applications of TIR were developed.
Cell volume. Cells grown on a coverglass were loaded with the fluid-phase
fluorescent indicator calcein. The time course of relative cell volunre was measured
directly from the TIR fluorescence signal. Applications to cell volume regulation
and water permeability were developed. 2. Solute mobility in membrane-adjacent
cytoplasm. Fluid-phase fluorescent indicators (BCECF and FITC-dextrans) near the
plasma membrane were excited selectively to measure rotational diffusion by
frequency-domain fluorimetry (Bicknese et al, Biophys. J. 65:1272-82,1993) and
translational diffusion by microsecond photobleaching recovery. 3. Calcium
signaling near the plasma membrane. Agonist-induced calcium activity in bulk and
membrane-adjacent cytoplasm was measured continuously by alternate TIR and
trans-illumination of 3T3 fibroblasts loaded with the indicator Calcium-green. 4.
Submicroscopic distance determination. From tieoretical modeling, Laplace transform
of multi-angle TIR images should provide information on spatial distribution of
intracellular fluorophores. This approach is being applied to image fluorescently
249a
1446
QUANTITATIVE ASPECTS OF DIGITAL MICROSCOPY APPLIED TO
CELLULAR LOCALIZATION OF HEPARIN IN SMOOTH MUSCLE
CELLS. ((
Johnston(l) D. Hanzel(l), B. Brandley(2) and J. Castellot(3))) 1:
Molecular Dynamics, Sunnyvale, CA. 2: Glycomed, Alameda, CA. 3: Tufts University
School of Medicine, Boston, MA.
High Resolution digital acquisition allows a great deal of flexibility in the types of questions that can be directed to microscopic samples. To eliminate subjective bias and
provide quantitative results we have approached microscopy with an automated digital
format. This mode can return quantitative data at high resolution over large fields.
The digital format makes accessible data including multispectral colocalization, seeding and connectivity, particle size and shape distribution and population analysis. We
have begun a program to investigate this approach using the confocal microscope. Scanning larger fields-of-view at lower spatial resolutions (e.g., low magnification objective)
defines large maps that allow alignment of high spatial resolution (defraction limited)
sampling. The selection of the field-of-view with low spatial resolution reduces the subjective nature of the selection of a "typical staining pattern". High resolution digital
scanning in three d! imensions contribute both to the objective" nature of the analysis and allow for quantitation of characteristics not historically available/accessible
The complex carbohydrate heparin is implicated in tumor growth and wound healing
by affecting angiogenesis, cell proliferation and motility. The internal localization of
heparin within vascular cells appears to be a good predictor of the sensitivity of those
cells to the action of heparin. Cells resistant to the antiproliferative action of heparin are able to sequester the heparin in large vacuoles whereas those cells sensitive to
the carbohydrate do not exhibit these structures. We have applied our approach to
QUANTITATIVE DIGITAL MICROSCOPY to the analysis of intracellular heparin
distribution.
labeled endosomes and skeletal components near the cell plasmn membrane.
1447
EVALUATION OF THE TOMCITY OF THE CANCER TREATMENT
DRUG CISPLATIN AT SINGLE CELL RESOLUTION BY ION
MICROSCOPY ((I. Gay, S. Chandra, and G.H. Morrison)) Department
of Chemistry, Cornell University, Ithaca, NY 14853 (Spon. by R.Wayne.)
1448
CHARACTER RECOGNrON OF RETINAL OUTGROWTH BASED ON FAST
FOURIER TRANSFORM (FF) OF DIITAL IMAGES. (N.G.Carri and E. GaNego
Uuesam). IMBICE and ClOp, Box 403,1900 La Plata, Argentina.
Cisplatin is used as a therapeutic drug for the treatment of various types of
cancers. One of the main problems with its use, however, has been the
evaluation of the drug toxicity. Ion microscopy provides a powerful
technique for the evaluation of the health status of cells. It is an imaging
image from a spatial context into an spatial frequency context. In the
Fourier spectrum of the images the explanted retina will appear as low
spatial frequencies while the fibres will be represented by high spatial
frequencies. An easily recognizeble image can be obtained from the FFT
spectrum. In others words what we have been working on is the study of
FFT of retinal images. The images were the outgrowth of chick refina E6
technique based on secondary ion mass spectrometry. It is capable of
localizing elements or molecules at a spatial resolution of approximately 0.5
im with a sensitivity reaching into the ppm range. It is possible to evaluate
toxicity by imaging the cellular K, Na, and Ca composition with ion
microscopy. LLC-PKi porcine kidney epithelial cells were treated with
various doses of cisplatin and then cryogenically prepared prior to ion
microscopic analysis. Injured cells characteristically showed high levels of
sodium and calcium and low levels of potassium, dependent on the cisplatin
dose given. Additionally, dead cells revealed higher levels of calcium in
their nuclei. We have also been able to detect the toxicity response in
correlation to various stages of the cell cycle by using bromodeoxyuridine
(BrdU) as a cell cycle marker which can be imaged independently and in
addition to K, Na, and Ca from the same cell. While other toxicity tests
only discriminate live versus dead cells, ion microscopy provides a
powerful approach for the evaluation of drug toxicity at the single cell level.
Supported by the Department of Energy and the National Institutes of
Health.
The use
of
FFT in digi
explanted in short-term
images give
assay.
us
the possibility to translate an
Retina were then cultured four days
on
collagn with the addition of trophic factors (TF) in the media. Retina
cultured under the effect of trophic factors gave dense outgrowth
elongating from the explant to the substrata. Controls show a discrete
outgrowth and easy to measure the length of fibers. However, it is
impossible to evaluate the pattem of the fibre network. This FFT pattern
gives as the possibility to evaluate the efficiency of TF. In this work we
have used digital images (phase contrast micoscopy) to characterize the
outgrowth in Fourier transformation. In the outgrowth network appears as
high spatial frequencies located in the periphery of the spectrum while the
explants are represented by of lower frequencies located centrally in the
spectrum. Our job is, by far, more simple, quicker and more accurate
than other techniques used for pattern recognition. The Optica Winner
Spectra will complete a more subtantial evaluation of the efficiency of
dosimetry on different trophics.
N.G. CA: E-ll CwdiMMK-MAI
d Fax:
4(21)253320
(IMBC) mi 1115 (COOp) CONICET,
Grwoe
S143141i411
P04D
A(149-15
Pre-College Science Education (1449-1450)
1449
1450
Precollege Science Education (CAPSE), University of Southern Califoria, Los
Angeles, CA 90033. (Spon. by H.C. Slavkin.)
ESTABLISHING
Foused, sategic, long-term commitment to profesional devlopment that
integrates science content, pedagogy and intuctional matrials results in a
coordinated, inquiry-basd schoolwide science intuctional program at twentyfour innerity elementary schools in Los Angeles. PRAXIS has worked with
twenty-four school teams of ree teachers and their principal for three years to
prepare the teams with sffiient scientific and leadership knowledge and skills to
make
significant gains
in siec
education
by: 1) Articlating K-6 science
education at eah school; 2) Providing on-site peer expert, resources and
trining; 3) Etlishing school-based instuctonal materials systems; 4)
Maximizing student exposure to hands-on sience; 5) Stimulating parent
involvement; and 6) Becoming models for otl schools School tams are
rained in life, phyical and earth sciences, and provded with a frmwwork for
schoolwide change in summer institutes and follow-up sessions. They acquire
in group dynamics, presentation statgies, leadership, lning styles and
resource acquisition in winter institute Sumn Institutes are
cted by 'Teaching Cadres' conssting of scientiss, secondary science
teachers and elementary currilum specialists who are oriented to the Califnia
Science Framework, St-adopted instrctiona mateials and e ional reform
philosophy and techniques. School teams receive salary poit credit for attending
insitutes, stipends for carrying out school-based change objectives, and are
igible to apply for grants to enhace thi
ools' science programs with a
skills
community
matching
inservicing and in
school
l
resouc
matels a
dedicated to teacher
tin.
planning,
Beging its third and final
year, schools in the PRAXIS powct
compledng the three projet objectives of
deveoping and implting a curriculum Content Matrix, organizing an
ins
tional mateials man
mt center and establishing an After-School
Stuet Science Club. Major funding for PRAXS is derived from the National
Science Foumdation, with added support from copate and family foundations.
are
AN
NRY
SCIENCE
AND
MATH
EDUCATION
PARTNER/MENTOR. PROGRA USING STUDENT AND FACULTY VOLUNTERS.
((R. L. DeHaan and F. H. Dabney)) Department of Anatomy and
Cell Biology, Emory University, Atlanta, GA 30322.
The Emory Elementary Science Education Partnership (ESEP)
Program provides elementary teachers with Emory faculty
mentors and student partners to help with hands-on teaching
of science. In the spring of 1994, we asked the principals
and teachers of four neighboring schools how the Emory
science community could be of help to them. Teachers asked
for contact with a working scientist and weekly in-classroom
help with active learning exercises from an undergraduate
science student. In response to questionnaires that we sent
to the Emory community and the schools, 47 faculty members
and 97 students volunteered to partner with 40 teachers. To
help define the goals and strategies of the program, we
established a steering committee that includes scientists,
educators, teachers, and school administrators, and a 14member student council. We have instituted a one semester
course that begins with three 3-hour training sessions to
familiarize partners and mentors with guided-discovery
teaching strategies and activities, and to sensitize them to
diverse learning modes. During the remainder of the semester
students devote 6 hours per week to in-school classroom teamteaching activities with their assigned teacher. Scientist
mentoring partners share information, donate spare equipment,
offer lectures and laboratory tours. Student journals,
statements, and pre-post testing are used for assessment.
250a
Pre-College Science Education (1451-1454). Monday
1451
1452
PRE-COLLEGE SaENCE MENTOR PROGRAM - AN EVOLVING
SaENCE EDUCATION PARTNERSHIP. ((S.H. Mayrand)) Science
Educadon Program, Worcester Foundation for ExperImental Biology,
Shrewsbury, MA 01545.
PRE-COLLEGE SCIENCE EDUCATION: RESEARCH AS AN
EDUCATIONAL TOOL ((M.C. Fields, 21st Century Biology Class)) The
Sidwell Friends School, 3825 Wisconsin Ave., NW, Washington D.C. 20016
Five years ago, In cooperadon with the iocal high school science teachers
and administrators, the Worcester Foundadon launched the Science Mentor
Program, which gives students the opportunity to meet two evenings a
month with sciendsts who engage them In hands-on projects In an Informial
setting. These encounters with scientists, and with the idnd of thIn1dng that
fuels the research process, helps students to appreciate the Importance of
standard laboratory
bask sciendfic principles In a way that textboox
exercises and lectures do not.
Science Mentors, recruited from the Worcester Foundation, local
blotechnology companies, insttutions of higher education and govenment
agencies, bring sinple experiments that allow students to acdvely explore
topics ranging from human genedcs to the environment. Through these
acMtides students begin to see the relevance of science to their daily lives
and, for some, to consider sclendfkc careers. Additionally, Informal
mentor/student Interactions often spawn In-depth discussions focusing on
Issues InvoMngsclence, ethics and soclety. The program has enjoyed much
success as evidenced by the duplication of our model by other
Massachusetts school systems, favorable attention by the local media, and
growing Institudonal and corporate support
,
1453
DEMONSTRATION OF BACIERIAL CELL TRANSFORMATION BY
ELEC[ROPORATION FOR THE HIGH SCHOOL SCIENCE
CURRICULUM ((R. A. Acey)) Dept. of Chenistry and Biochemistry, Cal.
State Univ., Long Beach, CA 90840, ((M. Ostresh)) BTX/Genetronics, Inc.,
San Diego, CA 92121 and ((T.F. Unger)) BioLogics, Inc., Irvine, CA 92718
The last sevemal years has seen a dramatic evolution in recombinant DNA
technology and its impact on society. A major goal of educators has been to
implement this technology int
the high school and cofl_ee un
aduate
science curiculunt We
here on a safe and elibe
nl
taches
that
the
tan
of
proceduem
bacterial cell
pnnciples
on and
n to in
provides students with hands on experience using ele
the
tasfioratio Students are supplied with a kit containing known amounts of
s, and pe
rd agar plates for
pre-aliquoted cells andplasmid, cell
of handlng. The HBlOl strain
with
ol
by
electr
ation using a IhnPnor Plus (BXGneonics). Int
n of
plasmid DNA imparts antibiotic restance to the transfomed cells. Aiquot of
untsunsrmed cells are plaed on agar with
without anpcillin to show that
fte oranisms are viable, yet sensitive to the antibiotic. Plabng of the
ee pwe
cells on agar cotiIng antibiotic reslts in cell growth,
donsaing the sucessl 'p-e of the plasmid by the bacteria.
Detrmn the number of drug resistant bace
colonies allows students to
calulte ta
ii
(transfiornants4pg DNA). Moreover, the
peCnage of oells tmafored is eaily calcuatd by compaison with an
equal number of cells not having undee procedure can
be coupleted in three consctie on
l ab periods without the need of
ease
or
I
sohistidi
equipment.
(Sponsored by R. D. Fields)
Motivating teen-agers while exposing them to the concepts and process of
scientific research is a challenging task. Our current system of lecture,
discussion and reading tends to present the teacher as the expert and often
fails to develop the creativity of students. The stanrd laboratory
investigations designed to fit into a ninety minute period while reiterating
facts do not encourage innovative thinking. Seniors at The Sidwell Friends
School have participated in an experimental course that discards the usual
method of teaching and replaces it with a research approach. Students
undertake a three-month laboratory investigation of neuron specific
enolase (NSE) in mouse neurons.
NSE is localized by
immunocytochemistry, the protein is identified by Western blot, and RNA
is extracted and reverse transcribed into cDNA for PCR amplification.
This exposes students to modern research techniques while promoting
critical thinking and problem solving that is student centered and teacher
facilitated. As a result, students gain an appreciation of scientific research
while they master key concepts from the core of cell biology, including
immunology, neuroscience, cytology, transcription, translation and DNA
replication. Students are no longer the passive recipients of knowledge,
but acquire it actively and through collaborative effort to complete the
research and produce a scientific poster. This poster presents the work
completed by students this past fall.
1454
"MEDICINES: THE INSIDE STORY" EDUCATION PROGRAM. AN
INTERDISCIPLINARY RESOURCE FOR HIGH SCHOOL SCIENCE
TEACHERS. (( T.M. Horn, J. Bennett, J. Berryman,
L.A. Pierce, and C. Raphael.)) Life Science and Biotechnology Laboratory, Thomas Jefferson High School for
Science and Technology, Alexandria, VA 22312. (Spon. by
R.A. Bloodgood.)
People all over the world use medicines to help restore
maintain health. Cellular molecules are the ultimate
targets of medicines. Thus, the concepts and methods of
inquiry of cell biology are invaluable to understanding
the actions of medications. This resource will enable high
school science teachers of biology, chemistry and health
professions, and their students, to follow in the footsteps
of the people who, through hard work, persistence, luck,
ingenuity, and prepared minds, discovered modern medicines.
The resource will contain labs, lessons, interviews, images
and cultural and historical documents. Teachers will be able
to access a variety of ways to model techniques and investioative approaches used by scientists. Information available
will include origin, identification, structure, and modes of
action and side effects of medicines familiar to high school
students. These classroom materials are being developed for
use as a CD-ROM. The education program is one facet of
Medicines: The Inside Story, a traveling museum exhibition,
symposium, and planetarium show sponsored by Glaxo Inc.
or
Symposium VI: Molecular Machinery of the Secretory Pathway (1455-1456). Tuesday
1455
MOLECULAR MECHANISMS OF INTRACELLULAR PROTEIN TRANSPORT.
((Jaaes E.
Rothaan)) Cellular Biocheaistry and Biophysics Program orial SloanKettering Cancer Center, 1275 York Avenue, New York, New York.
Reconstitution of intracellular protein transport in cell-free systems has
led to insights into the mechanisms responsible for vectorial transport by
vesicles. Transport between Golgi cisterna is iediated by COP-coated
vesicles whose principal subunits are complex of Coatomers and a small GTPbinding protein, ADP-Ribosylation Factor (ARF). Coatomer is composed of
seven distinct subunits. The concomitant budding of the transport vesicle
occurs In a series of steps. GTP-dependent binding of ARF to the membrane in
a
nucleotide exchange reaction is followed by binding of coatomer and
assembly into buds. Fatty-acyl-CoA is required for the buds to pinch off.
Coated vesicles then uncoat when the ARF-bound CTP is hydrolyzed. Uncoating
exposes a vesicle-born targetting molecule (termed v-SNARE) that docks the
vesicle as it pairs with its cognate t-SNARE at the target membrane. The
general fusion machinery, involving NSF and SNAP proteins, then assembles on
the SNARE complex and fusion is initiated upon ATP hydrolysis by NSF. This
mechanism is of great generality, as yeast temperature-sensitive mutants
defective In transport and fusion encode homologues of costomer and fusion
components discovered in animal cells, and similar components operate in
neurons at synapses.
carrier
1456
COMMUNICATION BETWEEN THE ER AND THE CELL NUCLEUS:
MONITORING PROTEIN FOLDING. ((M.-J. Gethingl.2. K. Moni2.4, W.
Ma2, and J.F. Sambrookl.3)) lDepartment of Biochemistry, 2Howard Hughes
Medical Institute, and 3McDermott Center, UT Southwestern Medical Center,
Dallas, TX 75235, and 4HSP Research Institute, Kyoto 600, Japan.
a
BiP, the endoplasmic reticulum (ER)-located member of the hsp7O family of
molecular chaperones, is required both for translocation of nascent proteins
across the ER membrane and for their subsequent folding and assembly in the
ER lumen. Transcription of the BiP gene is increased many fold when mutant
or unfolded proteins accumulate within the ER. This unfolded protein response
appears to be initiated by the decrease in the concentration of free BiP that occurs
when complexes are formed between BiP and unfolded proteins. In the yeast
Saccharomyces cerevisiae, BiP is encoded by the essential nuclear K4R2 gene.
Thus a novel intracellular sensing system must exist that monitors events in the
lumen of the ER and transduces signals across the ER membrane and to the
nucleus. To identify proteins involved in this signaling pathway, we devised a
genetic screen to isolate yeast mutants that are unable to activate the BiP gene
when unfolded proteins are present in the ER. Several of these mutants lie in a
gene (ERNIV) encoding a 11 15 amino acid polypeptide with an N-terminal signal
sequence and a single-transmembrane spanning domain approximately halfway
along its length. Ernlp is a type I integral membrane protein with its
glycosylated N-terminal domain in the lumen of the ER and its C-terminal
domain in the cytosol. Part of the cytosolic domain is homologous to the
catalytic domains of serine/threonine-specific protein kinases. Phosphorylation
by the Ernlp kinase could therefore be the first step in transcriptional activation
of KAR2 and other genes which contain UPR elements and encode proteins
resident in the ER that are required for protein folding.