advanced uses of fluorescent proteins in the investigation of cells

advanced uses of fluorescent proteins in the investigation of cells

advanced uses of fluorescent proteins
in the investigation of cells
Principles of Fluorescence Techniques & CONFOCAL 9
Fred S. Wouters
Genoa, June 25-29, 2007
CBG
Gottingen
Molecular neurophysiology
the Burgess nonsense book
being a complete collection of the humerous masterpieces of
Frank Gelett Burgess, Esq.
(published by Frederick A. Stokes Company, New York (1901)
Fluorescent Proteins
origins
colors
properties
labeling
problems
sensing
manipulating
multiplexing
reactive
oxygen
species
timer
dynamic
marking
FRETbased
conditional
properties
CALI
photobleaching
FRAP
FLIP
FLAP
photoconversion
complex
assays
reversible
photoswitching
pairs
resolft
pH, redox
chaperone
selective
killing
combination
fluorescent proteins
β-can structure
• rel. insensitive to environment
• proteolytically stable
• denaturation stable
• N- & C-terminus free
allow fusion
chromophore: aa 65-66-67
• post-translational oxidation
• single gene
• no co-factors required
Rainbow
Miyawaki (2005) Neuron 48: 189-199
Verkhusha (2004) Nat. Biotechnol. 22: 289-296
popularity of GFP
GFP OR "fluorescent protein"
GFP OR "fluorescent protein"
4000
4000
3000
3000
2000
2000
1000
1000
0
0
1990
1992
1994
1996
1998
2000
2002
microscopy
12
2004
microscopy
14
3
16
18
x10
increase in microscopy by GFP
0.7
x 1000
18
0.6
0.5
16
0.4
3
x10
0.3
14
0.2
12
0.1
0.0
1990
1992
1994
1996
1998
2000
2002
2004
1990
1992
1994
1996
1998
2000
2002
2004
Wouters (2006) Contemporary Physics 47: 239-255
Fluoraissance
and dynamics
Doing more with less colors
GFP+YFP
lifetime
YFP5
GFP
NA
NLS
Pepperkok (1999) Curr. Biol. 9: 269-272
NLS
B1
NLS
PDGFR
NA: N-acetylglucosaminyltransferase (Golgi+ER)
B1: Cyclin B1 (cytosol, microtubules)
Lifetime contrasting
CFP
SYFP2-SCFP3A
YFP
fusion
LT
single
2.0
2.7
phase lifetime (ns)
3A
3A
1A
1A
MIX
1.5
CFP
LT
1A
QYFRET = (1-E) • Q non-FRET
2.6
phase lifetime (ns)
SCFP3A-NES (τΦ=2.6 ns)
SCFP1A-NLS (τΦ=1.5 ns, QY=0.7)
Kremers (2006) Biochemistry 45: 6570-6580
QY=
τ obs
τ rad
changing lifetime by FRET
mutational screening for
changed lifetime
0
Lifetime (ns)
5
Lifetim
5
0
48
0 Brightness (a.u.) MAX
ess
B
e
Brightn
we
100
0
48
ll #
A
we
32
ll #
well #
32
5
1 1
well #
Lifetime (ns)
C
1 1
#
1. 2.634±0.002 ns (1%)
2. 3.743±0.003 ns (1%)
3. 1.380±0.002 ns (2%)
4. 1.548±0.002 ns (2%)
#
5. 1.794±0.002 ns (2%)
6. 2.212±0.002 ns (1%)
7. 2.868±0.005 ns (2%)
8. 4.225±0.006 ns (2%)
1 2 3 4 5 6 7 8
Sample (#)
0
8 groups of 96 wells:
EGFP, R6G+EGFP, R6G+KI (63, 50, 38, 25, 13,0 mM) 20 min.
A
C
14cm
B
D
cytoplasmic
0
E
Lifetime (ns)
5
0 Brightness (a.u.) MAX
9cm
nucleolar
targeting signal
Esposito A & Wouters FS (2004) Fluorescence Lifetime Imaging (Unit 4.14).
In Current Protocols in Cell Biology. J. Wiley & Sons, NY, USA.
±20,000 colonies of E. coli expressing EYFP on a 9x14 cm agar
plate. 1900 FOV, ±1 h.
Photobleaching tools: synchronizing populations
Diffusion
rates
immobile
fraction
velocity
Diffusion
continuities
Lippincott-Schwartz (2003) Science. 300: 87-91
aggregation of structurally-optimized tau
Hyper-aggregating tau mutant was created by
optimization of internal oligomerization sequences
±1/4 cells expressing
hyper-aggregating tau
exhibit WT tau immobilization
Iliev (2006) J. Biol. Chem. 281(48): 37195-204.
Fluorescence Localization After Photobleaching
rapid actin transport during cell protrusion
CFP-actin
FLAP
(CFP-YFP)
rFLAP
YFP-actin
phase
contrast
1-
YFP
CFP
protrusion
retraction
Zicha (2003) Science 300: 142-145
Cpre
Ypre
Cpost
Ypost
mix
mix
Directional transport of Synaptophysin-FLAP
Iliev (2007) J. Neurosci. Methods Nov 21 Epub ahead of print
photoconversion
GFP bleaching during observation under anearobic
conditions produces a red-emitting form
Elowitz (1997) Curr. Biol. 7: 809-812
GFP photoconversion put to (limited) use
Brunet (2006) Traffic. 7: 1283-1289
exc 514
exc 458
exc. 405
exc 514
∆F
post-bleach
pre-bleach
exc. 405
photoconversion of YFP:
bleaching of YFP leads to the
formation of a CFP-like photoproduct
432.5
452.5
472.5
492.5
512.5
532.5
552.5
572.5
592.5
Gertrude Bunt (Stuttgart University) and
Valentin (2005) Nat. Meth. 2: 801.
Photoconversion: another lucky observation
Kaede
Ando (2002) PNAS 99: 12651-12656
dsRed maturation mutant: timer
Drosophila neural fiber bundle formation
Verkushka (2001) J. Biol. Chem. 276: 29621-29624
expression in C. elegans
Terskikh (2000) Science 290: 1585-1588
Photoactivation of GFP
Lippincott-Schwartz (2003) Science. 300: 87-91
Patterson (2002) Science. 297: 1873-1877
photoactivatable mRFP
allows highly complex
multi-color dynamic
bioassays
Verkusha (2006) Chem. Biol. 12: 279-285
EYFP-Dopamine transporter (plasma
membrane) and PA-mRFP-Rab5
(endosomal surface)
photolabeling
photoconversion
photoactivation
photochromism
photoswitching
Dronpa
photoswitching
Ando (2004) Science 306: 1370-1373
and FRET
wtGFP
ratio Phluorin
ecliptic Phluorin
Miesenböck (1998) Nature 394:192-195
protonation sensing
sensing redox potentials
barrel destabilization
Hanson (2004) J. Biol. Chem. 279: 13044-13053
Dooley (2004) J. Biol. Chem. 279: 22284-22293
chaperone folding biosensor
chaperone
bacterial
phenotype
cdYFP fluorescence (AU)
cold ethanol shock in bact. culture
4
10
2
4
4
10
2
3
4
2
500
520
Liman (2005) Mol. Cell Biol. 25: 3715-3725
560
580
emission wavelength (nm)
-3
x10
expression in Cos cells
12
37 deg.
10
frequency
540
8
6
25 deg.
4
4
2
(AU)
0
0
2
4
6
cdYFP fluorescence (AU)
8
0
600
the folding biosensor is sensitive for Hsp70
-3
x10
Folding efficiency (cdYFP/Cy5)
WT CHO cells
80
60
40
Hsp70 co-expression
20
8
6
4
2
0
0
0
2
4
6
Folding efficiency (cdYFP/Cy5)
8
0
10
20
30
40
CFP-Hsp70 (AU)
50
5
(fold)
frequency
10
0
WT- CHO
CHO
transient overexpression of
Hsp70 chaperone
Cellular folding capacity in cellular ALS models
folding efficiency (fold)
Förster Resonance Energy Transfer (FRET)
FRET
Methods
ratio/ SE imaging
Acceptor PB
FLIM
intensities
"quenched" "sensitized"
donor
acceptor
emission
emission
photons are
emitted sooner
Molecular microscopy in modern biology
Pubmed
keyword hits
(per year)
GFP OR "fluorescent protein"
FRET
700
4000
600
Curr Biol
9: 1127-30
500
3000
EMBO J.
17: 7179-89
400
2000
300
200
1000
100
0
1990
1992
1994
1996
1998
2000
Localization
Dynamics
2002
2004
1990
1992
1994
1996
1998
2000
2002
2004
+ Biological function
= Cellular physiology
FRET-based physiological sensing
"actuator"
intermolecular
"sensor"
intramolecular
more FRET
no FRET
conformation
no FRET
FRET
interaction
proteolysis
FRET examples
Control
FRET
Lifetime
Distribution
1.0
Lifetime
1.0
0.6
0.6
0.4
0.4
0.2
0.2
0.0
0.0
2.2
2.3
2.4
2.5
2.7
2.6
Lifetime (ns)
Overlay
C
Distribution
D
Colocalization
100%
Lifetimes
E
α-Syn
0%
1.1
1.2
1.3
1.4
1.5
1.6 1.7
Lifetime (ns)
1.4
favorite FRET test subject: polystyrene beads, covalently
with GFP protein, chemically labeled with Cy3
α-Syn/Tau
FRET resolution translates to sub-nanometre resolution!!
Wouters (2006) Contemporary Physics 47: 239-255
see also: Bunt (2004) Int. Rev. Cytol. 237: 205-277
Esposito (2007) Neurobiol. Disease, in press
Lifetime distributions (ns)
0.8
EYFP-Tau
2.5
0.8
Cerulean-αSynuclein
A
B
Spectral Issues with FRET
100
1.2
60
1.0
0.8
0.6
-1
-1
ε (mM cm )
80
40
DE
SO
0.4
BT
20
0.2
0
0.0
300
350
400
450
500
wavelength (nm)
550
600
650
Relative emission intensity
CFP Exc.
CFP Em.
YFP Exc.
YFP Em.
FRET-pairs
• high overlap donor emission - acceptor excitation spectra
• low overlap donor emission - acceptor emission (high Stoke"s shift)
• high molar extinction coefficient acceptor
• high fluorescence quantum yield donor
• high photostability acceptor
• donor lifetime > acceptor lifetime
• NO dimerization!
Bunt G & Wouters FS (2004) Internat. Rev. Cytology, 237, 205-277
Esposito A & Wouters FS (2006) Fluorescence lifetime imaging:
Quality assurance and standards.
In Methods and Applications of Fluorescence, Springer Verlag (in press)
FRET-pairs
• high overlap donor emission - acceptor excitation spectra
• low overlap donor emission - acceptor emission (high Stoke"s shift)
• high molar extinction coefficient acceptor
• high fluorescence quantum yield donor
• high photostability acceptor
• donor lifetime > acceptor lifetime
• NO dimerization!
die Eierlegende Wollmilchsau!
(the egg-laying woolly milk-pig)
Bunt G & Wouters FS (2004) Internat. Rev. Cytology, 237, 205-277
Esposito A & Wouters FS (2006) Fluorescence lifetime imaging:
Quality assurance and standards.
In Methods and Applications of Fluorescence, Springer Verlag (in press)
chromoprotein FRET acceptor
1.0
EYFP
REACh 0.5
REACh 2
REACh 1
Emission
0.8
0.6
0.4
0.2
0.0
500
520
540
560
580
600
wavelength (nm)
GFP-PEST + Ubq-REACh1
GFP
τ
YFP REACh
Ganesan (2006) PNAS 103: 4089-4094
1.5
2.4 (ns)
490df30/530df35
540df10/580df25
546/488 ratio
QSY-gelsolin
Alexa-488 Actin Rhodamine-Actin
NO FRET
reference
FRET
emission quenched
Gelsolin actin uncapping at ruffles
Allen P.G. Nat. Cell Biol. 5, 972-979 (2003)
Intensity-based FRET using a dark acceptor
by application of a concentration reference dye
Exc
Exc
Em
Em
Exc
Exc
FRET
Em
GFP/Cy3 Ratio
Em
Emission Ratio reduced
GFP bandpass
Lifetime
2.6
(ns)
0.5
GFP/Cy5
Protein
ubiquitination
by lifetime
(FLIM)
and
normalised
donor quenching
(FqRET)
HA-PEST-GFP
GFP
PEST-GFP
REACh-Ubq
Ubq
no filter
Lifetime
REACh-Ubq
HA-PEST-GFP
low
GFP/Cy5
(AU)
high
Why have a dark acceptor?
1: spectral advantages
conventional
collect all fluorescence
photons
increase spectral
overlap: bigger R0
Why have a dark acceptor?
2: multiplexing FRET pairs
1
conventional: 1 FRET pair
CFP-YFP
1
2
2 FRET pair with
dark acceptors
acc 1
acc 2
Why have a dark acceptor?
3: no frustration of FRET (no build-up of FRET-incompetent DA species,
AND no contamination of FRET donor lifetime with non-FRET donor decay lifetime)
A
D
A
D
kFRET
kFRET
!
A
D
!
Acc needs
to depopulate
(emit), takes a
lifetime:
refractory for FRET
kFRET
donor emission
25 ns
5 ns
1 ns
200 ps
intensity FRET: Sens. A. em / D. em
= saturation
for each
saturation
level
each color
is a saturation
level
200 ps
1 ns
5 ns
25 ns
ideal case
ideal case
Build-up of the FRET-incompetent DA state causes :
• the emission of photons with normal, unreduced, lifetime
this has consequence for FLIM
• the emission of photons that should have been quenched by FRET
this has consequence for FLIM, but more so for intensity-based ratio methods
because the ratio is highly unlinear and the error thus "explodes"
this effect increases with acceptor lifetime and saturation-level of the donor!
Why have a dark acceptor?
4: no sensitized acceptor photobleaching therefore
no loss of FRET and persistent protection of donor
for photobleaching
A
D
kFRET
kFRET
D
x
This is the end of
the road.
Acceptor finito
A
D
a
poof!
The problem of sensitized acceptor photobleaching in FRET
photostability ratio
donor/acceptor
FRET efficiency
GFP-labeled beads incubated with increasing concentrations
of Rhodamin-6G leads to concentration-dependent FRET as
the average distance between molecules decreases
0%
100
10
1
Donor
emission
25%
50%
75%
0.1
0
0
sensitized
Acceptor
emission
99%
25
50
75
100
time (sec)
125
150
175
0
25
50
75
100
time (sec)
125
150
175
150
175
99%
75%
50%
25%
0%
0 (=donor decay)
0.1
1
10
100
0
25
50
75
100
time (sec)
125
150
175
0
25
50
75
100
time (sec)
125
and Other
even more scary: Killer Red FP
CALI
selective killing
• genetically-encoded
photosensitizer
• generates singlet oxygen ROS
mechanism ?
*
*
Bulina (2005) Nat. Biotechnol. 24: 95-99
killer-red Mito ±1 h.
Turbo-GFP cytoplasma
GFP and synthetic fluorophores
Integrin
Qdot
CFP
Fluorescein
Wouters (2006) Contemporary Physics 47: 239-255
bioorthogonal chemical labeling
GFP-APP, red Qdot-labeled sAPP
GFP-FAK
Cy3-ACP labeled APP
(G. Bunt, Stuttgart University)
REAsH-FAK
GFP-EGFR, Qdot-EGF orange and blue
Qdots followed each other by 20 min.
reviews: Prescher (2005) Nat. Chem. Biol. 1: 13-21
Ting (2005) Curr. Opin. Biotechnol. 16: 35-40
Miller (2005) Curr. Opin. Chem. Biol. 9: 56-61
Qdot-EGF during actin-driven retrograde filopodial transport
Arndt-Jovin (2003) Proc. SPIE 6096: 60960
fred.wouters@gwdg.de
FRET
upon photobleaching
c
n
c
n
even the "monomerized" forms aggregate
when expressed
in transgenic mice
as compared to
their transient expression
or their Aequoria
counterparts
Hirrlinger (2005) Mol. Cell. Neurosci. 30:291-303