Technical Journal Club: Intrabodies to Visualize Endogenous Proteins

Technical Journal Club:
Intrabodies to Visualize Endogenous
Proteins
Agnes Lau
Aguzzi Lab
Visualizing Proteins
1. Immunocytochemistry
-cells must be fixed and permeabilized
2. Fusion proteins (ie to GFP)
-can visualize proteins in a cell if sparsely distributed
-improper localization
-overexpression may have morphological or functional effects on cells
3. Intrabodies
Recombinant antibody-like proteins
Neuron June 2013
Science 2003
Activation of small GTPases is accompanied by a conformational change
Goal of this paper:
Design GTPase conformation sensors that do not interfere with function
Phage display
phage – viruses that infect bacterial cells
Foreign gene spliced into the gene for a coat protein of the virus
1. Library and make phage
population
2. Select specific phages,
usually affinity
chromatography
3. Wash and Elute
4. Infect bacteria to amplify
more phage
5. More rounds of selection
6. Obtain clones of phages
and determine DNA
sequence to identify
peptide
Phage display
Advantages:
• can screen 109 clones
• size of the protein to express does not appear to matter (41kDa)
• can display multi-subunit proteins
Disadvantages:
• diversity limited by ability to transfect the initial library into bacteria
Nizak et al Science 2003
1. scFv library
2. Phage display
-used a GTP-locked mutant of Rab6A tagged with biotin as selection
-3 rounds of selection
3. Selected 80 clones from E.coli randomly and induced scFv production from these
clones
4. Clones were analyzed by immunofluorescence for Golgi complex staining using
HeLa cells – 37 were positive
5. Sequencing of the strongest 19 clones revealed two unique antibodies: AA2 and
AH12
Nizak et al Science 2003
• AA2 antibody stains the Golgi (colocalizes with GalT (galactosyl transferase) in
the Golgi)
• AA2 antibody colocalizes with Rab6, even at peripheral sites (arrows)
Confirming AA2 antibody specificity
• Two spliced variants of Rab6:
Rab6A and Rab6A’
• GFP-tagged versions transfected
into HeLa cells
• AA2 detected both
• HeLa cells transfected with siRNA against
Rab6A and Rab6A’
• AA2 staining abolished, along with Rab6
Confirming AA2 antibody specificity
• Column of GST-Rab6A beads loaded
with GDP or GTPγS and incubated with
AA2 and brain homogenate containing
β GDI
• AA2 preferentially binds Rab6A-GTP
• Column of GST-Rab6A or GST-Rab11
beads loaded with GDP or GTPγS and
incubated with AA2 and Rab6IP1
• AA2 preferentially binds Rab6A-GTP
Confirming AA2 antibody specificity
IP of transfected HeLa cell lysates using
AA2 preferentially isolated GTP-locked
mutant
Rab6AQ72L – GTP-locked mutant
Rab6AT27N - GDP-locked mutant
Transfected HeLa cells identified
by GFP are outlined
Cells were stained with AA2 and
Rab6 antibody. AA2 only
recognizes GTP locked mutant
Q27L, whereas Rab6 recognizes all
Rab6A Q72L – GTP-locked mutant
Rab6A T27N - GDP-locked mutant
AA2 antibody in cells
Movie S2
Rab6 fused to CFP compared with AA2 fused to YFP
when co-expressed in HeLa cells
Nizak et al Conclusions
Recombinant antibody approach allows for rapid selection of scFvs as sensors for
a specific conformation of a protein.
Method will help design tools to identify the presence of pathological
conformations and detect/track/separate protein conformers
Next Generation of Intrabodies
Limitations of using scFvs:
I) Hard to work with due to instability
II) Low yields in bacterial expression systems
III) Problems folding properly in the reducing environment of cytoplasm
Non-immunoglobulin scaffolds :
I) Lack disulfide bonds
II) Can be expressed efficiently in bacteria and mammalian cells
III) Have improved stability
Fibronectin Type III domain (FN3)
Koide et al 1998
• characteristic of a subfamily (FN3 family) of the immunoglobulin superfamily
• ubiquitous domain – approximately 2% of all animal proteins
• FN3 family includes cell adhesion molecules, cell surface hormones receptors,
chaperonins, etc.
• FN3 is 94 residues and monomeric
• one of few members of immunoglobulin superfamily that does not have disulfide bonds
Karatan et al 2004
• FN3 domain has 7 anti-parallel β-strands
connected by 3 loops
• 5 of the 6 loops shown to tolerate insertions
of residues without destabilization,
indicating their usefulness as sites for
grafting/randomization of residues
Karatan et al 2004
• Randomized 5 residues on two loops of FN3 domain
• Phage display to select for binders of the SH3 domain of c-Src
• Resulting clone could be used for IPs
Conclusion: Successful isolation of affinity reagent for SH3 domain based on the
FN3 scaffold
Neuron June 2013
mRNA display
Terstappen et al 2007
1.
2.
3.
4.
DNA library
Synthesize mRNA pool
Synthesize 3’-puromycin oligonucleotides
Ligate mRNA and 3’-puromycin
oligonucleotides
5. In vitro translation
-rabbit reticulocyte lysate
6. Formation of RNA-protein fusion
7. Isolation of RNA-protein fusions
8. Generation of cDNA/mRNA-fusion
protein
9. Selection of functional fusions
10. PCR to identify
Synthesis of puromycin oligonucleotides
Liu et al 2003
Why puromycin?
Antibiotic that mimics aminoacyl end of tRNA so acts as translation
inhibitor when enters ribosome
mRNA display
Terstappen et al 2007
1.
2.
3.
4.
DNA library
Synthesize mRNA pool
Synthesize 3’-puromycin oligonucleotides
Ligate mRNA and 3’-puromycin
oligonucleotides
5. In vitro translation
-rabbit reticulocyte lysate??
6. Formation of RNA-protein fusion
7. Isolation of RNA-protein fusions
8. Generation of cDNA/mRNA-fusion
protein
9. Selection of functional fusions
10. PCR to identify
mRNA display
• Protein is covalently attached to the mRNA that encodes it
Roberts RW 1997 PNAS
mRNA display
Terstappen et al 2007
1.
2.
3.
4.
DNA library
Synthesize mRNA pool
Synthesize 3’-puromycin oligonucleotides
Ligate mRNA and 3’-puromycin
oligonucleotides
5. In vitro translation
-rabbit reticulocyte lysate??
6. Formation of RNA-protein fusion
7. Isolation of RNA-protein fusions
8. Generation of cDNA/mRNA-fusion
protein
9. Selection of functional fusions
10. PCR to identify
mRNA display
Advantages:
• entirely in vitro
• greater diversity - start with libraries of 1012 to 1014
• peptides and proteins synthesized as fusions commonly retain binding
properties of unfused polypeptides
• proteins ranging in size 1-30kDa can be synthesized as fusions
Disadvantages:
• limited by efficiency of translation reaction and efficiency of fusion
formation
• fusion efficiency decreased for larger proteins
Gross et al 2013
Intrabodies have not been used for imaging protein localization and
expression thus far.
• freely diffusable intrabodies in the cytoplasm will overwhelm the image
Expression of intrabodies should be the same or lower than the target protein
To solve the above problems, Gross et al have developed FingRs
(Fibronectin INtrabody Generated by mRNA display)
• intrabody that is transcriptionally regulated by the target protein and is
based on the FN3 scaffold
Gross et al 2013
Goal: Create reagents to label excitatory and inhibitory synapses in live neurons
Targets:
PSD-95 (post-synpatic density 95)
• marker of excitatory postsynaptic sites
• specifically targeted SH3-GK domain that mediates intra- and intermolecular
interactions
Gephyrin
• marker of inhibitory postsynaptic regions
• specifically targeted the G domain that mediates trimerization
Gross et al 2013
• mRNA display
• Library of >1012 proteins with randomization of 17 residues in the BC and FG
loops
• 6 rounds of selection
Choose FingRs with best intracellular labeling
COS cell assay
• Target localized to cytoplasmic face of Golgi apparatus by adding a Golgi-targeting
sequence
• Target is also fused to streptavidin and labeled with biotin-tagged rhodamine
Winner – tight cellular colocalization (ended up being 10-20% of clones)
Loser – diffuse staining, poor expression, or poor localization
Do FingRs label endogenous targets?
GFP-tagged FingRs from COS cell assay expressed in cortical neurons
Looking for punctate pattern of expression
Confirm interaction with endogenous target
GFP
Endogenous
antibody
protein
GFP-tagged
FingR
• cDNAs for each FingR incorporated into lentivirus that were used to infect cortical
neurons
• After 96 hours, cell lysates collected and exposed to anti-GFP antibody beads
• Isolated protein complexes analyzed by western
Binding of FingR does not disrupt functional interactions
PSD95
HA
antibody
PSD95.FingR GKAP-HA
GK domain of PSD95 (part of the initial target used for FingR selection) binds
GKAP (guanylate kinase-associated protein)
Interaction of PSD95.FingR with PSD95 does not interfere with binding between
PSD95 and GKAP
Excess FingR results in background signal
Overexpressed GFP-tagged FingRs in cortical neurons
Transcriptional Control of FingR levels
• Transcription repressor KRAB(A)
fused to ZINC-finger DNA-binding
domain, which is fused to FingR-GFP
• Zinc finger binding sites inserted into
DNA upstream of promoter controlling
FingR
If all targets are bound, excess FingR
moves to the nucleus and turns off
transcription via ZINC-finger binding
and KRAB(A)
Transcriptional regulation decreases background signal
Quantified ratio of
amount of FingR at nonsynaptic sites vs postsynaptic sites (Rn/s)
FingR levels in response to induction of target protein
GPHN-mKate: inducible by ecdysone
Induction of GPHN-mKate results in 110% increase in total GPHF, which is mirrored by
GPHN-FingR
FingR levels in response to inhibition of target protein
FingR levels in response to inhibition of target protein
Simultaneous use of different FingRs
• GPHN.FingR-mKate and PSD95.FingR-GFP
simultaneously expressed in cortical neurons
• GPHN.FingR-mKate fused to IL2RG2L zinc
finger
• PSD95.FingR-GFP fused to CCR5L zinc
finger
• Both expressed in punctate manner with little
overlap.
Use of FingRs in living cells
GPHN.FingR-GFP expressed in cortical neurons using a lentivirus
Visualize trafficking of Gephyrin
Movie
Specificity of FingR-binding
• Three close homologs of PSD-95 exist in the CNS that are also found at postsynaptic
sites: PSD-93, SAP-97, SAP-102
• Used COS cell assay
Specificity of FingR-binding
More stringent assay:
HA-tagged versions of SAP-102 and SAP-97 expressed in cortical neurons
where PSD-95 was knocked down with siRNA
Use of FingR in organotypic rat hippocampal slices
Slices from rats 8 days, transfected 2-3 days later and incubated for 7-8 days
Imaged live with two photon microscopy
PSD-95 concentrated in
dendritic spines
GPHN found in puncta on
dendritic shaft
Does FingR affect cell morphology or function?
Morphology of neurons was not
changed
Spine density did not differ
Measured spontaneous miniature
excitatory postsynaptic or inhibitory
currents for PSD95.FingR and
GPHF.FingR transfected neurons,
respectively
Use of FingRs in vivo
Transfected PSD95.FingRGFP into neurons of mouse
embryos using in utero
electroporation
Expression was examined at
7 weeks of age from
sections of perfused and
fixed brains.
Image E is from a live
animal through a cranial
window
Conclusions
Fibronectin intrabodies generated with mRNA display can be used to visualize
the localization of endogenous proteins in cell culture, organotypic slices and in
vivo.
FingRs do not appear to cause morphological or functional changes in neurons.
However, each new FingR generated must be examined for target specificity,
affinity, disruption of target function, disruption of functional interaction with
other proteins, success of transcriptional regulation, etc.
Thanks for your attention!!