technical journal club: intrabodies to visualize endogenous...

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

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 – viruses that infect bacterial cells Foreign gene spliced into the gene for a coat protein of the virus

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

Movie S2 Rab6 fused to CFP compared with AA2 fused to YFP when co-expressed in HeLa cells

AA2 antibody in 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

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

Next Generation of Intrabodies

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

1. DNA library 2. Synthesize mRNA pool 3. Synthesize 3’-puromycin oligonucleotides 4. Ligate mRNA and 3’-puromycin

oligonucleotides 5. In vitro translation -rabbit reticulocyte lysate

mRNA display

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

Terstappen et al 2007

Synthesis of puromycin oligonucleotides

Antibiotic that mimics aminoacyl end of tRNA so acts as translation inhibitor when enters ribosome

Why puromycin?

Liu et al 2003

1. DNA library 2. Synthesize mRNA pool 3. Synthesize 3’-puromycin oligonucleotides 4. Ligate mRNA and 3’-puromycin

oligonucleotides 5. In vitro translation -rabbit reticulocyte lysate??

mRNA display

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

Terstappen et al 2007

mRNA display

• Protein is covalently attached to the mRNA that encodes it

Roberts RW 1997 PNAS

1. DNA library 2. Synthesize mRNA pool 3. Synthesize 3’-puromycin oligonucleotides 4. Ligate mRNA and 3’-puromycin

oligonucleotides 5. In vitro translation -rabbit reticulocyte lysate??

mRNA display

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

Terstappen et al 2007

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

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

Gross et al 2013

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

• 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

GFP antibody

GFP-tagged FingR

Endogenous protein

Binding of FingR does not disrupt functional interactions

HA antibody

GKAP-HA

PSD95

PSD95.FingR

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 non-synaptic sites vs post-synaptic 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.FingR-GFP 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!!