Visualisering af protein-protein interaktion i nukleare og Cytoplasmiske Brøker af Co-immunopræcipitation og<em> In Situ</em> Proximity ligeringsassayet
Summary
Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.
Abstract
Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.
Introduction
Nuklear faktor 90 (NF90) er en multi-isoform protein med mange funktioner, herunder respons på virusinfektion, regulering af interleukin-2 post-transskription og regulering af miRNA biogenese 1-3. Rbm3 er et RNA-bindende protein, der er involveret i oversættelse og miRNA biogenese og kan induceres af forskellige faktorer, heriblandt hypotermi og hypoxi 4-6. For nylig fandt vi NF90 og Rbm3 i et proteinkompleks 7. Interaktionen af NF90 og Rbm3 er vigtigt at modulere protein kinase RNA-lignende endoplasmatisk reticulum kinase (PERK) aktivitet i udfoldet protein reaktion 7. Både NF90 og Rbm3 ligger overvejende i kernen, men en lille del af NF90 og Rbm3 shuttle ind i cytoplasmaet og binder der for hinanden for specifikke funktioner, f.eks at regulere PERK aktivitet. Derfor er det vigtigt at visualisere fordelingen af NF90-Rbm3 samspil med subcellulært afsnit, hvilket kan tydederes forskellige roller i respektive rum.
Årtier siden blev gær to-hybrid (Y2H) udviklet for at detektere interaktion mellem to proteiner 8. Men på grund af kunstig konstruktion af fusionerede proteiner, har falske positive resultater begrænset anvendelsen af denne metode. I lang tid, co-immunpræcipitering var den vigtigste teknik til at analysere protein-protein interaktioner, især i endogene betingelser 9. For at analysere co-immunpræcipiteret proteinkompleks, Western blot er den mest bekvemme metode, mens massespektrometri anvendes når super følsomhed og nøjagtighed ønskes. I de senere år har nærhed ligeringsassayet blevet udviklet som en ny fremgangsmåde til påvisning af protein-protein interaktioner i begge celler og væv in situ 10,11.
Her har vi sammenlignet den mest populære co-immunopræcipitering fremgangsmåde og relativt ny nærhed ligering assaymetode fange NF90-Rbm3 interaktion i subcellulære fraktioner. Vi drøftede også de fordele og begrænsninger ved begge teknikker.
Protocol
Representative Results
Discussion
Der er flere fordele såvel som mangler for begge metoder. Som en relativt ny teknik, en indlysende fordel ved nærhed ligeringsassayet er muligheden for at belyse protein-protein interaktioner på enkeltcelle-niveau i stedet for en batch af heterogene celler. Billeder med høj størrelsesorden og opløsning (fx ved konfokal mikroskop) giver mulighed for kvantificering ved at tælle enkelte fluorescerende pletter. I modsætning hertil kan den konventionelle kombination af co-immunoudfældning teknik med Western…
Declarações
The authors have nothing to disclose.
Acknowledgements
This study was supported by the Swiss National Science Foundation (SNSF, 31003A_163305).
Materials
| Dulbecco's Modified Eagle’s Medium (DMEM) | Sigma | D6429 | High glucose 4500 mg/L |
| Fetal bovine serum (FBS) | Gibco, Thermo Fisher Scientific | 10270106 | |
| Penicillin-Streptomycin (PenStrep) | BioConcept | 4-01F00-H | |
| NE-PER Nuclear and Cytoplasmic Extraction Reagents | Thermo Fisher Scientific | 78833 | |
| 1,4-Dithiothreitol (DTT) | Carl Roth | 6908.3 | |
| Dynabeads Protein G | Novex, Thermo Fisher Scientific | 10003D | |
| DRBP76 (NF90/NF110) antibody | BD Transduction Laboratories | 612154 | use 1:1000 for WB and 1:100 for ICC/PLA |
| RBM3 antibody | ProteinTech | 14363-1-AP | use 1:1000 for WB and 1:100 for ICC/PLA |
| Lamin A/C antibody | Cell Signaling Technology | #2032 | use 1:1000 for WB |
| anti-GAPDH antibody | Abcam | ab8245 | use 1:1000 for WB |
| normal rabbit IgG | Santa Cruz | sc-2027 | |
| anti-rabbit IgG, HRP-lined secodary antiboy | Cell Signaling Technology | #7074 | use 1:5000 for WB |
| anti-mouse HRP secondary antibody | Carl Roth | 4759.1 | use 1:5000 for WB |
| Clarity Western ECL Blotting Substrate | Bio-Rad | #1705060 | |
| NuPAGE Novex 4-12% Bis-Tris Gel | Novex, Thermo Fisher Scientific | NP0321BOX | |
| NuPAGE LDS Sample Buffer (4x) | Novex, Thermo Fisher Scientific | NP0007 | |
| 1,4-Dithiothreitol (DTT) | CarlRoth | 6908.1 | |
| NuPAGE MES SDS Running Buffer (20x) | Novex, Thermo Fisher Scientific | NP0002 | |
| NuPAGE Transfer Buffer (20x) | Novex, Thermo Fisher Scientific | NP00061 | |
| Amersham Hypond P 0.2 PVDF membrane | GE Healthcare Life Sciences | 10600021 | |
| Super RX X-ray film | Fujifilm | 4741029230 | |
| Poly-D-Lysine 8 Well Culture Slide | Corning BioCoat | 354632 | |
| Paraformaldehyde (PFA) | Sigma | P6148 | |
| Normal goat serum (NGS) | Gibco, Thermo Fisher Scientific | PCN5000 | |
| Goat anti-mouse IgG (H+L Antibody), Alexa Fluor 488 conjugate | Thermo Fisher Scientific | A-11001 | |
| Goat anti-rabbit IgG (H+L Antibody), Alexa Fluor 568 conjugate | Thermo Fisher Scientific | A-11011 | |
| 4′, 6-Diamidin-2-phenylindol (DAPI) | Sigma | D9542 | |
| Duolink PLA probe Anti-mouse PLUS | Sigma | DUO92001 | |
| Duolink PLA probe Anti-rabbit MINUS | Sigma | DUO92005 | |
| Duolink Detection Reagents Red | Sigma | DUO92008 | |
| Duolink Wash Buffers Fluorescence | Sigma | DUO82049 | |
| Duolink Mounting Medium with DAPI | Sigma | DUO82040 | |
| Mowiol 4-88 | Sigma | 81381 | |
| Microscope | Olympus | AX-70 | |
| CCD camera | SPOT | Insight 2MP Firewire | |
| X-ray film | Fujifilm | Super RX | |
| Film processing machine | Fujifilm | FPM-100A |
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