Techniques for detecting protein-protein interactions in living cells: principles, limitations, and recent progress
- 201 Downloads
Detecting protein-protein interactions (PPIs) provides fundamental information for understanding biochemical processes such as the transduction of signals from one cellular location to another; however, traditional biochemical techniques cannot provide sufficient spatio-temporal information to elucidate these molecular interactions in living cells. Over the past decade, several new techniques have enabled the identification and characterization of PPIs. In this review, we summarize three main techniques for detecting PPIs in vivo, focusing on their basic principles and applications in biological studies. We place a special emphasis on their advantages and limitations, and, in particular, we introduced some uncommon new techniques, such as single-molecule FRET (smFRET), FRET-fluorescence lifetime imaging microscopy (FRET-FLIM), cytoskeleton-based assay for protein-protein interaction (CAPPI) and single-molecule protein proximity index (smPPI), highlighting recent improvements to the established techniques. We hope that this review will provide a valuable reference to enable researchers to select the most appropriate technique for detecting PPIs.
Keywordsprotein-protein interactions in vivo techniques RET PCA co-localization
Unable to display preview. Download preview PDF.
This work was supported by the National Natural Science Foundation of China (31530084, 31761133009) and the Programme of Introducing Talents of Discipline to Universities (111 project, B13007).
- Branchini, B.R., Rosenberg, J.C., Ablamsky, D.M., Taylor, K.P., Southworth, T.L., and Linder, S.J. (2011). Sequential bioluminescence resonance energy transfer—fluorescence resonance energy transferbased ratiometric protease assays with fusion proteins of firefly luciferase and red fluorescent protein. Anal Biochem 414, 239–245.CrossRefGoogle Scholar
- Demir, F., Horntrich, C., Blachutzik, J.O., Scherzer, S., Reinders, Y., Kierszniowska, S., Schulze, W.X., Harms, G.S., Hedrich, R., Geiger, D., et al. (2013). Arabidopsis nanodomain-delimited ABA signaling pathway regulates the anion channel SLAH3. Proc Natl Acad Sci USA 110, 8296–8301.CrossRefGoogle Scholar
- Li, S., Armstrong, C.M., Bertin, N., Ge, H., Milstein, S., Boxem, M., Vidalain, P.O., Han, J.D., Chesneau, A., Hao, T., et al. (2004). A map of the interactome network of the metazoan C. elegans. Science 303, 540–543.Google Scholar
- Long, Y., Stahl, Y., Weidtkamp-Peters, S., Smet, W., Du, Y., Gadella Jr, T. W.J., Goedhart, J., Scheres, B., and Blilou, I. (2018). Optimizing FRETFLIM labeling conditions to detect nuclear protein interactions at native expression levels in living Arabidopsis roots. Front Plant Sci 9.Google Scholar
- Luker, K.E., Smith, M.C.P., Luker, G.D., Gammon, S.T., Piwnica-Worms, H., and Piwnica-Worms, D. (2004). Kinetics of regulated proteinprotein interactions revealed with firefly luciferase complementation imaging in cells and living animals. Proc Natl Acad Sci USA 101, 12288–12293.CrossRefGoogle Scholar
- Sambrook, J., and Russell, D.W. (2006). Detection of protein-protein interactions using the GST fusion protein pulldown technique. CSH Protocols 1, 275–276.Google Scholar
- Stirnweiss, A., Hartig, R., Gieseler, S., Lindquist, J.A., Reichardt, P., Philipsen, L., Simeoni, L., Poltorak, M., Merten, C., Zuschratter, W., et al. (2013). T cell activation results in conformational changes in the Src family kinase Lck to induce its activation. Sci Signal 6, ra13.CrossRefGoogle Scholar
- Sun, S.H., Yang, X.B., Wang, Y., and Shen, X.H. (2016). In vivo analysis of protein-protein interactions with bioluminescence resonance energy transfer (BRET): progress and prospects. Int J Mol Sci 17, 1–21.Google Scholar