Visualizing protein partnerships in living cells and organisms

Abstract

In recent years, scientists have expanded their focus from cataloging genes to characterizing the multiple states of their translated products. One anticipated result is a dynamic map of the protein association networks and activities that occur within the cellular environment. While in vitro-derived network maps can illustrate which of a multitude of possible protein-protein associations could exist, they supply a falsely static picture lacking the subtleties of subcellular location (where) or cellular state (when). Generating protein association network maps that are informed by both subcellular location and cell state requires novel approaches that accurately characterize the state of protein associations in living cells and provide precise spatiotemporal resolution. In this review, we highlight recent advances in visualizing protein associations and networks under increasingly native conditions. These advances include second generation protein complementation assays (PCAs), chemical and photo-crosslinking techniques, and proximity-induced ligation approaches. The advances described focus on background reduction, signal optimization, rapid and reversible reporter assembly, decreased cytotoxicity, and minimal functional perturbation. Key breakthroughs have addressed many challenges and should expand the repertoire of tools useful for generating maps of protein interactions resolved in both time and space.

Figure 1

Split reporters improved. (A) Schematic diagram of the split reporter approach to detect dual protein interactions. (B) Strategy for reversible light-controlled induction of the PIF-Phy interaction. (C) Split firefly luciferase enables visualization of the phosphorylation-mediated GSK3β-cMyc interaction. (D) Rationale for PET-based imaging by split thymidine kinase. Interacting fusion proteins reconstitute thymidine kinase, which phosphorylates radioactive PET probe [18F]-FHBG.

Figure 2

Crosslinking becomes sophisticated. (A) Rationale for chemical and photo-crosslinking strategies. Interacting proteins containing specific tags are crosslinked with a fluorescent reporter (top) or photo-crosslinked upon UV irradiation. (B) In bipartite tetracysteine display, a pair of CysCys tags are approxmated upon intra- or intermolecular protein association and bind bis-arsenicals such as ReAsH. (C) When applied with engineered tRNA synthetase machinery, diazirine lysine analogs enable UV-induced crosslinking of proximal biomolecules for complex stabilization and further structural analysis.

Figure 3

The benefit of being close. (A) General strategy for a proximity-induced ligation. Two proteins of interest (A, B) are fused to a modifying enzyme (Enz) or its substrate. Interacting proteins enable labeling of the substrate tag by the proximal enzyme. (B) Detecting neurexin1β (NRX)-neuroligin1 (NLG) interactions in live neurons with biotin ligase (BirA). Interaction of NRX and NLG increases the proximity of BirA to its acceptor peptide (AP) and biotin (B) transfer occurs. AlexaFluor-labeled monovalent streptavidin (mSA) then binds the ligated biotin, enabling direct visualization of the protein interaction. (C) In S-CROSS, bifunctional molecules consisting of a cyanine dye modified with two SNAP-tag or CLIP-tag substrates crosslink interacting proteins A and B to visualize their interaction in cells.