Detection and characterization of protein interactions in vivo by a simple live-cell imaging method

Abstract

Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc). The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation), a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50), exocytosis (exocyst complex) and endocytosis (Ede1-Syp1).

Figure 1. Representation of PICT approach.

Schematic representation of the assay. “−RAP” cells were treated with the vehicle; “+RAP” cells were treated with rapamycin; “Δ, +RAP” denotes that a gene has been deleted and the cells have been treated with rapamycin.

Figure 2. Analysis of the Ste5-Ste11-Ste50 MAPK cascade subcomplex with PICT.

(A) Schematic representation of the Ste5-Ste11-Ste50 assembly. (B) Recruitment of the Ste5-Ste11-Ste50 complex to Pil1-RFP-FKBP anchoring platforms. Ste11-FRB was used as bait. Bait recruitment upon addition of rapamycin was proved in a strain in which Ste11 was tagged with FRB and GFP (left panel). Co-recruitment of Ste50-GFP prey is shown in the right panel. (D) Matrix with representative cells in the GFP channel for each of the six combinations resulting from all components of the studied complex used as bait (FRB-tagged) and prey (GFP-tagged) in PICT assays. (B) and (C) are color-coded as in Figure 1. “RAP” cells were treated with the vehicle, “+RAP” cells were treated with rapamycin. In (C) “▵, +RAP” denotes that the indicated gene has been deleted and the cells have been treated with rapamycin.

Figure 3. Automated PICT assay.

(A) Schematic representation of strain generation. (B) Representative cells of the automated PICT assay from the screen for PPIs of Exo70. Cells were treated with the vehicle (−RAP) or rapamycin (+RAP). The upper images show the GFP channel and the bottom images show the RFP channel. Arrows highlight membrane sites where Sec3-GFP accumulates. The bars quantify the area of colocalization between Sec3-GFP and Pil1-RFP-FKBP from nine different fields of views (see Materials and Methods). (C) Summary of the screen. The Y-axis shows the ratio between the area of colocalization after and before Exo70-FRB recruitment. The X-axis shows the log (1/p) of the t-test between the area of colocalization before and after rapamycin treatment. All known exocyst components (orange circles) showed significant recruitment (log1/p>1.699; dashed line); other tested preys are represented as blue circles.

Figure 4. PICT-FRAP assay.

(A) PICT-FRAP assay to analyze stable and transient interactions. A schematic representation of PICT-FRAP assay color-coded as in Figure 1. Ste11-FRB and Ede1-FRB were used as bait and Ste50-GFP and Syp1-GFP as prey in the respective experiments. For each assay, a frame from the GFP channel is shown corresponding to an anchoring site before, immediately after photobleaching and at the end of the measurements. (B) PICT-FRAP of the Ste11–Ste50 interaction (left) and the Ede1–Syp1 interaction (right). The curves represent the mean ± SD, Ste11–Ste50 (n = 8) and Ede1–Syp1 (n = 12).