Probing cellular protein complexes using single-molecule pull-down

Abstract

Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

Figure 1

Schematic for SiMPull assay. Immunoprecipitated protein complexes are visualized using TIRF microscopy via (a) fluorophores-labeled antibody or (b) fluorescent protein tags. (c) Multi-color colocalization can distinguish between subcomplexes (e.g. AB+AC vs. ABC). (d) Photobleaching analysis can provide stoichiometric information. A simulated photobleaching trajectory for a trimeric protein. (e) TIRF images for YFP pulled down from cells expressing (His)₆-YFP (YFP) and control cells (Con) using His-tag or a control (Flag-tag) antibody. (−) indicates no antibody or sample. Scale bar is 5 µm. (f) Average number of fluorescent molecules per imaging area, N_f. Error bars denote s. d. (n > 20).

Figure 2

PKA pull-down. (a) Schematic of PKA construct. In WB, C-HA-YFP is pulled down via R-Flag-mCherry; on adding cAMP, PKA dissociates. (b) N_f for C-HA-YFP (C) as a function of lysates and antibodies demonstrate the specificity of pull-down. (c–e) PKA complex pull-down. (c) N_f for YFP (C) and mCherry (R) spots. (d) Images of single PKA complexes, YFP (left), mCherry (center) and overlay (right). (e) On adding cAMP, YFP spots decrease significantly. Photobleaching step distribution (f) for C-HA-YFP only lysate and (g) for C-HA-YFP pulled down via R-Flag-mCherry. Error bars denote s. d. (n > 20). Scale bar is 5 µm.

Figure 3

Applications of SiMPull assay. (a–c) β₂AR-YFP pull-down. (d–f) MAVS pull-down. Mitochondrial fraction from cells over-expressing YFP-MAVS was added either directly or after detergent solubilization (g–i) mTORC1 pull-down. Lysate from cells expressing Flag-mTOR, HA-Raptor or both was applied on chambers with Flag antibody, and probed through primary antibody against HA and labeled secondary antibody. (j–m) Endogenous PKA-AKAP complex pull-down from mouse brain extract. (k) WB shows AKAP immunoprecipitation with PKA antibody. (l) Immunofluorescence images of AKAP150 pulled down through PKA antibody. (c, f, i, m) show N_f. Scale bars are 5 µm. Error bars denote s. d. (n > 20).

Figure 4

PcrA pull-down and activity test (a) schematic, (b, c) labeled DNA binding to immunoprecipitated PcrA. Scale bar is 5 µm. Error bars represent s. d. (n > 20). (d) A typical time trace of repetitive reeling-in activity of PcrA monitored by FRET. The distribution of translocation times (Δt) and its mean, <Δt>, (e) for purified PcrA and (f) for PcrA pulled down from cell extracts, at 1 mM ATP concentration.