Direct visualization of single-molecule membrane protein interactions in living cells

Abstract

Interactions between membrane proteins are poorly understood despite their importance in cell signaling and drug development. Here, we present a co-immunoimmobilization assay (Co-II) enabling the direct observation of membrane protein interactions in single living cells that overcomes the limitations of currently prevalent proximity-based indirect methods. Using Co-II, we investigated the transient homodimerizations of epidermal growth factor receptor (EGFR) and beta-2 adrenergic receptor (β2-AR) in living cells, revealing the differential regulation of these receptors’ dimerizations by molecular conformations and microenvironment in a plasma membrane. Co-II should provide a simple, rapid, and robust platform for visualizing both weak and strong protein interactions in the plasma membrane of living cells.

Ligand-independent EGFR oligomers do not rely on the active state asymmetric kinase dimer

“…We observe concentration-dependent FRET efficiency values for several EGFR variants in the absence of ligand, which indicates dynamic equilibria between different EGFR association states and allows derivation of effective 2-dimensional dissociation constants. The homo-oligomerization strength of EGFR observed here, both in the presence and absence of ligand, is similar to previously published results (23,24). Mutations at the EGFR asymmetric kinase dimer interface alter the ensemble of ligand-independent EGFR oligomers but do not significantly alter their stability, implying that ligand-independent EGFR oligomers are distinct from the active state dimer.…”

“…The FRET efficiency for EGFR-Δ107-fp increased as a function of concentration and fit well to a monomer-dimer equilibrium model, with a two-dimensional Kd of 156 molecules/μm2 ( Fig. 2A) and a FRET efficiency value of 0.35 +/-0.06 (Table 1), consistent with previous observations (23,24). Addition of EGF or substitution of the EGFR extracellular domain with a constitutively dimeric immunoglobulin Fcdomain results in constant FRET efficiency values of 0.29 +/-0.03 and 0.29 +/-0.02, respectively, as well as constitutive receptor phosphorylation ( Supplementary Fig.…”

“…Consistent with previous observations, ligand-independent EGFR oligomer formation increases at higher EGFR concentrations, in accordance with the law of mass action ( Fig. 2) (23,24). To assess the activity of EGFR oligomers formed in the absence of ligand, we generated a panel of CHO cell lines stably expressing full length EGFR with EYFP at its C-terminus (EGFR-EYFP) and sorted cells to select cell lines expressing increasing EGFR-EYFP levels.…”

“…The reported levels of endogenous EGFR expression vary several orders of magnitude depending on cellular context (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). At least seven studies have reported that a fraction of cell-surface EGFR is dimeric in the absence of ligand (hereafter called 'ligand-independent dimers') (16)(17)(18)(19)(20)(21)(22) ( Supplementary Table S1), indicating that the receptor concentration in the plasma membrane might specify the ligandindependent oligomeric fraction in accordance with the law of mass action (23,24). Despite much study, the structure and signaling role of ligand-independent dimers remain unclear.…”

Characterization of Single Protein Dynamics in Cell Plasma Membrane Derived Polymer Cushioned Lipid Bilayers

“…[8][9][10][11] A lot has been learned from single-molecule tracking (SMT) in the plasma membranes. For example, nanoscopic membrane compartmentalization, 11 transient trapping of lipids 12 and protein-protein interactions 13 were revealed in live cells by high-speed SMT. The unique advantage of SMT is to measure dynamics of individual molecules without ensemble average, allowing for detecting heterogeneous behaviors in a large molecular population.…”