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

摘要

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. Author summary Protein–protein interactions govern cellular processes. The majority of these physical interactions previously identified are strong/permanent interactions, which typically remain unbroken even after purification. The weak/transient interactions between proteins have been implicated in the control of dynamic cellular process that maintain cellular homeostasis and trigger signaling cascades upon environmental changes. However, these interactions are poorly investigated, mainly due to the methodological limitations. Here, we have developed a co-immunoimmobilization assay called Co-II that enables the direct visualization of protein–protein interactions in the membrane of living cells at the single-molecule level. Co-II is based on the intuitive concept that if the protein of interest is immobilized, the interacting protein must be co-immobilized. The use of intrinsic protein diffusivity fundamentally overcomes the limitations of proximity-based methods. Using Co-II, we study the transient homodimerizations of EGFR and β2-AR in living cells, which have been implicated in several types of cancers and heart diseases. We show that the dimerization of these receptors is differently regulated by molecular conformations and the microenvironment in the plasma membrane.