Analysis of transient membrane protein interactions by single-molecule diffusional mobility shift assay

Abstract

Cell membrane: assaying protein interactions in cancer cells A new method for analyzing transient interactions between proteins in the outer membrane of living cells could help researchers better understand how cancer cells develop resistance to targeted drug therapies. A research team led by Do-Hyeon Kim and Sung Ho Ryu from Pohang University of Science and Technology, South Korea have developed a technique for investigating weak protein-protein interactions on the basis of changes in their rate of diffusion when bound to each other. Using this "single-molecule diffusional mobility shift assay," the researchers showed that a cancer-linked protein called EGFR tends to bind with a range of other proteins, depending on the type of cancer cell studied. EGFR is the target of several drug therapies, and the variability of its interactions may account for its ability to circumvent drug-induced inhibition in resistant cancer cells. Various repertoires of membrane protein interactions determine cellular responses to diverse environments around cells dynamically in space and time. Current assays, however, have limitations in unraveling these interactions in the physiological states in a living cell due to the lack of capability to probe the transient nature of these interactions on the crowded membrane. Here, we present a simple and robust assay that enables the investigation of transient protein interactions in living cells by using the single-molecule diffusional mobility shift assay (smDIMSA). Utilizing smDIMSA, we uncovered the interaction profile of EGFR with various membrane proteins and demonstrated the promiscuity of these interactions depending on the cancer cell line. The transient interaction profile obtained by smDIMSA will provide critical information to comprehend the crosstalk among various receptors on the plasma membrane.