Quantitative imaging of vesicle–protein interactions reveals close cooperation among proteins

Abstract

Membrane-bound vesicles such as extracellular vesicles (EVs) can function as biochemical effectors on target cells. Docking of the vesicles onto recipient plasma membranes depends on their interaction with cell-surface proteins, but a generalizable technique that can quantitatively observe these vesicle-protein interactions (VPIs) is lacking. Here, we describe a fluorescence microscopy that measures VPIs between single vesicles and cell-surface proteins, either in a surface-tethered or in a membrane-embedded state. By employing cell-derived vesicles (CDVs) and intercellular adhesion molecule-1 (ICAM-1) as a model system, we found that integrin-driven VPIs exhibit distinct modes of affinity depending on vesicle origin. Controlling the surface density of proteins also revealed a strong support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. An adsorption model accounting for multiple protein molecules was developed and captured the features of density-dependent cooperativity. We expect that VPI imaging will be a useful tool to dissect the molecular mechanisms of vesicle adhesion and uptake, and to guide the development of therapeutic vesicles. K E Y WO R D SCD9, cell-derived vesicles, ICAM-1, integrin, total internal reflection fluorescence microscopy, vesicleprotein interactions  INTRODUCTIONMembrane-bound vesicles can effectively elicit biochemical changes in cells. For example, extracellular vesicles (EVs), which are naturally secreted by virtually all types of cells, can adhere to the plasma membranes of target cells and trigger intracellular signalling, often accompanied by cargo transfer upon internalization. This process is therefore believed to enable effective Minkwon Cha and Sang Hyeok Jeong contributed equally to this work.