pHluo_M153R-CD63, a bright, versatile live cell reporter of exosome secretion and uptake, reveals pathfinding behavior of migrating cells

Small extracellular vesicles (EVs) called exosomes affect a variety of autocrine and paracrine cellular phenotypes, including cellular migration, immune activation, and neuronal function. Understanding the function of exosomes in these processes requires a variety of tools, including live cell imaging. We previously constructed a live-cell reporter, pHluorin-CD63, that allowed dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, there were some caveats to its use, including relatively low fluorescent expression in cells and the inability to make cell lines that stably express the protein. By incorporating a stabilizing mutation in the pHluorin moiety, M153R, pHluorin-CD63 now exhibits higher and stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, we demonstrate visualization of exosome secretion in 3D culture and identify a role for exosomes in promoting leader-follower behavior in 2D and 3D collective migration. By incorporating a further non-pH-sensitive red fluorescent tag, this reporter allows visualization of the entire exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This new reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.