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

Abstract

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.

Real-time imaging of multivesicular body–plasma membrane fusion to quantify exosome release from single cells

“…However, some applications, such as visualization of exosome deposition by migrating cells, might require higher CD63-pHluorin expression levels. To this end, Sung et al recently developed a CD63-pHluorinM153R mutant that offers increased stability and higher expression levels of the fusion protein, which could be used when the brightness of regular CD63-pHluorin is not sufficient 23 .…”

The versatile role of exosomes in human retroviral infections: from immunopathogenesis to clinical application

“…Furthermore, ceramide may be metabolized into sphingosine-1 phosphate, which binds to its receptor coupled with the G protein, in turn, this interaction contributes to exosome loading [18]. Besides, tetraspanin proteins such as CD63 [19], CD9, and CD81, CD82 [20], and other components like syndecan-syntenin-ALIX complex [21,22], VCAM-1 and α4 integrin [23,24], and phosphatidic acid (PA) [25] are involved in sorting different molecules into exosomes. Sorting of some molecules may be the result of together with exosomal and membrane cargo.…”

Extracellular Vesicles: Catching the Light in Zebrafish

“…In vivo imaging can be used to demonstrate trafficking to late-endosomal compartments by using EV markers tagged with a combination of pH-sensitive and non-pH sensitive markers (e.g. pHluorin-CD63-mScarlet [41]), in combination with pH-sensitive dyes, such as Lysotracker TM [3] or immuno-EM on previously identified recipient cell types/tissues by live-microscopy [2]. Functional cargo delivery in vivo can also be visualized using the Cre-stoplight system [42], or by loading EVs with other modulators of reporter genes.…”