Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Understanding the function of exosomes requires a variety of tools, including live imaging. A previous live-cell reporter, pHluorin-CD63, allows dynamic subcellular monitoring of exosome secretion in migrating and spreading cells. However, dim fluorescence and the inability to make stably-expressing cell lines limit its use.
Researchers from Vanderbilt University School of Medicine have incorporated a stabilizing mutation in the pHluorin moiety, M153R, which now exhibits higher, stable expression in cells and superior monitoring of exosome secretion. Using this improved construct, they visualize secreted exosomes in 3D culture and in vivo and identify a role for exosomes in promoting leader–follower behavior in 2D and 3D migration. Incorporating an additional non-pH-sensitive red fluorescent tag allows visualization of the exosome lifecycle, including multivesicular body (MVB) trafficking, MVB fusion, exosome uptake and endosome acidification. This reporter will be a useful tool for understanding both autocrine and paracrine roles of exosomes.
Cells exhibit pathfinding behavior on exosome trails
a Time-series images from live imaging of pHluo_M153R-CD63 (green)/mCherry-CaaX (magenta) dual-expressing HT1080 cells in 2D culture conditions. b Pathfinding index from (a) shown as a scatter-plot with median and quartile range. c Time-series images from live imaging of pHluo_M153R-CD63 (green)/mCherry-CaaX (magenta) dual-expressing HT1080 cells in 3D collagen gels (see Supplementary Movie 3). n = 151 cells from 27 movies from four independent experiments. Arrowheads indicate exosome trails. Arrows indicate protrusions. Scale bar, 50 µm. d Pathfinding index from (c) shown as a scatter-plot with median and quartile range. e Representative intravital images of pHluo_M153R-CD63 (green in merge) and collagen fibers (white in merge) in mice from eight independent experiments. f Representative intravital images of pHluo_M153R-CD63 in chicks from six independent experiments. g Half-life determination of pHluo_M153R-CD63 small EVs in vivo using the avian embryo model (n = 7 chick embryos from three independent experiments).