Abstract
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. Our 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. We 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, we 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.
Highlights
Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes
We adapted an approach from the synaptic vesicle field, that leverages the properties of a pH-sensitive GFP derivative, pHluorin, to observe dynamic vesicle fusion events19. pHluorin is virtually nonfluorescent under acidic conditions but fluoresces at neutral pH19, making it an ideal reporter to observe the fusion of acidic late endosomal multivesicular body (MVB) with the plasma membrane
To improve on our previous reporter, we tested whether a mutation previously shown to stabilize ratiometric pHluorin in bacterial fusion proteins, M153R22, would stabilize our superecliptic pHluorin-CD63 construct
Summary
Small extracellular vesicles called exosomes affect multiple autocrine and paracrine cellular phenotypes. Incorporating an additional non-pHsensitive 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. ESCRT-independent biogenesis mechanism involving membrane curvature induced by ceramide generation by neutral sphingomyelinase 2 (nSMase2) has been described[11] Tetraspanin proteins such as CD9, CD63, and CD81 are frequently used markers of exosomes and other small EVs and may be involved in ILV cargo selection and/or biogenesis[12]. Most previous studies used pH-insensitive fluorescent proteins such as GFP or RFP, leading to extremely bright fluorescence of internal endosomes This bright internal fluorescence limits the ability to observe fusion events of MVBs with the plasma membrane due to a poor signal-to-noise ratio. A similar reporter, with the pHluorin group placed 7 amino acids away from ours in the first extracellular loop of CD63, was used to study regulation of exosome secretion by G-protein-coupled receptor signaling[21]
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