LIVE-IMAGING OF MVB-PM FUSION

| Background: : Individual cancer cells secrete thousands of 40-100nm membrane vesicles called exosomes that presumably originate from multivesicular bodies (MVBs). Besides their molecular content, MVB fusion dynamics with the plasma membrane (PM) are expected to determine the physiology of tumor exosomes but this process remains poorly understood. Results: Here we labelled exosomes with a pH-sensitive optical reporter (CD63-pHluorin) and reveal with live-imaging the kinetics of SNARE-mediated fusion of peripheral MVBs with the PM. MVB-PM fusion events are uncommon (compared to rapid transport vesicle-PM fusion) but increase dramatically upon stimulation with cAMP-dependent protein kinase (PKA) and histamine treatment, supporting a direct role for G protein-coupled receptor (GPCR) signalling in exosome release. We identified the non-neuronal t-SNARE SNAP23 and Syntaxin-4 as mediators of MVB-PM fusion and inactivation of SNAP23 in invasive cancer cells reduced extracellular matrix remodelling and invasion. Moreover, increased SNAP23 expression was observed in tumor tissues and correlated with increased incidence of metastasis Conclusions: Together our findings demonstrate that MVB fusion with the plasma membrane is a dynamic, physiologically relevant process in cancer cells that can be modulated by soluble factors present in the tumor microenvironment.

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