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Calcium signaling recruits Rab27a and the exocyst complex to promote small extracellular vesicle release in skeletal muscle cells

Nelson, L. T., Boulestreau, J., Polgar, N.
10.64898/2026.10.22.761013 · was preprinted
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Abstract

Extracellular vesicles (EVs) are key mediators of intercellular communication with emerging therapeutic potential. Small EVs (~50-150 nm) released from skeletal muscle (SkM) following exercise improve insulin sensitivity and glucose tolerance in animal models, yet the molecular mechanisms governing their secretion remain poorly understood. Ca2+ signaling has been shown to regulate numerous membrane trafficking events ranging from vesicle transport, membrane repair, and exocytosis of cellular cargo. We investigated how Ca2+ signaling influences the intracellular trafficking and membrane delivery mechanisms necessary for EV release using an in vitro SkM cell model. We found that elevated intracellular Ca2+ stimulates EV secretion through a mechanism requiring activity of the octameric exocyst complex. Using proximity ligation assays, co-immunoprecipitation, structural modeling and proximity-dependent proteomics, we identified dynamic coordination between the exocyst complex and the small GTPase, Rab27a, during Ca2+-stimulated EV release. We found this coordination is modulated by Ca2+ signaling and perturbation of exocyst function. We then used direct binding assays to provide a structural basis for the proximity observed between GTP and GDP-loaded Rab27a and exocyst subunits. Additionally, proximity-dependent proteomics revealed that Ca2+ stimulation promoted the association of specific exocyst subunits with GTP-competent Rab27a, suggesting a nucleotide-state-dependent organization of this trafficking interface in living cells. Finally, we adapted a real-time reporter of EV exocytosis in SkM cells and identified Ca2+-responsive kinases that modulate EV secretion. Collectively, our findings support a model in which Ca2+ signaling reorganizes interactions between Rab27a and the exocyst to promote intracellular trafficking necessary for EV secretion in skeletal muscle cells.

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