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

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. Ca ²⁺ signaling has been shown to regulate numerous membrane trafficking events ranging from vesicle transport, membrane repair, and exocytosis of cellular cargo. We investigated how Ca ²⁺ 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 Ca ²⁺ 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 Ca ²⁺ -stimulated EV release. We found this coordination is modulated by Ca ²⁺ 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 Ca ²⁺ 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 Ca ²⁺ -responsive kinases that modulate EV secretion. Collectively, our findings support a model in which Ca ²⁺ signaling reorganizes interactions between Rab27a and the exocyst to promote intracellular trafficking necessary for EV secretion in skeletal muscle cells.