Skeletal muscle-derived extracellular vesicles (EVs): an integrated approach to study the role of protein lipidation in their biogenesis and structural organization

Background and Aims: Several cell types have the capacity to secrete small extracellular vesicles (EVs), as exosomes, which contain cell-specific collections of proteins, lipids, and genetic material. Recently, we and others have shown that skeletal muscle (SkM) cell can release Alix-positive exosomes, suggesting a new paradigm for understanding how muscles communicate with adipose tissue, the brain, or tumors. Our aims here are to understand how muscle cells generate these vesicles, to evaluate their heterogeneity and what their regulators are. Methods and Results: A skeletal muscle cell line was treated or not with specific inhibitors of protein lipidation, and then the SkM-derived exosomes were isolated using differential ultracentrifugation. To characterize exosomes and determine the role of protein lipidation (i.e., S-palmitoylation), we applied an integrated biological/biophysical approach. We were able to determine that Alix (exosomal marker) is S-palmitoylated and that palmitoylation inhibition altered its subcellular localization and protein interaction. We also proved that the inhibition of palmitoylation influences the number, size, heterogeneity of exosomes using dynamic light scattering (DLS), and Atomic Force Microscopy (AFM). Small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS) analyses showed that the structural organization of the lipid bilayer of palmitoylated-inhibited exosomes is qualitatively different compared to non-treated exosomes. Conclusion: Thus, we propose that S-palmitoylation might regulate the proper function of Alix in SkM EV biogenesis, support the interactions among the exosome-specific regulators/biomarkers, and maintain proper EV membrane structural organization. A better understanding of EV biogenesis and function would pave a way for a possible application of SkM-derived exosomes as a novel cell-free based therapy for muscle degenerative disease