The role of S-palmitoylation in the biogenesis of skeletal muscle-derived extracellular vesicles and the structural organization of their membrane

Introduction: Skeletal muscle (SkM) cells can release Alix-positive exosomes directly from their sarcolemma, suggesting a new paradigm for understanding how muscles communicate with adipose tissue, the brain or tumours. Our goals, here, were to understand how muscle cells generate these vesicles, to evaluate their heterogeneity and membrane structure, and to determine what regulates them. Methods: We have used an integrated biological/biophysical approach to determine whether protein lipidation (i.e. S-palmitoylation) affects the localization of extracellular vesicle (EV) modulator(s), EV biogenesis, cargo selection or other processes by (i) evaluating post-translational S-palmitoylation of EV proteins as well as their cell membrane and EV protein levels and (ii) characterizing EV heterogeneity and membrane structure in dispersion using dynamic light scattering (DLS), atomic force microscopy, small-angle X-ray scattering and small-angle neutron scattering. Results: By coupling these data, we were able to determine that Alix is S-palmitoylated and that palmitoylation inhibition altered its subcellular localization and protein interaction. Furthermore, the biophysical analyses of SkMEVs showed that the structural organization of the lipid bilayer of palmitoylated-inhibited exosomes is qualitatively different compared to non-treated exosomes. Conclusion: We propose that Spalmitoylation 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. Beside its biological relevance, our study sets the route for a comprehensive structural characterization of EV, which is expected to be crucial in EV-based therapies to be employed in the tissue regeneration field, for example, to help in recovery from muscle atrophy and/or injury. Italian Ministry for Education, University, and Research in the framework of the Flagship Project NanoMAX.