Characterization of skeletal muscle-derived extracellular vesicles (EVs): the effect of palmitoylation in EV biogenesis and the structural organization of their membrane

Several cell types have the capacity to secrete extracellular vesicles (EVs)with a size ranging from hundreds to tens of nanometers (Exosomes), which contain cell-specific collections of proteins, lipids, and genetic material. Skeletal muscle (SkM) cells can release Alix-positive EVs (Romancino et al., FEBS J. 2013), suggesting a new paradigm for understanding how muscles communicate with other organs, such as adipose tissue, bones, the brain, or tumors. Here,we have used an integrated biological/biophysical approach to determine whether protein lipidation (i.e., palmitoylation) affects the localization of EV modulator(s), EV biogenesis, and EV structural organization and heterogeneity. Protein palmitoylation was altered using a specific inhibitor (2-Br-Palmitate, 2BP). The biochemical analyses of exosome-specific biomarkers/regulatorsallowed us to determine that Alix is palmitoylated and that palmitoylation inhibition altered its subcellular localization. Also, we showed that palmitoylation inhibition altered the size and heterogeneity of EV, by using dynamic light scattering (DLS), that is here established as a routine technique to analyze and compare the size and diffusional properties of EVs in dispersion in different experimental settings. Small-angle X-ray scattering (SAXS) showed that the structural organization of the lipid bilayer of 2BP-treated EV, is qualitatively different compared to non-treated EV. Thus, we propose that palmitoylation might function to regulate the proper function of Alix in SkM-derived EVbiogenesis and to 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 the design of engineered exosomes tobe employed in the tissue regeneration field, e.g.to help in recovery from muscle atrophy and/or injury.