Characterization of skeletal muscle-derived extracellular nano-vesicles: protein lipidation for cargo selection and/or nano-vesicles biogenesis

Background: Several cell types have the capacity to secrete small vesicles that contain cell-specific collections of proteins, lipids, and genetic material. Research on secreted vesicles has been focusing primarily on the immune system and tumor cells. Recently, we have reported that skeletal muscle cell can release nano-vesicles; however, how muscle cells generate these vesicles and what their regulators are remain unknown. In this context, we focused on protein lipidation as possible post-translational modification (PTM) of specific regulators of extracellular vesicles biogenesis. Methods: We treated the muscle cell line, C2C12, with specific inhibitors of protein lipidation, then we isolated muscle-derived nano-vesicles using differential ultracentrifugation; to characterize them, we applied well-established colloid-chemical techniques, e.g. dynamic light scattering (DLS), coupled to immunoblot analyses of protein content. Results and Conclusions: To date, exosomes have mainly been studied with biological techniques; here, skeletal muscle-derived nano-vesicles were analyzed after inhibition of protein lipidation by DLS. Our analyses qualify DLS as valuable routine technique to analyze and compare the size and integrity of nano-vesicles in dispersion, in different experimental settings. We coupled this information with analyses of nano-vesicle specific biomarkers/regulators, after the inhibition of protein lipidation. Experimental issue: In this preliminary phase, we have used the following technical approach to determine whether protein lipidation level affects exosome biogenesis and/or cargo selection: 1) in silico prediction of different lipidation sites in proteins that are identified more often in exosomes; 2) altering protein lipidation, using a specific inhibitor; 3) analyses of cell membrane- and exosome-proteins, by non-reducing SDS-PAGE; and 4) analysing size and integrity of nano-vesicles in dispersion using dynamic light scattering (DLS). Thus, we would like to discuss and share our preliminary results regarding protein lipidation as possible post-translational modification (PTM) of specific regulators of extracellular vesicles biogenesis, in skeletal muscle cells.