Investigating the Impacts of Sphingomyelinases on Extracellular Vesicle Cargo Sorting

ABSTRACT: The biogenesis of extracellular vesicles (EVs) is a regulated process, driven by mechanisms at specific subcellular milieus. Sphingomyelinases (SMases), which metabolize sphingomyelin in membranes, play a role in EV biogenesis. Their metabolic product, ceramide, induces invaginations at the endosome or blebbing from the plasma membrane, both important in EV generation. Here, we sought to evaluate the impact of SMase inhibition on EV protein and RNA cargoes. For this, we treated human MCF7 cells with the neutral SMase (NSM) inhibitor GW4869 or the acid SMase (ASM) inhibitor FTY720. EVs were then purified from the conditioned media of control or inhibitor-treated cells and characterized by a variety of approaches, including LC-MS/MS and RNA-sequencing. SMase inhibition resulted in morphological and phenotypic changes in the heterogeneous EV population. Strikingly, NSM inhibition resulted in a depletion of nanoparticles, as well as a decrease in the RNA and protein content of EVs, with a marked reduction in endosomal, spliceosomal, and translation-related proteins. Furthermore, we observed a reduction in the overall RNA-binding proteins (RBPs) in EVs released by cells treated with the NSM-inhibitor. In contrast, the ASM-inhibitor treatment, which appears to reduce plasma membrane-derived vesicles, elicited an inverse response, leading to an increase in RBP and associated machineries within the released EV population. RNA sequencing of these EV revealed changes in the RNA biotypes composition, with an increase in protein coding transcripts. Interestingly, ASM-inhibitor resulting EVs induced increased cell migration and protein translation in recipient MCF10A cells. These results suggest that SMase-dependent vesiculation represents a major route of RBP and RNA trafficking outside the cell, via endosomal pathways.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Eric L'Ecuyer  

PROVIDER: MSV000093684 | MassIVE | Mon Dec 18 13:04:00 GMT 2023

REPOSITORIES: MassIVE