RIPK3 is released into Extracellular Vesicles during Necroptosis through a Rab27 independent mechanism

Extracellular vesicles (EVs) are important mechanisms used by cells to release biomolecules. A common necroptosis effector— mixed lineage kinase like (MLKL)--- was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. We performed mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Necroptosis increased the number of EVs released and altered the protein contents within the EVs. Comparing to EVs released by healthy cells, EVs released during necroptosis contained markedly higher number of unique proteins. Receptor interacting protein kinase 3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, MEFs derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis-associated EVs was sensitive to Ca2+ depletion or lysosomal disruption. Neither treatments affected the RIPK3-mediated MLKL phosphorylation. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.