Abstract
Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector—mixed lineage kinase domain 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. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry-based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a 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, mouse embryonic fibroblasts (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 were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3-mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation-mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune-complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.
| Original language | English |
|---|---|
| Article number | e12261 |
| Journal | Journal of Extracellular Vesicles |
| Volume | 11 |
| Issue number | 9 |
| DOIs | |
| Publication status | Published - 2022 Sept |
Bibliographical note
Funding Information:We acknowledge the generosity of Dr. Miguel C Seabra, Imperial College London and Dr. Ryan O'Connell, University of Utah for respectively generating Rab27DKO mice and sharing the breeder mice. We also acknowledge the help of Alissa Weaver, MD, PhD, of Vanderbilt University in EV isolation and characterization. This study was supported by the National Institutes of Health R01HL149404 (B.L), NIH R01GM125085 (Y.G), R01GM117058 (Y.G), and S10OD018475 (Y.G), American Heart Association 20TPA35490307 (B.L), 17PRE33670082 (KG), Vascular Surgery Research Training Program Grant T32 HL110853 (K.A.B and M.K.K), American Heart Association Predoctoral Fellowship (D.S.R). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Schematic figures were created with BioRender.com.
Funding Information:
We acknowledge the generosity of Dr. Miguel C Seabra, Imperial College London and Dr. Ryan O'Connell, University of Utah for respectively generating Rab27DKO mice and sharing the breeder mice. We also acknowledge the help of Alissa Weaver, MD, PhD, of Vanderbilt University in EV isolation and characterization. This study was supported by the National Institutes of Health R01HL149404 (B.L), NIH R01GM125085 (Y.G), R01GM117058 (Y.G), and S10OD018475 (Y.G), American Heart Association 20TPA35490307 (B.L), 17PRE33670082 (KG), Vascular Surgery Research Training Program Grant T32 HL110853 (K.A.B and M.K.K), American Heart Association Predoctoral Fellowship (D.S.R). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Schematic figures were created with BioRender.com.
Publisher Copyright:
© 2022 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.
All Science Journal Classification (ASJC) codes
- Histology
- Cell Biology