Date of Award

Summer 8-15-2016

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Cell Biology)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Extracellular vesicles (EVs) are important mediators of intercellular communication. Different types of EVs are released from cells by either fusion of late endsomal multivesicular bodies with the plasma membrane (exosomes) or direct budding from the plasma membrane (ectosomes). Topologically equivalent processes including intralumenal vesicle formation for degradation in the endosomal pathway and virus budding from the plasma membrane depend on the ATPase VPS4 and its Endosomal Sorting Complex Required for Transport (ESCRT)-III substrates for membrane fission and release. Whether this machinery is generally required for EV biogenesis has, however, been the subject of debate. Studies of the EV proteome from a variety of cell types consistently find ESCRT-III subunits, thus providing further impetus for studying their function in EV biogenesis. Detailed understanding of the mechanisms underlying EV biogenesis is critical to testing their proposed functions. Monitoring EV release, however, remains technically challenging. To this end, I developed a protocol for efficiently isolating EVs from small numbers of cultured cells. Using this together with complementary standard techniques, I found that inhibiting VPS4 reduced release of both protein and miRNA in EVs. Notably, VPS4 activity was required for the release of two proteins – the tetraspanins CD63 and CD9 – that preferentially reside on different membranes and are enriched on separate EV populations. I establish that CD63 is enriched on and preferentially marks endosome-derived exosomes while CD9 is a primary marker of plasma membrane-derived ectosomes. An important question in the cell biology of exosome release is what distinguishes exocytic from degradative MVBs. I identified Rab27b as a selective marker of a subset of less acidic and likely exocytic MVBs. Neutralizing endosomal pH increased Rab27b recruitment to CD63 containing MVBs, pointing toward an important role for pH in regulating Rab27b recruitment. Returning to the role of VPS4 and ESCRT-III, our data broadly implicate VPS4 and ESCRT-III in biogenesis of both exosomes and ectosomes. I established cell lines expressing fluorescent CD63 and CD9 singly or in combination in order to further probe their segregation to different EV populations. Intriguingly, inhibiting VPS4 increased the concentration of ESCRT-III protein recovered in EVs despite the significant decrease in total vesicle release. This suggests a role for ESCRT-III disassembly during EV fission. Altogether, the work presented here sets the stage for future studies aimed at understanding multiple aspects of exosome and ectosome biogenesis.

Language

English (en)

Chair and Committee

Phyllis I. Hanson

Committee Members

Robert P. Mecham, Michael M. Mueckler, Daniel S. Ory, Philip D. Stahl

Comments

Permanent URL: https://doi.org/doi:10.7936/K7ZK5F3D

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