Date of Award

2019

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Life requires biological membranes. Membrane-enclosed compartments separate and unite through dynamic fission and fusion reactions. These are catalyzed processes that are central in organismal biogenesis. This dissertation focuses on extracellular membrane fusions, which are central to several processes. (1) Enveloped viruses enter cells through membrane fusions. (2) Extracellular vesicles (EVs) also deliver molecules into cells through membrane fusions. (3) Entire cells also fuse together, generating fertilized zygotes, skeletal muscles, and giant cell macrophages.Mechanisms of extracellular membrane fusion are poorly understood. This dissertation aimed to further define these mechanisms. We focused on regulatory cofactors, including tetraspanins, transmembrane proteins that cluster into microdomains and promote cell-cell fusions by unclear mechanisms. We hypothesized that tetraspanin-enriched microdomains (TEMs) are platforms for virus, EV and cell membrane fusions, and that TEM composition regulates these fusion events.We evaluated this hypothesis using three systems. The first system measured virus-cell fusions, using Middle East Respiratory Syndrome Coronavirus (MERS-CoV) as a model. We found that TEMs are platforms for MERS-CoV fusion. TEMs contained the virus entry factors DPP4 (the MERS receptor) and TMPRSS2 (the MERS-activating protease). The tetraspanin CD9 was the architect of the MERS-CoV entry platform, bringing DPP4 and TMPRSS2 into close proximity.The second system evaluated vesicle-cell fusions, using EVs as a model. We determined that tetraspanins enable EV cargo transfer. In contrast, TEM resident interferon-induced transmembrane proteins (IFITMs), known inhibitors of virus-cell membrane fusion, suppressed transfers. Our results indicate that tetraspanins endow a subset of EVs with cargo transfer capacity.The third system evaluated cell-cell fusions, using monocytes as a model. Monocyte fusions form giant cell macrophages that resorb bone. IFITM5 is present on monocytes, and IFITM5 mutations link to severe bone disease. We determined that IFITM5 prevented monocytes from fusing into bone-resorbing cells, and one disease-causing mutation abrogated this restriction. Overexpressing the tetraspanin CD9, a facilitator of monocyte fusion, overcame IFITM5 restriction, indicating that tetraspanins influence IFITM activities.Overall, this dissertation identified tetraspanins and TEM resident proteins as central components in a variety of membrane fusion processes, illustrating how membrane dynamics in diverse environments are controlled by a limited set of common elements.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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