Date of Award

2023

Document Type

Dissertation

Degree Name

Ph.D.

Organizational Unit

College of Natural Science and Mathematics, Chemistry and Biochemistry

First Advisor

Michelle K. Knowles

Second Advisor

Schuyler van Engelenburg

Third Advisor

Yan Qin

Fourth Advisor

Dinah Loerke

Keywords

Exocytosis, Membranes, Multivesicular endosomes, Phosphatidic acid, Phospholipase D, Supported lipid bilayers

Abstract

Exocytosis is an essential process for intercellular communication in eukaryotic cells. This process involves significant changes in membrane curvature, and lipids and curvature-sensing proteins can assist these processes. One protein in particular, phospholipase D1 (PLD1), and its product, the lipid phosphatidic acid (PA), are involved in multiple exocytic processes. However, PLD1 and PA’s role in this process has remained unclear. In this work, PLD1 and the production of PA were visualized during exocytosis, and PA localization to regions of membrane curvature was established. Together, these results support the hypothesis that PLD1 production of PA stabilizes negative curvature during membrane fusion.

We demonstrate the localization of PLD1 to multiple exocytic proteins during different forms of exocytosis. We investigate PLD1 localization to secretory exocytic machinery and cargo; using a pH-sensitive probe for secretory vesicles in PC12 cells, we observe this localization to sites of exocytosis. PLD1 is present on secretory vesicles throughout the process from trafficking, to docking and finally to fusion. A fluorescently tagged PA binding protein, PASS, was then used to visualize the change of PA localization or production during this process. With PLD inhibitors we identify that PLD1 specifically produces PA after vesicles dock and during fusion.

In parallel, PLD1 was observed during exosome secretion from multivesicular endosomes (MVEs) in A549 cells. PLD1 localizes to late endosomes, especially MVEs. PLD1 is also present on MVEs during the entire process, but it is only required to produce PA on docking MVEs. PLD1 inhibition also increases the density of lysosomes near the surface, indicating a role in late endosome fate.

Finally, to understand why PA is essential in these processes, we explored PA accumulation to curvature in vitro. A tubulated supported lipid bilayer assay was developed to identify curvature preference of lipids. PA was found to stabilize the formation of these regions of curvature and to localize to the inner, negatively curved leaflet of liposomes.

Copyright Date

8-2023

Copyright Statement / License for Reuse

All Rights Reserved
All Rights Reserved.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Broderick L. Bills

Provenance

Received from author

File Format

application/pdf

Language

English (eng)

Extent

125 pgs

File Size

5.5 MB

Discipline

Biochemistry, Cellular biology, Molecular biology

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