Date of Award

12-15-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Applied Science

First Advisor

John Bush

Abstract

Vesicular trafficking is a fundamental process essential to cell viability and dictates roles in both cellular and tissue function in multicellular organisms. To further understand various vesicular trafficking processes, our lab focuses on the moderators of these processes, Rab GTPases, using the model organism Dictyostelium discoideum. This dissertation focuses explicitly on the uncharacterized protein Rab32C, a homolog of human Rab32. While several Rab family members have been well defined, the functionality of Rab32C remains poorly understood. This project investigated both the subcellular localization and functional role of Rab32C with biochemical, imaging, and molecular assays. Results from this study provide evidence of Rab32C's involvement in osmoregulation, developmental, and calcium homeostasis pathways. It was demonstrated that Rab32C-overexpressing (OE) mutant cells exhibited colocalization with the contractile vacuole network and lysosomal-related organelles, suggesting involvement with these pathways. Osmotic stress assays further support this, with Rab32C OE mutants altering osmotic regulation upon exposure to hypotonic environments, leading to exaggerated swelling, vacuole formation, and ultimately, cell death. Moreover, media inoculated with calcium chloride induced rapid apoptosis in Rab32C Q87L mutants, suggesting disruption of calcium homeostasis. Developmental assays with added calcium supplementation revealed an altered phenotype among Rab32C OE, Q87L, and N142K mutant cell lines compared to wildtype cells. Specifically, Rab32C OE and Q87L mutants developed significantly longer stalks, while N142K mutants displayed inhibited stalk formation. Collectively, these findings, along with others, elucidate Rab GTPase diversity and underscore Rab32C’s unique role as a Rab protein in osmoregulation and calcium homeostasis.

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