Date of Award

5-27-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Applied Science

First Advisor

John Bush

Abstract

Dictyostelium discoideum is a simple eukaryotic amoeba that has proven useful in studying various aspects of endosomal and lysosomal membrane trafficking, as well as development. The Rab family of small molecular weight GTPases has been implemented in several cellular processes to include various vesicle trafficking roles, cell-cell signaling, and development. This study focuses on the localization and function of two Dictyostelium GTPases, RabS, and Rab2 which we hypothesized to both localize to the Golgi, and the contractile vacuole system. Expression of GFP-tagged DdRab2, and DdRabS proteins revealed similar localization to ER-Golgi membranes as well as the contractile vacuole system. Microscopic approaches indicate that the DdRab2 and DdRabS localize and are essential for the proper structure of ER-Golgi membranes as well as the CV system. Dominant negative (DN) forms of these proteins show fractionation of the ER-Golgi membranes supporting a role in the structure and function of these organelles. This is the first evidence of Rab GTPases co-localizing to ER-Golgi membranes, and the CV system in Dictyostelium. Although both DdRab2 and DdRabS localized similarly within the cell, their cellular functions differed. Functional analysis of DdRab2 revealed a role in vesicular transport, specifically in phagocytosis, and pinocytosis. Furthermore, constitutively active (CA), and dominant negative (DN) forms of Rab2 displayed an increased rate of multicellular development and cell-cell cohesion as well as a reduced rate of programmed cell death. Our data suggests that similar to other eukaryotes, DdRab2 may indirectly play a role in the phagocytic removal of cell corpses. Functional analysis of DdRabS also revealed a role in vesicular transport, specifically, endocytosis, and endocytic recycling. Both DdRabS overexpressing, and DN cell lines over-secreted lysosomal glycosidase enzymes. These data support a role for DdRabS in trafficking along the endocytic, phagocytic, and particularly the biosynthetic pathways. Furthermore, it was observed that cells overexpressing DdRabS could not complete the developmental cycle, and are defective in aggregation from both decreased cell-cell cohesion, and decreased sensitivity to cAMP. These cells also proved to have an increase in induced cell death of nearly 55%. This data demonstrates a novel Rab GTPase pathway between the Golgi and CV. Furthermore, we suggest that the abundance of these Rabs to their respective cellular location significantly affects various cellular processes to include vesicular trafficking, cell signaling, cell-cell contact, and differentiation.

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