Audhya Lab
Rab-type GTPases
rabs
Entry of cells into mitosis is accompanied by a dramatic reorganization of several organelles including the ER, Golgi, and endosomal system. Previous studies have shown that members of the Rab family of small G proteins function as critical regulators of membrane organization, but their roles in organelle restructuring during development remain largely uncharacterized. We have identified the subset of essential Rab-type GTPases necessary for C. elegans embryogenesis. To explore the roles of these essential proteins upon mitotic entry, we have conducted partial depletions and focused on post-fertilization membrane reorganization. This analysis has uncovered new roles for several Rab-type GTPases that have been previously overlooked. For example, we found that depletion of the endosomal Rab-type GTPase Rab5 results in a pronounced defect in nuclear membrane disassembly during mitosis that is independent of its well-characterized roles in endocytosis. Direct analysis of post-fertilization membrane dynamics revealed a specific failure in ER reorganization that normally accompanies nuclear membrane disassembly. These observations have both provided clues to the mechanisms underlying ER remodeling during cell cycle progression and suggest a function for ER remodeling in the disassembly of the nuclear envelope. Strikingly, expression of a constitutively active form of Rab5 abnormally potentiates ER reorganization in both C. elegans and cultured HeLa cells, suggesting a direct and conserved role for Rab5 signaling in this process. To support these in vivo findings, we have also used ER enriched membranes isolated from Xenopus eggs to assemble ER tubules in vitro. Addition of Rab GDI, which specifically inhibits Rab-type GTPases, blocks ER tubule formation, while Rho GDI, an inhibitor of related Rho-type GTPases, fails to have any effect. Together these data demonstrate a new function for Rab5 in membrane reorganization, and underscore the importance of crosstalk between different organelles.

To determine relevant effectors in Rab5-mediated membrane remodeling during early development, we have initially undertaken a biochemical approach. Using comparative mass spectrometry analysis, we have identified a set of C. elegans embryonic proteins that specifically interact with Rab5-GTP, but not Rab5-GDP. These include numerous known Rab5 effectors, validating our approach, as well as three new proteins that may regulate ER and/or endosome membrane dynamics during development. We are currently using RNAi and fluorescence-based functional assays for organelle remodeling to uncover the roles of these new factors. Of particular interest, we have identified a conserved protein phosphatase important for mitotic progression that binds to active Rab5, suggesting a link between membrane remodeling and cell cycle progression. Additionally, we have successfully purified the 5 other Rab-type GTPases necessary for embryogenesis, and plan to identify new effector molecules for each using a similar mass spectrometry-based approach, followed by functional analysis in vivo. Our studies will provide a comprehensive analysis of Rab-type GTPase function in membrane dynamics during oocyte fertilization and early embryogenesis and establish a key framework that catalyzes future investigation of related events in mammalian development.