The atypical Rho GTPase RhoD and its role in cellular dynamics

Detta är en avhandling från Stockholm : Karolinska Institutet, Dept of Microbiology, Tumor and Cell Biology

Sammanfattning: Despite its discovery 20 years ago, the accumulated knowledge about the Rho member RhoD is scarce. Instead, the vast majority of studies concerning Rho GTPases has focused on the three classical members RhoA, Cdc42 and Rac1. In contrast, RhoD is considered an atypical Rho GTPase, with an aberrant GTP/GDP cycling and an unknown regulation. Like most Rho GTPases, altered RhoD protein levels result in actin cytoskeleton reorganization. We found that increased RhoD protein levels lead to a less dynamic actin cytoskeleton, while RhoD silencing leads to more pronounced actin-containing structures, such as stress fibers, cortical actin and ruffles, depending on cell type. Actin-dependent processes, such as cell migration and cell proliferation, are significantly affected in absence of RhoD in fibroblasts. Moreover, we have shown that endogenous RhoD, as well as its interaction partner WHAMM, localize to the Golgi apparatus. Silencing or overexpression of RhoD or WHAMM leads to a dispersion of the Golgi apparatus, suggesting a role of these proteins in Golgi homeostasis. In addition, protein transport from the ER to the plasma membrane is delayed both when overexpressing and silencing RhoD and WHAMM, as measured by the VSV-G protein transport assay. One of the first described functions of RhoD was its regulatory role in endosome fusion and trafficking. We found that the localization to vesicles is independent of the nucleotide-bound status of RhoD. However, only the GTP-bound RhoD can localize to the plasma membrane. In contrast, RhoD must be inactivated for fusion of RhoD positive vesicles. Deleting the unique N-terminal of RhoD leads to an altered distribution and characteristics of RhoD positive vesicles. Taken together, this thesis elucidates the role of RhoD in three different dynamic cellular processes; reorganization of the actin cytoskeleton, Golgi homeostasis and vesicle transport and fusion.

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