AbstractsBiology & Animal Science

Small GTPases of the Rho family are key regulators of the actin and microtubule cytoskeleton, whereby many cellular functions including cell migration, adhesion and polarity, as well as cell cycle progression are controlled. Increasing evidence suggests that Rho proteins are also critically involved in the regulation of membrane trafficking pathways within exocytosis and endocytosis. Although the molecular mechanisms are not well understood, Rho GTPases apparently have to govern and finely tune cytoskeletal remodeling, in order to support the formation, fusion and motility of transport carriers. However, the identity of their regulators, the guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) that ensure the balanced GTPase activation in space and time is largely elusive. The ‘Deleted in Liver Cancer’ (DLC1/2/3) proteins are a structurally conserved subfamily of RhoGAP proteins that act as negative regulators of Rho GTPases. In addition to the catalytically active GAP domain, all DLC proteins contain a sterile alpha motif (SAM) and steroidogenic acute regulatory protein-related lipid transfer (START) domain. Expression of the best studied member, DLC1, is frequently lost in various types of human cancers and a tumor suppressive function associated with its RhoGAP activity has been established in vivo. Although DLC3 was also observed to be downregulated in several cancer cell lines and primary tumors, the cellular functions of DLC3 are still poorly characterized. So far, GAP activity for RhoA has only been demonstrated in vitro and, associated with its localization at cell-cell contacts, a Rho-regulatory role in adherens junction stability was described. Thus, the aims of this thesis were to further investigate the subcellular localization of DLC3 and to shed light on the role of DLC3 in the regulation of Rho-mediated cellular processes, in particular endocytic membrane trafficking. This study provides convincing evidence that DLC3 is a functional, Rho-specific GAP protein in living cells and that its loss enhances perinuclear RhoA activity. DLC3 is recruited to Rab8-positive membrane tubules and required for the integrity of the Rab8 and Golgi compartments. Depletion of DLC3 impairs the transport of internalized transferrin to the endocytic recycling compartment, which is restored by the simultaneous downregulation of RhoA and RhoB. As a consequence, DLC3 loss interferes with epidermal growth factor receptor (EGFR) degradation and causes prolonged receptor signaling. Furthermore, it was found that DLC3-depleted cells show reduced surface N-cadherin levels, leading to decreased cell aggregation. Together, these findings identify DLC3 as a novel component of the endocytic trafficking machinery, wherein it maintains organelle integrity and regulates membrane transport via the control of local Rho activity. Die kleinen GTPasen der Rho-Familie sind Schlüsselregulatoren des Aktin- und Mikrotubulizytoskeletts, wodurch sie viele zelluläre Funktionen, wie z.B. Zellmigration, Zelladhäsion und…