Recruitment of Podosome Components Involved in the Remodelling of the Actin Cytoskeleton

Podosomes are dynamic, actin-rich structures that are involved in cell adhesion and extracellular matrix degradation; they are composed of a densely packed actin core surrounded by a ring structure made of components commonly found in focal adhesions. Podosome formation is characterized by the recruitment of AFAP-110, p190RhoGAP, and cortactin, which have specific roles in Src activation, local down-regulation of RhoA activity, and actin polymerization, respectively. However, the precise function of p190RhoGAP in podosome formation is not clear yet. By employing siRNA-mediated expression knockdown and expressing a catalytically inactive point mutant, I provide evidence that p190RhoGAP is required for podosome formation. It is well documented that Src-induced interaction of p190RhoGAP with p120RasGAP regulates p190RhoGAP activity and subcellular localization. In this thesis, I show that p190RhoGAP is constitutively associated in a complex with p120RasGAP in vascular smooth muscle cells, and that p120RasGAP translocates to podosomes upon PDBu stimulation. Nevertheless, siRNA-mediated knockdown of p120RasGAP expression does not impair p190RhoGAP recruitment or podosome formation, indicating that p120RasGAP is not essential for podosome formation. The molecular mechanism that underlies the specific recruitment of critical podosome components to sites of podosome formation remains unknown. The scaffold protein Tks5 is localized to podosomes in Src-transformed fibroblasts and in vascular smooth muscle cells, and may serve as a specific recruiting adapter for various components during podosome formation. I show here that induced mislocalization of Tks5 to the surface of mitochondria leads to a major subcellular redistribution of AFAP-110, p190RhoGAP, and cortactin, and to inhibition of podosome formation in vascular smooth muscle cells. Analysis of a series of similarly mistargeted deletion mutants of Tks5 indicates that the fifth SH3 domain is essential for this recruitment. A Tks5-GFP mutant lacking the PX domain also inhibits podosome formation and induces the redistribution of AFAP-110, p190RhoGAP, and cortactin to the perinuclear area. Together these findings demonstrate that Tks5 plays a central role in the recruitment of AFAP-110, p190RhoGAP, and cortactin to drive podosome formation. Evidence from osteoclasts and tumour cells cultured on different substrates indicates that the physical parameters of the underlying substrate influence the ability of cells to form podosomes or the related structures invadopodia. However, it is unclear how vascular smooth muscle cells respond to contact with different types of substrates. Thus, the last part of this thesis is dedicated to determine how podosome-forming vascular smooth muscle cells respond to alterations in the properties of the underlying substrate. I show here that A7r5 cells cultured on cross-linked gelatin degrade matrix by forming invadopodia-like structures. This is the first time that a cell type is reported to be capable of forming both podosomes and invadopodia in different conditions