Rho GTPase signaling in cell adhesion and migration

Cytoskeletal dynamics is regulated by the family of Rho-like small GTPases. We have been focusing on the Rac1 GTPase, a key member of this family, that is known for its induction of actin polymerization and regulation of integrin and cadherin-based cell adhesion. Over the past years we have identified a series of novel Rac1-interacting proteins and have identified the biology that accompanies these interactions. In the past year, we have published recent findings on two of these new interactors, CD2AP and Caveolin-1, that bind to the Rac1 hypervariable C-terminal region.

CD2AP is an adapter protein which is critical for the formation and maintenance of a specialized cell-cell contact between kidney podocyte foot processes, the slit diaphragm. Here, CD2AP links the cell adhesion protein nephrin to the actin cytoskeleton. In addition, CMS/CD2AP binds actin-regulating proteins, such as CAPZ and cortactin, and has been implicated in the internalization of growth factor receptors. CD2AP interacts specifically with the C-terminal domain of Rac1 but not with that of other Rho family members. Efficient interaction between Rac1 and CD2AP requires both the proline-rich domain and the poly-basic region in the Rac1 C terminus, and at least two of the three N-terminal SH3 domains of CD2AP. CD2AP co-localizes with Rac1 to membrane ruffles, and small interfering RNA-based experiments showed that CD2AP links Rac1 to CAPZ and cortactin. Finally, expression of constitutive active Rac1 recruits CD2AP to cell-cell contacts and we found that CD2AP participates in the control of the intercellular barrier function.

In addition to CD2AP, Rac1 binds the membrane-associated adapter protein Caveolin-1. We found that Rac1 activity promotes Cav1 accumulation at Rac1-positive peripheral adhesions in adherent cells. Using Cav1-deficient mouse fibroblasts and depletion of Cav1 expression in human epithelial and endothelial cells, mediated by small interfering RNA and short hairpin RNA, we found that loss of Cav1 induces an increase in Rac1 protein and its activated GTP-bound form. Cav1 controls Rac1 protein levels by regulating the poly-ubiquitylation and degradation of activated Rac1 in an adhesion-dependent fashion. Finally, we show that Rac1 ubiquitylation is not required for effector binding, but regulates the dynamics of Rac1 at the periphery of the cell. These data extend the canonical model of Rac1 inactivation and uncover Cav1-regulated poly-ubiquitylation as an additional mechanism to control Rac1 signaling.The regulation of Rac1 signaling by ubiquitylation is a novel aspect that we will pursue in the coming years. Identification of the Rac1 E3 ubiquitin ligase represents an essential yet very challenging aspect of this work. In addition, we plan to define the role for mono-ubiquitylated Rac1, that appears to function as a separate protein species, in endothelial cell function and leukocyte transendothelial migration.

Recently, we have also embarked on a research line focusing on special adhesion structures called podosomes (see figure). These are very dynamic circular adhesion structures that are well known for their formation in myeloid cells, but also occur in primary human endothelial cells. The podosome proteome as well as the regulation of these structures is currently subject of study.

podosomes-in-human-dendritic-cells 
Podosomes in human dendritic cells (blue, nuclei; green, vinculin; red, F-actin)

Key publications