Quantitative imaging of focal adhesion dynamics and their regulation by HGF and Rap1 signaling.

Cell migration is crucial in development, tissue repair and immunity and frequently aberrant in pathological processes including tumor metastasis. Focal adhesions (FAs) are integrin-based adhesion complexes that form the link between the cytoskeleton and the extracellular matrix and are thought to orchestrate cell migration. Understanding the regulation of FAs by (oncogenic) signaling pathways may identify strategies to target pathological cell migration. Here we describe the development of a robust FA tracker that enables the automatic, multi-parametric analysis of FA dynamics, morphology and composition from time-lapse image series generated by total internal reflection fluorescence (TIRF) microscopy. In control prostate carcinoma cells, this software recapitulates previous findings that relate morphological characteristics of FAs to their lifetime and their cellular location. We then investigated how FAs are altered when cell migration is induced by the metastasis-promoting hormone HGF and subsequently inhibited by activation of the small GTPase Rap1. We performed a detailed analysis of individual FA parameters, which identified FA size, sliding and intensity as primary targets of Rap1. HGF did not have strong effects on any of the FA parameters within the first hours of its addition. Subsequent Bayesian network inference (BNI), using all measured parameters as input, revealed little correlation between changes in cell migration and FA characteristics in this prostate carcinoma cell line. Instead BNI indicated a concerted coordination of cell size and FA parameters. Thus our results did not reveal a direct relation between the regulation of cell migration and the regulation of FA dynamics.

Rasip1 mediates Rap1 regulation of Rho in endothelial barrier function through ArhGAP29.

Rap1 is a small GTPase regulating cell-cell adhesion, cell-matrix adhesion, and actin rearrangements, all processes dynamically coordinated during cell spreading and endothelial barrier function. Here, we identify the adaptor protein ras-interacting protein 1 (Rasip1) as a Rap1-effector involved in cell spreading and endothelial barrier function. Using Förster resonance energy transfer, we show that Rasip1 interacts with active Rap1 in a cellular context. Rasip1 mediates Rap1-induced cell spreading through its interaction partner Rho GTPase-activating protein 29 (ArhGAP29), a GTPase activating protein for Rho proteins. Accordingly, the Rap1-Rasip1 complex induces cell spreading by inhibiting Rho signaling. The Rasip1-ArhGAP29 pathway also functions in Rap1-mediated regulation of endothelial junctions, which controls endothelial barrier function. In this process, Rasip1 cooperates with its close relative ras-association and dilute domain-containing protein (Radil) to inhibit Rho-mediated stress fiber formation and induces junctional tightening. These results reveal an effector pathway for Rap1 in the modulation of Rho signaling and actin dynamics, through which Rap1 modulates endothelial barrier function.

Ezrin is required for efficient Rap1-induced cell spreading.

The Rap family of small GTPases regulate the adhesion of cells to extracellular matrices. Several Rap-binding proteins have been shown to function as effectors that mediate Rap-induced adhesion. However, little is known regarding the relationships between these effectors, or about other proteins that are downstream of or act in parallel to the effectors. To establish whether an array of effectors was required for Rap-induced cell adhesion and spreading, and to find new components involved in Rap-signal transduction, we performed a small-scale siRNA screen in A549 lung epithelial cells. Of the Rap effectors tested, only Radil blocked Rap-induced spreading. Additionally, we identified a novel role for Ezrin downstream of Rap1. Ezrin was necessary for Rap-induced cell spreading, but not Rap-induced cell adhesion or basal adhesion processes. Furthermore, Ezrin depletion inhibited Rap-induced cell spreading in several cell lines, including primary human umbilical vein endothelial cells. Interestingly, Radixin and Moesin, two proteins with high homology to Ezrin, are not required for Rap-induced cell spreading and cannot compensate for loss of Ezrin to rescue Rap-induced cell spreading. Here, we present a novel function for Ezrin in Rap1-induced cell spreading and evidence of a non-redundant role of an ERM family member.

Metalloprotease ADAM10 is required for Notch1 site 2 cleavage.

Notch signaling is controlled by ligand binding, which unfolds a negative control region to induce proteolytic cleavage of the receptor. First, a membrane-proximal cleavage is executed by a metalloprotease, removing the extracellular domain. This allows gamma-secretase to execute a second cleavage within the Notch transmembrane domain, which releases the intracellular domain to enter the nucleus. Here we show that the ADAM10 metalloprotease Kuzbanian, but not ADAM17/tumor necrosis factor alpha-converting enzyme, plays an essential role in executing ligand-induced extracellular cleavage at site 2 (S2) in cells and localizes this step to the plasma membrane. Importantly, genetic or pharmacological inhibition of metalloproteases still allowed extracellular cleavage of Notch, indicating the presence of unknown proteases with the ability to cleave at S2. Gain of function mutations identified in human cancers and in model organisms that map to the negative control region alleviate the requirement for ligand binding for extracellular cleavage to occur. Because cancer-causing Notch1 mutations also depend on (rate-limiting) S2 proteolysis, the identity of these alternative proteases has important implications for understanding Notch activation in normal and cancer cells.