FAK, PDZ-RhoGEF and ROCKII cooperate to regulate adhesion movement and trailing-edge retraction in fibroblasts

Abstract

A key step in cell migration is the dynamic formation and disassembly of adhesions at the front and the concomitant movement and release of adhesions in the rear of the cell. Fibroblasts maintained in the absence of serum have stable adhesions within the rear of the cell and exhibit reduced trailing-edge retraction resulting in an elongated cell phenotype. Addition of lysophosphatidic acid (LPA) induced the movement of adhesions and retraction of the trailing edge, thus mimicking tail retraction in a migrating cell. Focal adhesion kinase (FAK), guanine nucleotide exchange factors (GEF) for Rho and the Rho effector Rho kinase II (ROCKII) are crucial for the regulation of adhesion movement and trailing-edge retraction. Downregulation of FAK by small interfering RNAs or small hairpin RNAs blocked LPA-induced adhesion movement and restoration of cell shape. This phenotype was rescued by the ectopic expression of PDZ-RhoGEF or a RhoA-effector-domain mutant that activates ROCK. Knockdown of PDZ-RhoGEF or ROCKII inhibited LPA-induced trailing-edge retraction and adhesion movement. Moreover, overexpressed PDZ-RhoGEF co-immunoprecipitated with FAK and localized to FAK-containing adhesions. These studies support a model in which FAK and PDZ-RhoGEF cooperate to induce Rho/ROCKII-dependent focal adhesion movement and trailing-edge retraction in response to LPA.

Regulation of Focal Adhesion Kinase Activation, Breast Cancer Cell Motility, and Amoeboid Invasion by the RhoA Guanine Nucleotide Exchange Factor Net1

“…RhoA drives cell migration in part by stimulating actomyosin contraction, which in the rear of the cell results in disassembly of focal adhesions and retraction of the trailing edge. Lack of trailing edge retraction classically results in long extensions in the rear of the migrating cell (31,35,36). To monitor the contribution of each Net1 isoform to trailing edge retraction, we assessed the effects of their knockdown on cells migrating into a cleared area.…”

“…This value was then divided by the width of the cell in the nucleus, perpendicular to the first axis (31). For analysis of FAK activation after wounding or replating, images were acquired with a constant exposure time, and the background subtraction was performed uniformly for all samples and time points.…”

Integrin α6β4 cooperates with LPA signaling to stimulate Rac through AKAP-Lbc-mediated RhoA activation

“…Notably, early work suggested that the primary action of RhoA was to mediate trailing edge retraction in migrating cells (58). In accordance with these concepts, PDZ-RhoGEF operates preferentially at the rear of migrating cells to activate Rho and promote tail retraction in cooperation with ROCKII (20). However, studies using a fluorescence resonance energy transfer reporter for assessing RhoA activity demonstrated that RhoA activity is found at the leading edge (24,40).…”

Force-induced cell polarisation is linked to RhoA-driven microtubule-independent focal-adhesion sliding

“…GEF-H1 activation, however, is less likely to contribute to the force-induced RhoA activation per se, because RhoA activity still increases upon force application when MT dynamics are inhibited by taxol. Other guaninenucleotide exchange factors (GEFs), such as p190RhoGEF and PDZ RhoGEF, that localise to FAs and are involved in regulating RhoA activity (Iwanicki et al, 2008;Lim et al, 2008;Tomar and Schlaepfer, 2009) might be potential candidates with respect to the reorganisation of FAs and the upregulation of RhoA activity upon stretching forces.…”