Spatiotemporal activation of Cdc42 and RhoA in carcinoma cells undergoing motility

Abstract

Cell motility is a fundamental property of cells and is essential for physiological and pathological events. In carcinoma cells, the initial steps in cell motility require a biphasic actin polymerization response leading to the extension of a lamellipodia containing a zone of F-actin at the leading edge. Phosphoinositide-3 kinase is a main regulator of actin polymerization and is required for lamellipod formation. The signaling of PI3K to the actin cytoskeleton depends on the production of phosphatidylinositol-(3,4,5)P 3, which targets the pleckstrin homology domains of GTP exchange factors for Rho GTPases to the plasma membrane. Rho GTPases, in turn, can activate PI3K, suggesting a positive feedback loop between PI3K and Rho GTPases at the leading edge of cells. Rho GTPase activation results in the activation of Arp2/3, which is essential for lamellipod protrusion through its actin nucleation and branching activity.;The classical roles of Rho GTPases during cell motility were based on studies done in fibroblasts using constitutively active and dominant negative forms of Rho GTPases, which showed that Rac1, Cdc42 and RhoA regulate the formation of lamellipodia, filopodia and stress fibers, respectively. Previous work from our laboratory has shown that the inhibition of Rac1 does not affect actin polymerization in carcinoma cells. In this thesis, I examine the role of Cdc42 in actin polymerization leading to lamellipod formation.;In order to examine the kinetics of activation of Cdc42, I used a recently developed Cdc42 biosensor (MeroCBD) that monitors the activation of endogenous Cdc42 in single cells. When stimulated with EGF, carcinoma cells showed a rapid increase in activated Cdc42 that is primarily localized to the leading edge of the cells. Cdc42 activation is co-temporal with the previously defined actin polymerization transients at 1 and 3 min in carcinoma cells. SiRNA-mediated knock-down of Cdc42 expression caused a decrease in EGF-mediated cell protrusion and lamellopodia formation, and reduced cell motility in time-lapse studies. These changes correlated with a decrease in barbed end formation and Arp2/3 localization at the cell edge, and images obtained by rotary shadowing scanning electron microscopy showed a marked defect in actin filament branching. Upstream of Arp2/3, EGF-stimulated targeting of both N-WASP and WAVE-2 to the leading edge was defective in Cdc42 knock-down cells, as was activation of Rac, PI 3-kinase, and membrane recruitment of IRSp53. I concluded that in carcinoma cells, Cdc42 directly regulates lamellipodia required for cell motility.;RhoA is another Rho GTPase that plays a positive role in the regulation of cell motility. Initially, the role of RhoA during cell motility was thought to be restricted to the generation of contractile force and focal adhesion turnover needed for tail retraction. This is achieved through its downstream effector, the serine/threonine kinase p160ROCK, which leads to myosin light chain phosphorylation and increased actin-myosin contractility. Based on the antagonism between RhoA/ROCK and Rac, it was originally postulated that RhoA activity at the front of a migrating cell was incompatible with membrane protrusion.;In contrast, recent reports have described a zone of RhoA/ROCK activation at the leading edge of cells. I used a FRET-based RhoA biosensor to show that RhoA activation localizes to the leading edge of EGF-stimulated carcinoma cells. Inhibition of Rho or ROCK did not inhibit protrusion, yet markedly inhibited cell motility. This reduced motility coincided with an increase in Rac-dependent focal complexes at the cell edge, but a complete loss of focal complex maturation into focal adhesions. Focal adhesions play a key role in anchoring the cells during cell motility. Thus, Rho and ROCK are critical for motility via its effects on focal complex maturation at the front of the moving cell.;This thesis re-examines the role of Cdc42 and RhoA in carcinoma cell motility. Cdc42 was found to localize to the leading edge where it regulates actin polymerization leading to lamellipod formation, whereas RhoA localizes behind the leading edge, where it regulates focal adhesion formation needed for cell motility.