Cortactin Promotes Migration and Platelet-derived Growth Factor-induced Actin Reorganization by Signaling to Rho-GTPases

Abstract

Dynamic actin rearrangements are initiated and maintained by actin filament nucleators, including the Arp2/3-complex. This protein assembly is activated in vitro by distinct nucleation-promoting factors such as Wiskott-Aldrich syndrome protein/Scar family proteins or cortactin, but the relative in vivo functions of each of them remain controversial. Here, we report the conditional genetic disruption of murine cortactin, implicated previously in dynamic actin reorganizations driving lamellipodium protrusion and endocytosis. Unexpectedly, cortactin-deficient cells showed little changes in overall cell morphology and growth. Ultrastructural analyses and live-cell imaging studies revealed unimpaired lamellipodial architecture, Rac-induced protrusion, and actin network turnover, although actin assembly rates in the lamellipodium were modestly increased. In contrast, platelet-derived growth factor-induced actin reorganization and Rac activation were impaired in cortactin null cells. In addition, cortactin deficiency caused reduction of Cdc42 activity and defects in random and directed cell migration. Reduced migration of cortactin null cells could be restored, at least in part, by active Rac and Cdc42 variants. Finally, cortactin removal did not affect the efficiency of receptor-mediated endocytosis. Together, we conclude that cortactin is fully dispensable for Arp2/3-complex activation during lamellipodia protrusion or clathrin pit endocytosis. Furthermore, we propose that cortactin promotes cell migration indirectly, through contributing to activation of selected Rho-GTPases. INTRODUCTIONCell migration is a complex process requiring the coordinated activities of multiple cellular machines, driving actin polymerization, actin-myosin II-based force generation, and coupling to the extracellular matrix. However, the relative contribution of each of these machines to the different steps in the motility cycle is just beginning to emerge. Irrespective of the complexity of coordination of these activities, it is commonly agreed that protrusion at the cell front is initiated by localized actin polymerization, to form structures such as lamellipodia or ruffles (Small et al., 2002;Pollard and Borisy, 2003).The best characterized factors driving the nucleation of actin filaments in vertebrate cells are the Arp2/3-complex and formins (Pollard, 2007). Arp2/3-complex activity is considered essential for processes as diverse as lamellipodium protrusion, actin assembly during clathrin-mediated endocytosis, podosome formation, and different types of hostpathogen interaction (Goley and Welch, 2006;Linder, 2007).Activators of Arp2/3-complex are termed nucleation promoting factors (NPFs) and are roughly subdivided into class I and II composed of classical members Wiskott-Aldrich syndrome protein (WASP)/WASP family Verprolin-homologous protein (WAVE) proteins and cortactin, respectively (Welch and Mullins, 2002). This distinction is derived, at least in part, from their mode of interaction with actin: the WH2 domains of class I and tande...

How distinct Arp2/3 complex variants regulate actin filament assembly

“…This is even more remarkable given the multitude of potential activities ascribed to cortactin including (aside from branch stabilization and coronin regulation) the promotion of N-WASP dissociation from the Arp2/3 complex 11 . Interestingly, increased actin assembly rates and Arp2/3 complex flux were previously also observed in cortactin-deficient lamellipodia 10 , which are Arp2/3-12, 13 but not N-WASP-dependent 4, 5 , indicating that those effects are independent of the NPF at play. Inspecting those velocity data in isolation, it is tempting to speculate that cortactin specifically selects for the preferred usage of Arp2/3 high and Arp2/3 low complexes over mixed ones, but independent experiments would be needed to solidify such ideas.…”

“…In brief, cortactin knockdown had no effect on tail lengths when looking at wild type cells expressing mixtures of Arp2/3 complex variants (Fig. 1c), reminiscent of the lack of evident changes in lamellipodial dimension and structure in cortactin-deficient fibroblasts 10 . However, cortactin knockdown in cells depleted of Arp2/3 high and Arp2/3 low complexes converted the short tails of the former and long tails of the latter to a regular length (Fig.…”

Cortactin-Binding Protein 2 Modulates the Mobility of Cortactin and Regulates Dendritic Spine Formation and Maintenance

“…Rac1, a small GTPase protein, is known to promote actin branching and polymerization through the pathway containing the insulin receptor substrate p53 (IRSp53), neural-WiskottAldrich syndrome protein or WASP family verprolin-homologous protein, and the Arp2/3 complex (for review, see Ethell and Pasquale, 2005). Recently, cortactin was also shown to regulate cellular mobility and actin cytoskeleton dynamics via regulation of Rac1 (Lai et al, 2009). Thus, cortactin may use two mechanisms to regulate actin cytoskeleton dynamics: direct binding to F-actin and the Arp2/3 complex, and via control of Rac1 activity (Fig.…”

Induction of membrane circular dorsal ruffles requires co-signalling of integrin–ILK-complex and EGF receptor

“…In concert with RTK signals, active Rac could participate in DR induction (Buccione et al, 2004). In addition, several Src substrates have been implicated in DR formation, such as cortactin (Lai et al, 2009), Abl (Plattner et al, 1999) and Cbl (Sirvent et al, 2008). In this study we used a SILAC-based phosphoproteomics screen to detect proteins differentially phosphorylated by EGF in the presence or absence of functional integrin-ILK signalling.…”