Cortactin Releases the Brakes in Actin- Based Motility by Enhancing WASP-VCA Detachment from Arp2/3 Branches

Abstract

Cortactin is involved in invadopodia and podosome formation [1], pathogens and endosome motility [2], and persistent lamellipodia protrusion [3, 4]; its overexpression enhances cellular motility and metastatic activity [5-8]. Several mechanisms have been proposed to explain cortactin's role in Arp2/3-driven actin polymerization [9, 10], yet its direct role in cell movement remains unclear. We use a biomimetic system to study the mechanism of cortactin-mediated regulation of actin-driven motility [11]. We tested the role of different cortactin variants that interact with Arp2/3 complex and actin filaments distinctively. We show that wild-type cortactin significantly enhances the bead velocity at low concentrations. Single filament experiments show that cortactin has no significant effect on actin polymerization and branch stability, whereas it strongly affects the branching rate driven by Wiskott-Aldrich syndrome protein (WASP)-VCA fragment and Arp2/3 complex. These results lead us to propose that cortactin plays a critical role in translating actin polymerization at a bead surface into motion, by releasing WASP-VCA from the new branching site. This enhanced release has two major effects: it increases the turnover rate of branching per WASP molecule, and it decreases the friction-like force caused by the binding of the moving surface with respect to the growing actin network.

Co-assembly, spatiotemporal control and morphogenesis of a hybrid protein–peptide system

“…The release of the stored elastic energy produces a stress that opens the membrane. This phenomenon may be similar to that reported by Sykes and co-workers 34 , in which local rupture of an actin membrane resulted in symmetry breaking and the release of elastic energy accumulated during the formation of the membrane 35,36 . In the system described by Sykes and co-workers, elastic stress resulted from the polymerization of a branched actin network 34 ; however, in our case the stress may emerge from the PAK3-driven ELP5 opening and subsequent ELP5/PAK3 interactions within the membrane.…”

Interactions with Actin Monomers, Actin Filaments, and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks

“…7H). Dimeric WASP-VCA was recently shown by TIRF microscopy to increase Arp2/3 complex-mediated branching in the presence of cortactin (22). However, it is unknown whether the biochemical differences between WASP and N-WASP influence the potency or mechanism of synergy.…”

“…WASP proteins are thought to provide a transient connection between membranes and the polymerizing actin networks by binding the barbed ends of polymerizing filaments, nascent branch junctions, or both (75,76). Using the displacement mechanism to synergistically activate Arp2/3 complex with WASP family proteins, cortactin can not only increase rates of branching nucleation but may also decrease the lifetime of WASP-mediated connections between the polymerizing network and the membrane (22). The importance of WASP-mediated connections to polymerizing actin networks has been demonstrated using in vitro bead motility assays (75), and an important question to resolve will be the role of these connections in modulating actin network-membrane interactions in vivo.…”

Toward the reconstitution of synthetic cell motility

“…Yet the same surfacebound NPFs also inherently prevent the translation of the polymerization forces into motion, essentially because the NPF molecule has to be in contact with the network during the formation of the new branch. In a recent study it was shown that cortactin relaxes this internal inhibition by enhancing the release of the NPFs from the new branch, thereby decreasing the networksurface binding time, required for fast and persistent movement 28 ( Fig. 2A(c,d)).…”

“…Cortactin stimulate the release of the surface-bound NPFs from the new branch, thereby increasing the turnover rate of branching per NPFs molecule and decreasing the network-membrane binding time, required for fast protrusion rates. [28][29][30] The branched nucleation process is followed by filament elongation that pushes the membrane forward. In response to this deformation, the membrane applies resistive elastic forces on the growing filaments.…”