Cell wound repair requires the coordinated action of linear and branched actin nucleation factors
Authors: Justin Hui; Mitsutoshi Nakamura; Susan Parkhurst
Affiliation: Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA
Keywords: a. cytoskeleton; a. cytoskeleton
During cellular wound repair, actin and other cytoskeletal proteins act in concert to coordinate wound closure in a timely fashion. A critical step is to form an actomyosin ring that contracts to close the wound. This process is dynamically regulated in part by Rho family GTPases, which through their different effectors can regulate both linear and branched actin nucleators to facilitate cytoskeletal dynamics. We have previously shown that knockdown of the formin Diaphanous, a linear actin nucleator and downstream effector of Rho, results in prolonged wound closure and the inability to form a proper actomyosin ring. However, when all linear nucleation factors are removed, some actin organization at the wound persists. We investigated the contribution of branched nucleation factors to cell wound repair. We show that the Wiskott-Aldrich Syndrome (WAS) protein family members (WASp, Wash, and SCAR), and their cofactor Arp2/3, play non-redundant roles in regulating actin organization and architecture of the contractile actomyosin ring. Notably, we observe stark differences in the spatial and temporal recruitment patterns to wounds among the WAS proteins during the wound repair process. In particular, WASp is recruited early, SCAR recruitment peaks late, and Wash is present throughout the process. Further, individual knockdown of the WAS proteins resulted in different actomyosin ring architectures, including differences in mesh density and filament orientation. Dynamically, WASp knockdown exhibits slower contraction whereas SCAR contracts faster and Wash contracts similarly to wildtype. Interestingly, in the absence of branched actin, wounds exhibited an abundance of elongated linear filaments that colocalize with Diaphanous, as well as other unique actin structures, which colocalize with Myosin. When both linear and branched nucleation factors are simultaneously knocked down, we observe a significant decrease of these elongated filaments and the absence of previously seen actin structures. We also inhibited myosin activity in an Arp3 knockdown and observed a surprising wound repair phenotype consisting of spiraling linear filaments and excessive actin bundling. Our results emphasize the strong requirement for balance and crosstalk among linear and branched actin nucleators, as well as myosin, to facilitate proper actin filament architecture, organization, and dynamic contractile closure of the actomyosin ring during cell wound repair.