Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscle

Abstract

Contractile stimulation has been shown to initiate actin polymerization in smooth muscle tissues, and this actin polymerization is required for active tension development. We evaluated whether neuronal Wiskott-Aldrich syndrome protein (N-WASp)-mediated activation of the actin-related proteins 2 and 3 (Arp2/3) complex regulates actin polymerization and tension development initiated by muscarinic stimulation in canine tracheal smooth muscle tissues. In vitro, the COOH-terminal CA domain of N-WASp acts as an inhibitor of N-WASp-mediated actin polymerization; whereas the COOH-terminal VCA domain of N-WASp is constitutively active and is sufficient by itself to catalyze actin polymerization. Plasmids encoding EGFP-tagged wild-type N-WASp, the N-WASp VCA and CA domains, or enhanced green fluorescent protein (EGFP) were introduced into tracheal smooth muscle strips by reversible permeabilization, and the tissues were incubated for 2 days to allow for expression of the proteins. Expression of the CA domain inhibited actin polymerization and tension development in response to ACh, whereas expression of the wild-type N-WASp, the VCA domain, or EGFP did not. The increase in myosin light-chain (MLC) phosphorylation in response to contractile stimulation was not affected by expression of either the CA or VCA domain of N-WASp. Stimulation of the tissues with ACh increased the association of the Arp2/3 complex with N-WASp, and this association was inhibited by expression of the CA domain. The results demonstrate that 1) N-WASp-mediated activation of the Arp2/3 complex is necessary for actin polymerization and tension development in response to muscarinic stimulation in tracheal smooth muscle and 2) these effects are independent of the regulation of MLC phosphorylation.

The Plastic Nature of the Vascular Wall: A Continuum of Remodeling Events Contributing to Control of Arteriolar Diameter and Structure

“…For vascular smooth muscle cells, vasoconstrictors and growth factors are common humoral factors that induce changes in tension development by the cytoskeleton through crossbridge cycling and structural remodeling (43, 44, 136, 137, 163). As discussed above, increasing evidence indicates that the contractile process in smooth muscle is accompanied by changes in actin polymerization and the recruitment of structural proteins (including, vinculin, paxillin, talin and ␣-actinin) to the cytoplasmic side of focal adhesion sites (68, 161,165,[180][181][182][183]. It has been shown that these changes strengthen and stiffen the cytoskeleton, increasing the mechanical efficiency of the cell for the transmission of force, while reducing the elasticity of the cell over time.…”

The effects of the small GTPase RhoA on the muscarinic contraction of airway smooth muscle result from its role in regulating actin polymerization

“…Contractile agonists have been shown to stimulate actin polymerization in a number of smooth muscle cells and tissues, and the critical role of actin polymerization in regulating active tension development in smooth muscle is well documented (4,8,12,16,18,22,29,38,63,64,67). The inhibition of actin polymerization using either pharmacologic or molecular approaches has been shown to depress tension development in many smooth muscles with little or no effect on MLC phosphorylation or cross-bridge cycling (16, 38, 42, 43, 49, 51, 57-59, 64, 65).…”

Reversal of Aging‐Induced Increases in Aortic Stiffness by Targeting Cytoskeletal Protein‐Protein Interfaces

“…Previous studies on animal models have shown an involvement of the N‐WASP protein from the WASL gene in the regulation of actin polymerization and contractility in smooth muscle of mammalian airways,36 but effects on aortic stiffness have not been determined. Thus, N‐WASP was an attractive candidate molecule to pursue with a decoy peptide strategy to determine effects on stiffness.…”

“…The CA domain of N‐WASP is a subset of the C‐terminal VCA domain by which N‐WASP activates the Arp 2/3 complex, inducing actin polymerization at branches from the side of parent actin filaments 36, 37. The CA domain binds to G‐actin, and because it lacks the V domain, acts as a dominant negative inhibitor of N‐WASP‐mediated actin polymerization in airway smooth muscle,36 but its effects in vascular smooth muscle are unknown.…”

“…The CA domain binds to G‐actin, and because it lacks the V domain, acts as a dominant negative inhibitor of N‐WASP‐mediated actin polymerization in airway smooth muscle,36 but its effects in vascular smooth muscle are unknown. In order to test the novel idea that the CA peptide and other decoy cytoskeletal peptides might function as a useful approach to inhibit aortic stiffness and vascular contractility, we synthesized a series of constructs using the generic design illustrated in Figure 2A to incorporate the TAT cell permeation tag38, 39 and a fluorescent tag together with the peptide.…”