Glia maturation factor-? phosphorylation at Tyr-104 regulates actin dynamics and contraction in human airway smooth muscle.
ABSTRACT: Actin dynamics plays an essential role in regulating airway smooth muscle contraction. The mechanisms that regulate actin dynamics in smooth muscle are not completely understood. Glia maturation factor (GMF) is a protein that has been reported to inhibit actin nucleation and to induce actin network debranching in vitro. The role of GMF in human smooth muscle cells and tissues has not been investigated. In this study, knockdown of GMF-? by RNA interference enhanced actin polymerization and contraction in human airway smooth muscle (HASM) cells and tissues without affecting myosin phosphorylation (another important biochemical change during contractile activation). Activation of HASM cells and tissues with acetylcholine induced dissociation of GMF-? from Arp2 of the Arp2/3 complex. Acetylcholine stimulation also increased GMF-? phosphorylation at Tyr-104. GMF-? phosphorylation at this residue was mediated by c-Abl tyrosine kinase. The GMF-? mutant Y104F (phenylalanine substitution at Tyr-104) had higher association with Arp2 in HASM cells upon contractile activation. Furthermore, expression of mutant Y104F GMF-? attenuated actin polymerization and contraction in smooth muscle. Thus, we propose a novel mechanism for the regulation of actin dynamics and smooth muscle contraction. In unstimulated smooth muscle, GMF-? binds to the Arp2/3 complex, which induces actin disassembly and retains lower levels of F-actin. Upon contractile stimulation, phosphorylation at Tyr-104 mediated by c-Abl tyrosine kinase leads to the dissociation of GMF-? from Arp2/3, by which GMF-? no longer induces actin disassembly. Reduced actin disassembly renders F-actin in higher level, which facilitates smooth muscle contraction.
Project description:The lamellipodium is an important structure for cell migration containing branched actin nucleated via the Arp2/3 complex. The formation of branched actin is relatively well studied, but less is known about its disassembly and how this influences migration. GMF is implicated in both Arp2/3 debranching and inhibition of Arp2/3 activation. Modulation of GMF?, a ubiquitous GMF isoform, by depletion or overexpression resulted in changes in lamellipodial dynamics, branched actin content, and migration. Acute pharmacological inhibition of Arp2/3 by CK-666, coupled to quantitative live-cell imaging of the complex, showed that depletion of GMF? decreased the rate of branched actin disassembly. These data, along with mutagenesis studies, suggest that debranching (not inhibition of Arp2/3 activation) is a primary activity of GMF? in vivo. Furthermore, depletion or overexpression of GMF? disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMF? plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration.
Project description:Lamellipodia are dynamic actin-rich cellular extensions that drive advancement of the leading edge during cell migration. Lamellipodia undergo periodic extension and retraction cycles, but the molecular mechanisms underlying these dynamics and their role in cell migration have remained obscure. We show that glia-maturation factor (GMF), which is an Arp2/3 complex inhibitor and actin filament debranching factor, regulates lamellipodial protrusion dynamics in living cells. In cultured S2R(+) cells, GMF silencing resulted in an increase in the width of lamellipodial actin filament arrays. Importantly, live-cell imaging of mutant Drosophila egg chambers revealed that the dynamics of actin-rich protrusions in migrating border cells is diminished in the absence of GMF. Consequently, velocity of border cell clusters undergoing guided migration was reduced in GMF mutant flies. Furthermore, genetic studies demonstrated that GMF cooperates with the Drosophila homolog of Aip1 (flare) in promoting disassembly of Arp2/3-nucleated actin filament networks and driving border cell migration. These data suggest that GMF functions in vivo to promote the disassembly of Arp2/3-nucleated actin filament arrays, making an important contribution to cell migration within a 3D tissue environment.
Project description:Cell locomotion and endocytosis are powered by the rapid polymerization and turnover of branched actin filament networks nucleated by Arp2/3 complex. Although a large number of cellular factors have been identified that stimulate Arp2/3 complex-mediated actin nucleation, only a small number of studies so far have addressed which factors promote actin network debranching. Here, we investigated the function of a conserved homolog of ADF/cofilin, glia maturation factor (GMF). We found that S. cerevisiae GMF (also called Aim7) localizes in vivo to cortical actin patches and displays synthetic genetic interactions with ADF/cofilin. However, GMF lacks detectable actin binding or severing activity and instead binds tightly to Arp2/3 complex. Using in vitro evanescent wave microscopy, we demonstrated that GMF potently stimulates debranching of actin filaments produced by Arp2/3 complex. Further, GMF inhibits nucleation of new daughter filaments. Together, these data suggest that GMF binds Arp2/3 complex to both "prune" daughter filaments at the branch points and inhibit new actin assembly. These activities and its genetic interaction with ADF/cofilin support a role for GMF in promoting the remodeling and/or disassembly of branched networks. Therefore, ADF/cofilin and GMF, members of the same superfamily, appear to have evolved to interact with actin and actin-related proteins, respectively, and to make mechanistically distinct contributions to the remodeling of cortical actin structures.
Project description:Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 ?M affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.
Project description:Actin filament polymerization plays a critical role in the regulation of smooth muscle contraction. However, our knowledge regarding modulation of the actin cytoskeleton in smooth muscle just begins to accumulate. In this study, stimulation with acetylcholine (ACh) induced an increase in the association of the adapter protein c-Abl interactor 1 (Abi1) with neuronal Wiskott-Aldrich syndrome protein (N-WASP) (an actin-regulatory protein) in smooth muscle cells/tissues. Furthermore, contractile stimulation activated N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor. Abi1 knockdown by lentivirus-mediated RNAi inhibited N-WASP activation, actin polymerization, and contraction in smooth muscle. However, Abi1 silencing did not affect myosin regulatory light chain phosphorylation at Ser-19 in smooth muscle. In addition, c-Abl tyrosine kinase and Crk-associated substrate (CAS) have been shown to regulate smooth muscle contraction. The interaction of Abi1 with c-Abl and CAS has not been investigated. Here, contractile activation induced formation of a multiprotein complex including c-Abl, CAS, and Abi1. Knockdown of c-Abl and CAS attenuated the activation of Abi1 during contractile activation. More importantly, Abi1 knockdown inhibited c-Abl phosphorylation at Tyr-412 and the interaction of c-Abl with CAS. These results suggest that Abi1 is an important component of the cellular process that regulates N-WASP activation, actin dynamics, and contraction in smooth muscle. Abi1 is activated by the c-Abl-CAS pathway, and Abi1 reciprocally controls the activation of its upstream regulator c-Abl.
Project description:Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.
Project description:Profilin-1 (Pfn-1) is an actin-regulatory protein that has a role in modulating smooth muscle contraction. However, the mechanisms that regulate Pfn-1 in smooth muscle are not fully understood. Here, stimulation with acetylcholine induced an increase in the association of the adapter protein cortactin with Pfn-1 in smooth muscle cells/tissues. Furthermore, disruption of the protein/protein interaction by a cell-permeable peptide (CTTN-I peptide) attenuated actin polymerization and smooth muscle contraction without affecting myosin light chain phosphorylation at Ser-19. Knockdown of cortactin by lentivirus-mediated RNAi also diminished actin polymerization and smooth muscle force development. However, cortactin knockdown did not affect myosin activation. In addition, cortactin phosphorylation has been implicated in nonmuscle cell migration. In this study, acetylcholine stimulation induced cortactin phosphorylation at Tyr-421 in smooth muscle cells. Phenylalanine substitution at this position impaired cortactin/Pfn-1 interaction in response to contractile activation. c-Abl is a tyrosine kinase that is necessary for actin dynamics and contraction in smooth muscle. Here, c-Abl silencing inhibited the agonist-induced cortactin phosphorylation and the association of cortactin with Pfn-1. Finally, treatment with CTTN-I peptide reduced airway resistance and smooth muscle hyperreactivity in a murine model of asthma. These results suggest that the interaction of cortactin with Pfn-1 plays a pivotal role in regulating actin dynamics, smooth muscle contraction, and airway hyperresponsiveness in asthma. The association of cortactin with Pfn-1 is regulated by c-Abl-mediated cortactin phosphorylation.
Project description:Abl is a nonreceptor tyrosine kinase that is required for smooth muscle contraction. However, the mechanism by which Abl regulates smooth muscle contraction is not completely understood. In the present study, Abl underwent phosphorylation at Tyr412 (an index of Abl activation) in smooth muscle in response to contractile activation. Treatment with a cell-permeable decoy peptide, but not the control peptide, attenuated Abl phosphorylation during contractile stimulation. Treatment with the decoy peptide did not affect the association of Abl with the cytoskeletal protein vinculin and the spatial location of vinculin in smooth muscle. Inhibition of Abl phosphorylation by the decoy peptide attenuated the agonist-induced phosphorylation of Crk-associated substrate (CAS), an adapter protein participating in the signaling processes that regulate force development in smooth muscle. Additionally, previous studies have shown that contractile stimulation triggers the dissociation of CAS from the vimentin network, which is important for cytoskeletal signaling and contraction in smooth muscle. In this report, the decrease in the amount of CAS in cytoskeletal vimentin in response to contractile activation was reversed by the Abl inhibition with the decoy peptide. Moreover, force development and the enhancement of F-actin-to-G-actin ratios (an indication of actin polymerization) upon contractile activation were also attenuated by the Abl inhibition. However, myosin phosphorylation induced by contractile activation was not affected by the inhibition of Abl. These results suggest that Abl regulates the dissociation of CAS from the vimentin network, actin polymerization, and contraction by modulating CAS phosphorylation in smooth muscle.
Project description:The Arp2/3 complex mediates formation of complex cellular structures such as lamellipodia by nucleating branched actin filaments. Arp2/3-complex activity is precisely controlled by over a dozen regulators, yet the structural mechanism by which regulators interact with the complex is unknown. GMF is a recently discovered regulator of the Arp2/3 complex that can inhibit nucleation and disassemble branches. We solved the structure of the 240-kDa assembly of Mus musculus GMF and Bos taurus Arp2/3 complex and found that GMF binds the barbed end of Arp2, overlapping with the proposed binding site of WASP-family proteins. The structure suggests that GMF can bind branch junctions in the manner that cofilin binds filament sides, consistent with a modified cofilin-like mechanism for debranching by GMF. The GMF-Arp2 interface reveals how the ADF-H actin-binding domain in GMF is exploited to specifically recognize Arp2/3 complex and not actin.
Project description:Vinculin localizes to membrane adhesion junctions where it links actin filaments to the extracellular matrix by binding to the integrin-binding protein talin at its head domain (Vh) and to actin filaments at its tail domain (Vt). Vinculin can assume an inactive (closed) conformation in which Vh and Vt bind to each other, masking the binding sites for actin and talin, and an active (open) conformation in which the binding sites for talin and actin are exposed. We hypothesized that the contractile activation of smooth muscle tissues might regulate the activation of vinculin and thereby contribute to the regulation of contractile tension. Stimulation of tracheal smooth muscle tissues with acetylcholine (ACh) induced the recruitment of vinculin to cell membrane and its interaction with talin and increased the phosphorylation of membrane-localized vinculin at the C-terminal Tyr-1065. Expression of recombinant vinculin head domain peptide (Vh) in smooth muscle tissues, but not the talin-binding deficient mutant head domain, VhA50I, inhibited the ACh-induced recruitment of endogenous vinculin to the membrane and the interaction of vinculin with talin and also inhibited vinculin phosphorylation. Expression of Vh peptide also inhibited ACh-induced smooth muscle contraction and inhibited ACh-induced actin polymerization; however, it did not affect myosin light chain phosphorylation, which is necessary for cross-bridge cycling. Inactivation of RhoA inhibited vinculin activation in response to ACh. We conclude that ACh stimulation regulates vinculin activation in tracheal smooth muscle via RhoA and that vinculin activation contributes to the regulation of active tension by facilitating connections between actin filaments and talin-integrin adhesion complexes and by mediating the initiation of actin polymerization.