Agrin triggers the clustering of raft-associated acetylcholine receptors through actin cytoskeleton reorganization

Background information. Cholesterol/sphingolipid‐rich membrane microdomains or membrane rafts have been implicated in various aspects of receptor function such as activation, trafficking and synapse localization. More specifically in muscle, membrane rafts are involved in AChR (acetylcholine receptor) clustering triggered by the neural factor agrin, a mechanism considered integral to NMJ (neuromuscular junction) formation. In addition, actin polymerization is required for the formation and stabilization of AChR clusters in muscle fibres. Since membrane rafts are platforms sustaining actin nucleation, we hypothesize that these microdomains provide the suitable microenvironment favouring agrin/MuSK (mu scle‐s pecific k inase) signalling, eliciting in turn actin cytoskeleton reorganization and AChR clustering. However, the identity of the signalling pathways operating through these microdomains still remains unclear. Results. In this work, we attempted to identify the interactions between membrane raft components and cortical skeleton that regulate, upon signalling by agrin, the assembly and stabilization of synaptic proteins of the postsynaptic membrane domain at the NMJ. We provide evidence that in C2C12 myotubes, agrin triggers the association of a subset of membrane rafts enriched in AChR, the ‐MuSK and Cdc42 (cell division cycle 42) to the actin cytoskeleton. Disruption of the liquid‐ordered phase by methyl‐β‐cyclodextrin abolished this association. We further show that actin and the actin‐nucleation factors, N‐WASP (neuronal Wiscott—Aldrich syndrome protein) and Arp2/3 (actin‐related protein 2/3) are transiently associated with rafts on agrin engagement. Consistent with these observations, pharmacological inhibition of N‐WASP activity perturbed agrin‐elicited AChR clustering. Finally, immunoelectron microscopic analyses of myotube membrane uncovered that AChRs were constitutively associated with raft nanodomains at steady state that progressively coalesced on agrin activation. These rearrangements of membrane domains correlated with the reorganization of cortical actin cytoskeleton through concomitant and transient recruitment of the Arp2/3 complex to AChR‐enriched rafts. Conclusions. The present observations support the notion that membrane rafts are involved in AChR clustering by promoting local actin cytoskeleton reorganization through the recruitment of effectors of the agrin/MuSK signalling cascade. These mechanisms are believed to play an important role in vivo in the formation of the NMJ.     

A Second Las17 Monomeric Actin‐Binding Motif Functions in Arp2/3‐Dependent Actin Polymerization During Endocytosis

During clathrin‐mediated endocytosis (CME), actin assembly provides force to drive vesicle internalization. Members of the Wiskott–Aldrich syndrome protein (WASP) family play a fundamental role stimulating actin assembly. WASP family proteins contain a WH2 motif that binds globular actin (G‐actin) and a central‐acidic motif that binds the Arp2/3 complex, thus promoting the formation of branched actin filaments. Yeast WASP (Las17) is the strongest of five factors promoting Arp2/3‐dependent actin polymerization during CME. It was suggested that this strong activity may be caused by a putative second G‐actin‐binding motif in Las17. Here, we describe the in vitro and in vivo characterization of such Las17 G‐actin‐binding motif (LGM) and its dependence on a group of conserved arginine residues. Using the yeast two‐hybrid system, GST‐pulldown, fluorescence polarization and pyrene‐actin polymerization assays, we show that LGM binds G‐actin and is necessary for normal Arp2/3‐mediated actin polymerization in vitro. Live‐cell fluorescence microscopy experiments demonstrate that LGM is required for normal dynamics of actin polymerization during CME. Further, LGM is necessary for normal dynamics of endocytic machinery components that are recruited at early, intermediate and late stages of endocytosis, as well as for optimal endocytosis of native CME cargo. Both in vitro and in vivo experiments show that LGM has relatively lower potency compared to the previously known Las17 G‐actin‐binding motif, WH2. These results establish a second G‐actin‐binding motif in Las17 and advance our knowledge on the mechanism of actin assembly during CME.      

Scar1 and the related Wiskott–Aldrich syndrome protein, WASP, regulate the actin cytoskeleton through the Arp2/3 complex

Background: The actin-related proteins Arp2 and Arp3 are part of a seven-protein complex which is localized in the lamellipodia of a variety of cell types, and in actin-rich spots of unknown function. The Arp2/3 complex enhances actin nucleation and causes branching and crosslinking of actin filaments in vitro; in vivo it is thought to drive the formation of lamellipodia and to be a control center for actin-based motility. The Wiskott–Aldrich syndrome protein, WASP, is an adaptor protein implicated in the transmission of signals from tyrosine kinase receptors and small GTPases to the actin cytoskeleton. Scar1 is a member of a new family of proteins related to WASP, and it may also have a role in regulating the actin cytoskeleton. Scar1 is the human homologue of Dictyostelium Scar1, which is thought to connect G-protein-coupled receptors to the actin cytoskeleton. The mammalian Scar family contains at least four members. We have examined the relationships between WASP, Scar1, and the Arp2/3 complex.Results: We have identified WASP and its relative Scar1 as proteins that interact with the Arp2/3 complex. We have used deletion analysis to show that both WASP and Scar1 interact with the p21 subunit of the Arp2/3 complex through their carboxyl termini. Overexpression of carboxy-terminal fragments of Scar1 or WASP in cells caused a disruption in the localization of the Arp2/3 complex and, concomitantly, induced a complete loss of lamellipodia and actin spots. The induction of lamellipodia by platelet-derived growth factor was also suppressed by overexpression of the fragment of Scar1 that binds to the Arp2/3 complex.Conclusions: We have identified a conserved sequence domain in proteins of the WASP family that binds to the Arp2/3 complex. Overexpression of this domain in cells disrupts the localization of the Arp2/3 complex and inhibits lamellipodia formation. Our data suggest that WASP-related proteins may regulate the actin cytoskeleton through the Arp2/3 complex.     

Analysis of functional surfaces on the actin nucleation promoting factor Dip1 required for Arp2/3 complex activation and endocytic actin network assembly

Arp2/3 complex nucleates branched actin filaments that drive processes like endocytosis and lamellipodial protrusion. WISH/DIP/SPIN90 (WDS) proteins form a class of Arp2/3 complex activators or nucleation promoting factors (NPFs) that, unlike WASP family NPFs, activate Arp2/3 complex without requiring preformed actin filaments. Therefore, activation of Arp2/3 complex by WDS proteins is thought to produce the initial actin filaments that seed branching nucleation by WASP-bound Arp2/3 complexes. However, whether activation of Arp2/3 complex by WDS proteins is important for the initiation of branched actin assembly in cells has not been directly tested. Here, we used structure-based point mutations of the Schizosaccharomyces pombe WDS protein Dip1 to test the importance of its Arp2/3-activating activity in cells. Six of thirteen Dip1 mutants caused severe defects in Arp2/3 complex activation in vitro, and we found a strong correlation between the ability of mutants to activate Arp2/3 complex and to rescue endocytic actin assembly defects caused by deleting Dip1. These data support a model in which Dip1 activates Arp2/3 complex to produce actin filaments that initiate branched actin assembly at endocytic sites. Dip1 mutants that synergized with WASP in activating Arp2/3 complex in vitro showed milder defects in cells compared to those that did not, suggesting that in cells the two NPFs may coactivate Arp2/3 complex to initiate actin assembly. Finally, the mutational data reveal important complementary electrostatic contacts at the Dip1–Arp2/3 complex interface and corroborate the previously proposed wedge model, which describes how Dip1 binding triggers structural changes that activate Arp2/3 complex.     

Direct regulation of Arp2/3 complex activity and function by the actin binding protein coronin

Mechanisms for activating the actin-related protein 2/3 (Arp2/3) complex have been the focus of many recent studies. Here, we identify a novel mode of Arp2/3 complex regulation mediated by the highly conserved actin binding protein coronin. Yeast coronin (Crn1) physically associates with the Arp2/3 complex and inhibits WA- and Abp1-activated actin nucleation in vitro. The inhibition occurs specifically in the absence of preformed actin filaments, suggesting that Crn1 may restrict Arp2/3 complex activity to the sides of filaments. The inhibitory activity of Crn1 resides in its coiled coil domain. Localization of Crn1 to actin patches in vivo and association of Crn1 with the Arp2/3 complex also require its coiled coil domain. Genetic studies provide in vivo evidence for these interactions and activities. Overexpression of CRN1 causes growth arrest and redistribution of Arp2 and Crn1p into aberrant actin loops. These defects are suppressed by deletion of the Crn1 coiled coil domain and by arc35-26, an allele of the p35 subunit of the Arp2/3 complex. Further in vivo evidence that coronin regulates the Arp2/3 complex comes from the observation that crn1 and arp2 mutants display an allele-specific synthetic interaction. This work identifies a new form of regulation of the Arp2/3 complex and an important cellular function for coronin.     

How tropomyosin regulates lamellipodial actin‐based motility: a combined biochemical and reconstituted motility approach

At the leading edge of migrating cells, protrusive forces are developed by the assembly of actin filaments organised in a lamellipodial dendritic array at the front and a more distal lamellar linear array. Whether these two arrays are distinct or functionally linked and how they contribute to cell migration is an open issue. Tropomyosin severely inhibits lamellipodium formation and facilitates the lamellar array while enhancing migration, by a mechanism that is not understood. Here we show that the complex in vivo effects of tropomyosin are recapitulated in the reconstituted propulsion of neural Wiskott–Aldrich syndrome protein (N‐WASP)‐functionalised beads, which is based on the sole formation of a dendritic array of actin‐related protein (Arp)2/3‐branched filaments. Actin‐depolymerising factor (ADF) and tropomyosin control the length of the actin tail. By competing with Arp2/3 during filament branching, tropomyosin displays opposite effects on propulsion depending on the surface density of N‐WASP. Tropomyosin binding to the dendritic array is facilitated following filament debranching, causing its enrichment at the rear of the actin tail, like in vivo. These results unveil the mechanism by which tropomyosin generates two morphologically and dynamically segregated actin networks from a single one.     

p41-Arc subunit of human Arp2/3 complex is a p21-activated kinase-1-interacting substrate

The formation of new branched actin filament networks at the cell cortex of migrating cells is choreographed by the actin-related protein (Arp) 2/3 complex. Despite the fundamental role of the Arp2/3 complex in actin nucleation and branching, upstream signals that control the functions of p41-Arc, a putative regulatory component of the mammalian Arp2/3 complex, remain unidentified. Here we show that p41-Arc interacts with p21-activated kinase 1 (Pak1) both in vitro and in vivo. Pak1 phosphorylation of p41-Arc regulates its localization with the Arp2/3 complex in the cortical nucleation regions of cells. Pak1 phosphorylates p41-Arc on threonine 21 in the first WD repeat, and its mutation has functional implications in vivo. Threonine 21 phosphorylation by Pak1 is required for both constitutive and growth-factor-induced cell motility. Pak1 regulation of p41-Arc activation status represents a novel mechanism by which signalling pathways may influence the functions of the Arp2/3 complex, leading to motility in mammalian cells.     

Collapsin Response Mediator Protein-1 Regulates Arp2/3-dependent Actin Assembly

Listeria monocytogenes is a bacterial parasite that uses host proteins to assemble an Arp2/3-dependent actin comet tail to power its movement through the host cell. Initiation of comet tail assembly is more efficient in cytosol than it is under defined conditions, indicating that unknown factors contribute to the reaction. We therefore fractionated cytosol and identified CRMP-1 as a factor that facilitates Arp2/3-dependent Listeria actin cloud formation in the presence of Arp2/3 and actin alone. It also scored as an important factor for Listeria actin comet tail formation in brain cytosol. CRMP-1 does not nucleate actin assembly on its own, nor does it directly activate the Arp2/3 complex. Rather, CRMP-1 scored as an auxiliary factor that promoted the ability of Listeria ActA protein to activate the Arp2/3 complex to trigger actin assembly. CRMP-1 is one member of a family of five related proteins that modulate cell motility in response to extracellular signals. Our results demonstrate an important role for CRMP-1 in Listeria actin comet tail formation and open the possibility that CRMP-1 controls cell motility by modulating Arp2/3 activation.     

Immunological evidence for the presence of plant homologues of the actin- related protein Arp3 in tobacco and maize: subcellular localization to actin-enriched pit fields and emerging root hairs

Summary. The actin-nucleating and -organizing Arp2/3 protein complex is well known to be conserved throughout the eukaryotic kingdom. For higher plants, however, only limited evidence is available for the presence of the Arp2/3 complex so far. Using heterologous antibodies against the Dictyostelium discoideum and Schizosaccharomyces pombe proteins and a bovine peptide, we found immunological evidence for the presence of Arp3 homologues in plants. First, proteins with a molecular mass of about 47–50 kDa were clearly recognized in extracts of both a dicotyledonous plant (tobacco) and a monocotyledonous plant (maize) in immunoblots with the anti-Arp3 antibodies. Second, immunolocalization with these Arp3 antibodies was performed on different plant cells, selected for their diverse actin organizations and functions. On isolated plasma membrane ghosts derived from tobacco leaf protoplasts, a putative Arp3 was localized along cortical actin filaments. In the inner cortex of maize roots, Arp3 was localized to actin-rich plasmodesmata and pit fields and to multivesicular bodies in the cytoplasm. During root hair formation, distinct site-specific localization was found at the protruding apical plasma membrane portions of these tip-growing cells.Correspondence and reprints: Department of Biology, Universitaire Instelling Antwerpen, Universiteitsplein 1, 2610 Wilrijk, Belgium.Received January 3, 2003; accepted February 7, 2003; published online August 26, 2003     

Generation of branched actin networks: assembly and regulation of the N‐WASP and WAVE molecular machines

The Arp2/3 complex is a molecular machine that generates branched actin networks responsible for membrane remodeling during cell migration, endocytosis, and other morphogenetic events. This machine requires activators, which themselves are multiprotein complexes. This review focuses on recent advances concerning the assembly of stable complexes containing the most‐studied activators, N‐WASP and WAVE proteins, and the level of regulation that is provided by these complexes. N‐WASP is the paradigmatic auto‐inhibited protein, which is activated by a conformational opening. Even though this regulation has been successfully reconstituted in vitro with isolated N‐WASP, the native dimeric complex with a WIP family protein has unique additional properties. WAVE proteins are part of a pentameric complex, whose basal state and activated state when bound to the Rac GTPase were recently clarified. Moreover, this review attempts to put together diverse observations concerning the WAVE complex in the conceptual frame of an in vivo assembly pathway that has gained support from the recent identification of a precursor.     

Arp2/3 Complex from Acanthamoeba Binds Profilin and Cross-links Actin Filaments

The Arp2/3 complex was first purified from Acanthamoeba castellanii by profilin affinity chromatography. The mechanism of interaction with profilin was unknown but was hypothesized to be mediated by either Arp2 or Arp3. Here we show that the Arp2 subunit of the complex can be chemically cross-linked to the actin-binding site of profilin. By analytical ultracentrifugation, rhodamine-labeled profilin binds Arp2/3 complex with a K_d of 7 μM, an affinity intermediate between the low affinity of profilin for barbed ends of actin filaments and its high affinity for actin monomers. These data suggest the barbed end of Arp2 is exposed, but Arp2 and Arp3 are not packed together in the complex exactly like two actin monomers in a filament. Arp2/3 complex also cross-links actin filaments into small bundles and isotropic networks, which are mechanically stiffer than solutions of actin filaments alone. Arp2/3 complex is concentrated at the leading edge of motile Acanthamoeba, and its localization is distinct from that of α-actinin, another filament cross-linking protein. Based on localization and actin filament nucleation and cross-linking activities, we propose a role for Arp2/3 in determining the structure of the actin filament network at the leading edge of motile cells.     

ARPC1/Arc40 mediates the interaction of the actin-related protein 2 and 3 complex with Wiskott-Aldrich syndrome protein family activators

The actin-related protein 2 and 3 (Arp2/3) complex is a seven-subunit protein complex that nucleates actin filaments at the cell cortex. Despite extensive cross-linking, crystallography, genetic and biochemical studies, the contribution of each subunit to the activity of the complex remains largely unclear. In this study we characterized the function of the 40-kDa subunit, ARPC1/Arc40, of the yeast Arp2/3 complex. We showed that this subunit is indeed a stable component of the Arp2/3 complex, but its highly unusual electrophoretic mobility eluded detection in previous studies. Recombinant Arc40 bound the VCA domain of Wiskott-Aldrich syndrome protein family activators at a K(d) of 0.45 mum, close to that of the full complex with VCA (0.30 microm), and this interaction was dependent on the conserved tryptophan at the COOH terminus of VCA. Using a newly constructed Delta arc40 yeast strain, we showed that loss of Arc40 severely reduced the binding affinity of the Arp2/3 complex with VCA as well as the nucleation activity of the complex, suggesting that Arc40 contains an important contact site of the Arp2/3 complex with VCA. The Delta arc40 cells exhibited reduced growth rate, loss of actin patches, and accumulation of cables like actin aggregates, phenotypes typical of other subunit nulls, suggesting that Arc40 functions exclusively within the Arp2/3 complex.     

Insertions within the Actin Core of Actin-related Protein 3 (Arp3) Modulate Branching Nucleation by Arp2/3 Complex

The Arp2/3 (actin-related protein 2/3) complex nucleates branched actin filaments involved in multiple cellular functions, including endocytosis and cellular motility. Two subunits (Arp2 and Arp3) in this seven-subunit assembly are closely related to actin and upon activation of the complex form a “cryptic dimer” that stably mimics an actin dimer to nucleate a new filament. Both Arps contain a shared actin core structure, and each Arp contains multiple insertions of unknown function at conserved positions within the core. Here we characterize three key insertions within the actin core of Arp3 and show that each one plays a distinct role in modulating Arp2/3 function. The β4/β5 insert mediates interactions of Arp2/3 complex with actin filaments and “dampers” the nucleation activity of the complex. The Arp3 hydrophobic plug plays an important role in maintaining the integrity of the complex but is not absolutely required for formation of the daughter filament nucleus. Deletion of the αK/β15 insert did not constitutively activate the complex, as previously hypothesized. Instead, it abolished in vitro nucleation activity and caused defects in endocytic actin patch assembly in fission yeast, indicating a role for the αK/β15 insert in the activated state of the complex. Biochemical characterization of each mutant revealed steps in the nucleation pathway influenced by each Arp3-specific insert to provide new insights into the structural basis of activation of the complex.     

A novel synthetic small molecule YH‐306 suppresses colorectal tumour growth and metastasis via FAK pathway

Cell migration and invasion are key processes in the metastasis of cancer, and suppression of these steps is a promising strategy for cancer therapeutics. The aim of this study was to explore small molecules for treating colorectal cancer (CRC) and to investigate their anti‐metastatic mechanisms. In this study, six CRC cell lines were used. We showed that YH‐306 significantly inhibited the migration and invasion of CRC cells in a dose‐dependent manner. In addition, YH‐306 inhibited cell adhesion and protrusion formation of HCT116 and HT‐29 CRC cells. Moreover, YH‐306 potently suppressed uninhibited proliferation in all six CRC cell lines tested and induced cell apoptosis in four cell lines. Furthermore, YH‐306 inhibited CRC colonization in vitro and suppressed CRC growth in a xenograft mouse model, as well as hepatic/pulmonary metastasis in vivo. YH‐306 suppressed the activation of focal adhesion kinase (FAK), c‐Src, paxillin, and phosphatidylinositol 3‐kinases (PI3K), Rac1 and the expression of matrix metalloproteases (MMP) 2 and MMP9. Meanwhile, YH‐306 also inhibited actin‐related protein (Arp2/3) complex‐mediated actin polymerization. Taken together, YH‐306 is a candidate drug in preventing growth and metastasis of CRC by modulating FAK signalling pathway.     

Molecular dynamics simulations of Arp2/3 complex activation

Actin-related protein 2 and 3 (Arp2/3) complex forms a dendritic network of actin filaments during endocytosis and cellular locomotion by nucleating branches on the sides of preexisting actin filaments. Reconstructions of electron tomograms of branch junctions show how Arp2/3 complex anchors the branch, with Arp2 and Arp3 serving as the first two subunits of the branch. Our aim was to characterize the massive conformational change that moves Arp2 ∼30 Å from its position in crystal structures of inactive Arp2/3 complex to its position in branch junctions. Starting with the inactive crystal structure, we used atomistic-scale molecular dynamics simulations to drive Arp2 toward the position observed in branch junctions. When we applied forces to Arp2 while restraining Arp3, one block of structure (Arp2, subunit ARPC1, the globular domain of ARPC4 and ARPC5) rotated counterclockwise by 30° around a pivot point in an α-helix of ARPC4 (Glu⁸¹-Asn¹⁰⁰) to align Arp2 next to Arp3 in a second block of structure including ARPC3 and the globular domains of ARPC2. This active structure buried more surface area than the inactive conformation. The complex was stable in all simulations. In most simulations, collisions of subdomain 2 of Arp2 with Arp3 impeded the movement of Arp2.     

Diaphanous regulates SCAR complex localization during Drosophila myoblast fusion

From Drosophila to man, multinucleated muscle cells form through cell-cell fusion. Using Drosophila as a model system, researchers first identified, and then demonstrated, the importance of actin cytoskeletal rearrangements at the site of fusion. These actin rearrangements at the fusion site are regulated by SCAR and WASp mediated Arp2/3 activation, which nucleates branched actin networks. Loss of SCAR, WASp or both leads to defects in myoblast fusion. Recently, we have found that the actin regulator Diaphanous (Dia) also plays a role both in organizing actin and in regulating Arp2/3 activity at the fusion site. In this Extra View article, we provide additional data showing that the Abi-SCAR complex accumulates at the fusion site and that excessive SCAR activity impairs myoblast fusion. Using constitutively active Dia constructs, we provide additional evidence that Dia functions upstream of SCAR activity to regulate actin dynamics at the fusion site and to localize the Abi-SCAR complex.