Exo70 stimulates the arp2/3 complex for lamellipodia formation and directional cell migration

Directional cell migration requires the coordination of actin assembly and membrane remodeling. The exocyst is an octameric protein complex essential for exocytosis and plasma membrane remodeling [1, 2]. A component of the exocyst, Exo70, directly interacts with the Arp2/3 complex, a core nucleating factor for the generation of branched actin networks for cell morphogenesis and migration [3-9]. Using in?vitro actin polymerization assay and time-lapse total internal reflection fluorescence microscopy, we found that Exo70 functions as a kinetic activator of the Arp2/3 complex that promotes actin filament nucleation and branching. We further found that the effect of Exo70 on actin is mediated by promoting the interaction of the Arp2/3 complex with WAVE2, a member of the N-WASP/WAVE family of nucleation promoting factors. At the cellular level, the stimulatory effect of Exo70 on the Arp2/3 complex is required for lamellipodia formation and maintaining directional persistence of cell migration. Our findings provide a novel mechanism for regulating actin polymerization and branching for effective membrane protrusion during cell morphogenesis and migration.     

Cortactin: A multifunctional regulator of cellular invasiveness

Branched actin assembly is critical for a variety of cellular processes that underlie cell motility and invasion, including cellular protrusion formation and membrane trafficking. Activation of branched actin assembly occurs at various subcellular locations via site-specific activation of distinct WASp family proteins and the Arp2/3 complex. A key branched actin regulator that promotes cell motility and links signaling, cytoskeletal and membrane trafficking proteins is the Src kinase substrate and Arp2/3 binding protein cortactin. Due to its frequent overexpression in advanced, invasive cancers and its general role in regulating branched actin assembly at multiple cellular locations, cortactin has been the subject of intense study. Recent studies suggest that cortactin has a complex role in cellular migration and invasion, promoting both on-site actin polymerization and modulation of autocrine secretion. Diverse cellular activities may derive from the interaction of cortactin with site-specific binding partners.Key words: cortactin, migration, invasion, lamellipodia, invadopodia, cancer, actin, actin assembly, scaffold, membrane trafficking, secretion     

A novel role for 14-kDa phosphohistidine phosphatase in lamellipodia formation

Cell migration involves dynamic regulation of the actin cytoskeleton, which exhibits rapid actin polymerization at the leading edge of migrating cells. This process relies on regulated recruitment of actin nucleators and actin-binding proteins to the leading edge to polymerize new actin filaments. Many of these proteins have been identified, including the actin-related protein (Arp) 2/3 complex, which has emerged as the core player in the initiation of actin polymerization. However, the functional coordination of these proteins is unclear. Previously, we have demonstrated that the 14-kDa phosphohistidine phosphatase (PHP14) is involved in cell migration regulation and affects actin cytoskeleton reorganization. Here, we show that PHP14 may regulate actin remodeling directly and play an important role in dynamic regulation of the actin cytoskeleton. We observed a colocalization of PHP14 with Arp3 and F-actin at the leading edge of migrating cells. Moreover, PHP14 was recruited to the actin remodeling sites in parallel with Arp3 during lamellipodia formation. Furthermore, PHP14 knockdown impaired Arp3 localization at the leading edge of lamellipodia, as well as lamellipodia formation. Most importantly, we found that PHP14 was a novel F-actin-binding protein, displaying an Arp2/3-dependent localization to the leading edge. Collectively, our results indicated a crucial role for PHP14 in the dynamic regulation of the actin cytoskeleton and cell migration.     

From WRC to Arp2/3: Collective molecular mechanisms of branched actin network assembly

Branched actin networks have emerged as major force-generating structures driving the protrusions in various distinct cell types and processes, ranging from lamellipodia operating in mesenchymal and epithelial cell migration or tails pushing intracellular pathogens and vesicles to developing spine heads on neurons. Many key molecular features are conserved among all those Arp2/3 complex-containing, branched actin networks. Here, we will review recent progress in our molecular understanding of the core biochemical machinery driving branched actin nucleation, from the generation of filament primers to Arp2/3 activator recruitment, regulation and turnover. Due to the wealth of information on distinct, Arp2/3 network-containing structures, we are largely focusing—in an exemplary fashion—on canonical lamellipodia of mesenchymal cells, which are regulated by Rac GTPases, their downstream effector WAVE Regulatory Complex and its target Arp2/3 complex. Novel insight additionally confirms that WAVE and Arp2/3 complexes regulate or are themselves tuned by additional prominent actin regulatory factors, including Ena/VASP family members and heterodimeric capping protein. Finally, we are considering recent insights into effects exerted by mechanical force, both at the branched network and individual actin regulator level.     

GMFβ controls branched actin content and lamellipodial retraction in fibroblasts

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 GMFβ, 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 GMFβ 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 GMFβ in vivo. Furthermore, depletion or overexpression of GMFβ disrupted the ability of cells to directionally migrate to a gradient of fibronectin (haptotaxis). These data suggest that debranching by GMFβ plays an important role in branched actin regulation, lamellipodial dynamics, and directional migration.     

Caldesmon inhibits Arp2/3-mediated actin nucleation

The Arp2/3 complex greatly accelerates actin polymerization, which is thought to play a major role in cell motility by inducing membrane protrusions including ruffling movements. Membrane ruffles contain a variety of actin-binding proteins, which would modulate Arp2/3-dependent actin polymerization. However, their exact roles in actin polymerization remain to be established. Because caldesmon is present in membrane ruffles, as well as in stress fibers, it may alter Arp2/3-mediated actin polymerization. We have found that caldesmon greatly retards Arp2/3-induced actin polymerization. Kinetic analyses have revealed that caldesmon inhibits the nucleation process, whereas it does not largely reduce elongation. Caldesmon is found to inhibit binding of Arp2/3 to F-actin, which apparently reduces the ability of F-actin as a secondary activator of Arp2/3-mediated nucleation. We also have found that the inhibition of the binding between actin and caldesmon either by Ca(2+)/calmodulin or by phosphorylation with cdc2 kinase reverses the inhibitory effect of caldesmon on Arp2/3-induced actin polymerization. Our results suggest that caldesmon may be a key protein that modulates membrane ruffling and that this may involve changes in caldesmon phosphorylation and/or intracellular calcium concentrations during signal transduction.     

IQGAP1 stimulates actin assembly through the N-WASP-Arp2/3 pathway

IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, beta-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.     

N-WASP inhibitor wiskostatin nonselectively perturbs membrane transport by decreasing cellular ATP levels

Wiskott-Aldrich syndrome protein (WASP) and WAVE stimulate actin-related protein (Arp)2/3-mediated actin polymerization, leading to diverse downstream effects, including the formation and remodeling of cell surface protrusions, modulation of cell migration, and intracytoplasmic propulsion of organelles and pathogens. Selective inhibitors of individual Arp2/3 activators would enable more exact dissection of WASP- and WAVE-dependent cellular pathways and are potential therapeutic targets for viral pathogenesis. Wiskostatin is a recently described chemical inhibitor that selectively inhibits neuronal WASP (N-WASP)-mediated actin polymerization in vitro. A growing number of recent studies have utilized this drug in vivo to uncover novel cellular functions for N-WASP; however, the selectivity of wiskostatin in intact cells has not been carefully explored. In our studies with this drug, we observed rapid and dose-dependent inhibition of N-WASP-dependent membrane trafficking steps. Additionally, however, we found that addition of wiskostatin inhibited numerous other cellular functions that are not believed to be N-WASP dependent. Further studies revealed that wiskostatin treatment caused a rapid, profound, and irreversible decrease in cellular ATP levels, consistent with its global effects on cell function. Our data caution against the use of this drug as a selective perturbant of N-WASP-dependent actin dynamics in vivo. phosphatidylinositol 4,5-bisphosphate; cytoskeleton; membrane traffic; Arp2/3; actin comets     

The Conformational State of Actin Filaments Regulates Branching by Actin-related Protein 2/3 (Arp2/3) Complex

Actin is a highly ubiquitous protein in eukaryotic cells that plays a crucial role in cell mechanics and motility. Cell motility is driven by assembling actin as polymerizing actin drives cell protrusions in a process closely involving a host of other actin-binding proteins, notably the actin-related protein 2/3 (Arp2/3) complex, which nucleates actin and forms branched filamentous structures. The Arp2/3 complex preferentially binds specific actin networks at the cell leading edge and forms branched filamentous structures, which drive cell protrusions, but the exact regulatory mechanism behind this process is not well understood. Here we show using in vitro imaging and binding assays that a fragment of the actin-binding protein caldesmon added to polymerizing actin increases the Arp2/3-mediated branching activity, whereas it has no effect on branch formation when binding to aged actin filaments. Because this caldesmon effect is shown to be independent of nucleotide hydrolysis and phosphate release from actin, our results suggest a mechanism by which caldesmon maintains newly polymerized actin in a distinct state that has a higher affinity for the Arp2/3 complex. Our data show that this new state does not affect the level of cooperativity of binding by Arp2/3 complex or its distribution on actin. This presents a novel regulatory mechanism by which caldesmon, and potentially other actin-binding proteins, regulates the interactions of actin with its binding partners.     

Control of actin assembly and disassembly at filament ends

The most important discovery in the field is that the Arp2/3 complex nucleates assembly of actin filaments with free barbed ends. Arp2/3 also binds the sides of actin filaments to create a branched network. Arp2/3's nucleation activity is stimulated by WASP family proteins, some of which mediate signaling from small G-proteins. Listeria movement caused by actin polymerization can be reconstituted in vitro using purified proteins: Arp2/3 complex, capping protein, actin depolymerizing factor/cofilin, and actin. actin depolymerizing factor/cofilin increases the rate at which actin subunits leave pointed ends, and capping protein caps barbed ends.     

Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis

The dynamic regulation of actin polymerization plays crucial roles in cell morphology and endocytosis. The mechanistic details of these processes and the proteins involved are not fully understood, especially in neurons. PICK1 is a PDZ-BAR-domain protein involved in regulated AMPA receptor (AMPAR) endocytosis in neurons. Here, we demonstrate that PICK1 binds filamentous (F)-actin and the actin-nucleating Arp2/3 complex, and potently inhibits Arp2/3-mediated actin polymerization. RNA interference (RNAi) knockdown of PICK1 in neurons induces a reorganization of the actin cytoskeleton resulting in aberrant cell morphology. Wild-type PICK1 rescues this phenotype, but a mutant PICK1, PICK1(W413A), that does not bind or inhibit Arp2/3 has no effect. Furthermore, this mutant also blocks NMDA-induced AMPAR internalization. This study identifies PICK1 as a negative regulator of Arp2/3-mediated actin polymerization that is critical for a specific form of vesicle trafficking, and also for the development of neuronal architecture.     

The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.     

Arp2/3 is a negative regulator of growth cone translocation

Arp2/3 is an actin binding complex that is enriched in the peripheral lamellipodia of fibroblasts, where it forms a network of short, branched actin filaments, generating the protrusive force that extends lamellipodia and drives fibroblast motility. Although it has been assumed that Arp2/3 would play a similar role in growth cones, our studies indicate that Arp2/3 is enriched in the central, not the peripheral, region of growth cones and that the growth cone periphery contains few branched actin filaments. Arp2/3 inhibition in fibroblasts severely disrupts actin organization and membrane protrusion. In contrast, Arp2/3 inhibition in growth cones minimally affects actin organization and does not inhibit lamellipodia protrusion or de novo filopodia formation. Surprisingly, Arp2/3 inhibition significantly enhances axon elongation and causes defects in growth cone guidance. These results indicate that Arp2/3 is a negative regulator of growth cone translocation.     

Recruitment of the Arp2/3 complex to vinculin: coupling membrane protrusion to matrix adhesion

Cell migration involves many steps, including membrane protrusion and the development of new adhesions. Here we have investigated whether there is a link between actin polymerization and integrin engagement. In response to signals that trigger membrane protrusion, the actin-related protein (Arp)2/3 complex transiently binds to vinculin, an integrin-associated protein. The interaction is regulated, requiring phosphatidylinositol-4,5-bisphosphate and Rac1 activation, and is sufficient to recruit the Arp2/3 complex to new sites of integrin aggregation. Binding of the Arp2/3 complex to vinculin is direct and does not depend on the ability of vinculin to associate with actin. We have mapped the binding site for the Arp2/3 complex to the hinge region of vinculin, and a point mutation in this region selectively blocks binding to the Arp2/3 complex. Compared with WT vinculin, expression of this mutant in vinculin-null cells results in diminished lamellipodial protrusion and spreading on fibronectin. The recruitment of the Arp2/3 complex to vinculin may be one mechanism through which actin polymerization and membrane protrusion are coupled to integrin-mediated adhesion.     

Involvement of the Arp2/3 complex and Scar2 in Golgi polarity in scratch wound models

Cell motility and cell polarity are essential for morphogenesis, immune system function, and tissue repair. Many animal cells move by crawling, and one main driving force for movement is derived from the coordinated assembly and disassembly of actin filaments. As tissue culture cells migrate to close a scratch wound, this directional extension is accompanied by Golgi apparatus reorientation, to face the leading wound edge, giving the motile cell inherent polarity aligned relative to the wound edge and to the direction of cell migration. Cellular proteins essential for actin polymerization downstream of Rho family GTPases include the Arp2/3 complex as an actin nucleator and members of the Wiskott-Aldrich Syndrome protein (WASP) family as activators of the Arp2/3 complex. We therefore analyzed the involvement of the Arp2/3 complex and WASP-family proteins in in vitro wound healing assays using NIH 3T3 fibroblasts and astrocytes. In NIH 3T3 cells, we found that actin and Arp2/3 complex contributed to cell polarity establishment. Moreover, overexpression of N-terminal fragments of Scar2 (but not N-WASP or Scar1 or Scar3) interfere with NIH 3T3 Golgi polarization but not with cell migration. In contrast, actin, Arp2/3, and WASP-family proteins did not appear to be involved in Golgi polarization in astrocytes. Our results thus indicate that the requirement for Golgi polarity establishment is cell-type specific. Furthermore, in NIH 3T3 cells, Scar2 and the Arp2/3 complex appear to be involved in the establishment and maintenance of Golgi polarity during directed migration.     

The putative Arabidopsis arp2/3 complex controls leaf cell morphogenesis

The evolutionarily conserved Arp2/3 complex has been shown to activate actin nucleation and branching in several eukaryotes, but its biological functions are not well understood in multicellular organisms. The model plant Arabidopsis provides many advantages for genetic dissection of the function of this conserved actin-nucleating machinery, yet the existence of this complex in plants has not been determined. We have identified Arabidopsis genes encoding homologs of all of the seven Arp2/3 subunits. The function of the putative Arabidopsis Arp2/3 complex has been studied using four homozygous T-DNA insertion mutants for ARP2, ARP3, and ARPC5/p16. All four mutants display identical defects in the development of jigsaw-shaped epidermal pavement cells and branched trichomes in the leaf. These loss-of-function mutations cause mislocalization of diffuse cortical F-actin to the neck region and inhibit lobe extension in pavement cells. The mutant trichomes resemble those treated with the actin-depolymerizing drug cytochalasin D, exhibiting stunted branches but dramatically enlarged stalks due to depolarized growth suggesting defects in the formation of a fine actin network. Our data demonstrate that the putative Arabidopsis Arp2/3 complex controls cell morphogenesis through its roles in cell polarity establishment and polar cell expansion. Furthermore, our data suggest a novel function for the putative Arp2/3 complex in the modulation of the spatial distribution of cortical F-actin and provide evidence that the putative Arp2/3 complex may activate the polymerization of some types of actin filaments in specific cell types.     

Involvement of Arp2/3 complex in MCP-1-induced chemotaxis

The migrating monocyte shows dynamic actin polymerization in response to MCP-1. We investigated the involvement of the actin-related protein 2 and 3 complex (Arp2/3 complex) during chemotaxis of a human monocyte cell line (THP-1). To clarify whether the Arp2/3 complex directly polymerizes actin in response to MCP-1 stimulation, THP-1 cells were transfected with complementary DNA constructs encoding ScarWA. In ScarWA-transfected cells, neither recruitment of Arp2/3 complex at the leading edge nor actin polymerization was detected. Indeed, migration induced by MCP-1 was almost completely blocked. At the same time, transfection also interfered with the recruitment of integrin beta-1 at the leading edge and reduced affinity binding to fibronectin. Immunoprecipitation with an anti-Arp2 antibody showed that integrin beta-1 and WASP were co-precipitated under the condition of MCP-1 stimulation. These results indicate that interaction between the Arp2/3 complex and WASP stimulates actin polymerization and integrin beta-1-mediated adhesion during MCP-1-induced chemotaxis of THP-1 cells.     

Focal adhesion kinase controls actin assembly via a FERM-mediated interaction with the Arp2/3 complex

Networks of actin filaments, controlled by the Arp2/3 complex, drive membrane protrusion during cell migration. How integrins signal to the Arp2/3 complex is not well understood. Here, we show that focal adhesion kinase (FAK) and the Arp2/3 complex associate and colocalize at transient structures formed early after adhesion. Nascent lamellipodia, which originate at these structures, do not form in FAK-deficient cells, or in cells in which FAK mutants cannot be autophosphorylated after integrin engagement. The FERM domain of FAK binds directly to Arp3 and can enhance Arp2/3-dependent actin polymerization. Critically, Arp2/3 is not bound when FAK is phosphorylated on Tyr 397. Interfering peptides and FERM-domain point mutants show that FAK binding to Arp2/3 controls protrusive lamellipodia formation and cell spreading. This establishes a new function for the FAK FERM domain in forming a phosphorylation-regulated complex with Arp2/3, linking integrin signalling directly with the actin polymerization machinery.     

Acceleration of yeast actin polymerization by yeast Arp2/3 complex does not require an Arp2/3-activating protein

The Arp2/3 complex creates filament branches leading to an enhancement in the rate of actin polymerization. Work with Arp complexes from different sources indicated that it was inactive by itself, required an activating factor such as the Wiskott-Aldrich syndrome protein (WASP), and might exhibit a preference for ATP or ADP-P(i) actin. However, with yeast actin, P(i) release is almost concurrent with polymerization, eliminating the presence of an ADP-P(i) cap. We thus investigated the ability of the yeast Arp2/3 complex (yArp2/3) to facilitate yeast actin polymerization in the presence and absence of the Arp2/3-activating factor Las17p WA. yArp2/3 significantly accelerates yeast actin but not muscle actin polymerization in the absence of Las17p WA. The addition of Las17p WA further enhances yeast actin polymerization by yArp2/3 and allows the complex to now assist muscle actin polymerization. This actin isoform difference is not observed with bovine Arp2/3 complex, because the neural WASP VCA fragment is required for polymerization of both actins. Observation of individual branching filaments showed that Las17p WA increased the persistence of filament branches. Compared with wild type actin, the V159N mutant actin, proposed to be more ATP-like in behavior, exhibited an enhanced rate of polymerization in the presence of the yArp2/3 complex. yArp2/3 caused a significant rate of P(i) release prior to observation of an increase in filament mass but while branched structures were present. Thus, yeast F-actin can serve as a primary yArp2/3-activating factor, indicating that a newly formed yeast actin filament has a topology, unlike that of muscle actin, that is recognized specifically by yArp2/3.     

Activation of Arp2/3 complex by Wiskott-Aldrich Syndrome protein is linked to enhanced binding of ATP to Arp2

In response to signaling, the Arp2/3 complex (actin-related proteins 2 and 3 complex) is activated by binding the C-terminal (WA) domain of proteins of the Wiskott-Aldrich Syndrome family to promote the formation of a branched actin filament array, responsible for cell protrusion. The Arp2/3 complex exists in different structural/functional states: the inactive Arp2/3, the activated WA.Arp2/3 complex, the ternary G-actin.WA.Arp2/3 complex, which branches the filaments. This work addresses the role of ATP binding in Arp2/3 function. Using photo-cross-linking, hydrodynamic, and fluorescence techniques, we show that in the inactive Arp2/3 complex only one rapidly exchangeable ATP is tightly bound to Arp3 with an affinity of 10(8) m(-1). Upon activation of the Arp2/3 complex by WA, ATP binds to Arp2 with high affinity (10(7) m(-1)), implying that a large structural change of Arp2 is linked to Arp2/3 activation. ATP is rapidly exchangeable on Arp2 and Arp3 in WA.Arp2/3 and G-actin.WA.Arp2/3 complexes. ATP is not hydrolyzed in inactive Arp2/3, in WA.Arp2/3, nor in G-actin.WA.Arp2/3. Arp2 has a greater specificity than Arp3 for ATP versus ATP analogs. Using functional assays of actin polymerization in branched filaments, we show that binding of ATP to Arp2 is required for filament branching.