The formin homology 1 domain modulates the actin nucleation and bundling activity of Arabidopsis FORMIN1

The organization of actin filaments into large ordered structures is a tightly controlled feature of many cellular processes. However, the mechanisms by which actin filament polymerization is initiated from the available pool of profilin-bound actin monomers remain unknown in plants. Because the spontaneous polymerization of actin monomers bound to profilin is inhibited, the intervention of an actin promoting factor is required for efficient actin polymerization. Two such factors have been characterized from yeasts and metazoans: the Arp2/3 complex, a complex of seven highly conserved subunits including two actin-related proteins (ARP2 and ARP3), and the FORMIN family of proteins. The recent finding that Arabidopsis thaliana plants lacking a functional Arp2/3 complex exhibit rather modest morphological defects leads us to consider whether the large FORMIN family plays a central role in the regulation of actin polymerization. Here, we have characterized the mechanism of action of Arabidopsis FORMIN1 (AFH1). Overexpression of AFH1 in pollen tubes has been shown previously to induce abnormal actin cable formation. We demonstrate that AFH1 has a unique behavior when compared with nonplant formins. The activity of the formin homology domain 2 (FH2), containing the actin binding activity, is modulated by the formin homology domain 1 (FH1). Indeed, the presence of the FH1 domain switches the FH2 domain from a tight capper (Kd approximately 3.7 nM) able to nucleate actin filaments that grow only in the pointed-end direction to a leaky capper that allows barbed-end elongation and efficient nucleation of actin filaments from actin monomers bound to profilin. Another exciting feature of AFH1 is its ability to bind to the side and bundle actin filaments. We have identified an actin nucleator that is able to organize actin filaments directly into unbranched actin filament bundles. We suggest that AFH1 plays a central role in the initiation and organization of actin cables from the pool of actin monomers bound to profilin.     

EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.     

The world according to Arp: regulation of actin nucleation by the Arp2/3 complex.

The coordination of cell shape change and locomotion requires that actin polymerization at the cell cortex be tightly controlled in response to both intracellular and extracellular cues. The Arp2/3 complex - an actin filament nucleating and organizing factor - appears to be a central player in the cellular control of actin assembly. Recently, a molecular pathway leading from key signalling molecules to actin filament nucleation by the Arp2/3 complex has been discovered. In this pathway, the GTPase Cdc42 acts in concert with WASP family proteins to activate the Arp2/3 complex. These findings have led to a more complete picture of the mechanism of actin filament generation and organization during cell motility.     

Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization

The 70 C-terminal amino acids of Wiskott-Aldrich syndrome protein (WASp WA) activate the actin nucleation activity of the Arp2/3 complex. WASp WA binds both the Arp2/3 complex and actin monomers, but the mechanism by which it activates the Arp2/3 complex is not known. We characterized the effect of WASp WA on actin polymerization in the absence and presence of the human Arp2/3 complex. WASp WA binds actin monomers with an apparent K(d) of 0.4 microM, inhibiting spontaneous nucleation and subunit addition to pointed ends, but not addition to barbed ends. A peptide containing only the WASp homology 2 motif behaves similarly but with a 10-fold lower affinity. In contrast to previously published results, neither WASp WA nor a similar region of the protein Scar1 significantly depolymerizes actin filaments under a variety of conditions. WASp WA and the Arp2/3 complex nucleate actin filaments, and the rate of this nucleation is a function of the concentrations of both WASp WA and the Arp2/3 complex. With excess WASp WA and <10 nM Arp2/3 complex, there is a 1:1 correspondence between the Arp2/3 complex and the concentration of filaments produced, but the filament concentration plateaus at an Arp2/3 complex concentration far below the cellular concentration determined to be 9.7 microM in human neutrophils. Preformed filaments increase the rate of nucleation by WASp WA and the Arp2/3 complex but not the number of filaments that are generated. We propose that filament side binding by the Arp2/3 complex enhances its activation by WASp WA.     

Inhibition of the Arp2/3 complex-nucleated actin polymerization and branch formation by tropomyosin

The actin filament network immediately under the plasma membrane at the leading edge of rapidly moving cells consists of short, branched filaments, while those deeper in the cortex are much longer and are rarely branched. Nucleation by the Arp2/3 complex activated by membrane-bound factors (Rho-family GTPases and PIP(2)) is postulated to account for the formation of the branched network. Tropomyosin (TM) binds along the sides of filaments and protects them from severing proteins and pointed-end depolymerization in vitro. Here, we show that TM inhibits actin filament branching and nucleation by the Arp2/3 complex activated by WASp-WA. Tropomyosin increases the lag at the outset of polymerization, reduces the concentration of ends by 75%, and reduces the number of branches by approximately 50%. We conclude that TM bound to actin filaments inhibits their ability to act as secondary activators of nucleation by the Arp2/3 complex. This is the first example of inhibition of branching by an actin binding protein. We suggest that TM suppresses the nucleation of actin filament branches from actin filaments in the deep cortex of motile cells. Other abundant actin binding proteins may also locally regulate the branching nucleation by the Arp2/3 complex in cells.     

Yeast actin patches are networks of branched actin filaments

Cortical actin patches are the most prominent actin structure in budding and fission yeast. Patches assemble, move, and disassemble rapidly. We investigated the mechanisms underlying patch actin assembly and motility by studying actin filament ultrastructure within a patch. Actin patches were partially purified from Saccharomyces cerevisiae and examined by negative-stain electron microscopy (EM). To identify patches in the EM, we correlated fluorescence and EM images of GFP-labeled patches. Patches contained a network of actin filaments with branches characteristic of Arp2/3 complex. An average patch contained 85 filaments. The average filament was only 50-nm (20 actin subunits) long, and the filament to branch ratio was 3:1. Patches lacking Sac6/fimbrin were unstable, and patches lacking capping protein were relatively normal. Our results are consistent with Arp2/3 complex-mediated actin polymerization driving yeast actin patch assembly and motility, as described by a variation of the dendritic nucleation model.     

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.     

Cortactin binding to F-actin revealed by electron microscopy and 3D reconstruction

Cortactin and WASP activate Arp2/3-mediated actin filament nucleation and branching. However, different mechanisms underlie activation by the two proteins, which rely on distinct actin-binding modules and modes of binding to actin filaments. It is generally thought that cortactin binds to "mother" actin filaments, while WASP donates actin monomers to Arp2/3-generated "daughter" filament branches. Interestingly, cortactin also binds WASP in addition to F-actin and the Arp2/3 complex. However, the structural basis for the role of cortactin in filament branching remains unknown, making interpretation difficult. Here, electron microscopy and 3D reconstruction were carried out on F-actin decorated with the actin-binding repeating domain of cortactin, revealing conspicuous density on F-actin attributable to cortactin that is located on a consensus-binding site on subdomain-1 of actin subunits. Strikingly, the binding of cortactin widens the gap between the two long-pitch filament strands. Although other proteins have been found to alter the structure of the filament, the cortactin-induced conformational change appears unique. The results are consistent with a mechanism whereby alterations of the F-actin structure may facilitate recruitment of the Arp2/3 complex to the "mother" filament in the cortex of cells. In addition, cortactin may act as a structural adapter protein, stabilizing nascent filament branches while mediating the simultaneous recruitment of Arp2/3 and WASP.     

Activation of Arp2/3 complex: addition of the first subunit of the new filament by a WASP protein triggers rapid ATP hydrolysis on Arp2

In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly—with no detectable lag—upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2–Arp3–actin nucleus. This actin monomer becomes the first monomer of the new daughter filament.     

Actin filaments align into hollow comets for rapid VASP-mediated propulsion

For cells, the growth of a dense array of branched actin filaments organized by the actin-related proteins 2 and 3 (Arp2/3) complex at the plasma membrane offers an explanation as to how movement is produced, and this arrangement is considered to be optimal for motility. Here, we challenged this assumption by using an in vitro system of polystyrene beads in cell extracts that contained a complex mix of actin polymerization proteins as in vivo. We employed the surface of the bead as a reactor where we mixed two different actin polymerization-activating factors, the Arp2/3 complex and the vasodilator-stimulated phosphoprotein (VASP), to examine their contribution to actin-based movement and filament organization. We varied the coating of the bead surface but left the extracts identical for all assays. We found that the degree of filament alignment in the actin comet tails depended on the surface ratio of VASP to Arp2/3. Alignment of actin filaments parallel to the direction of bead movement in the presence of VASP was accompanied by an abrupt 7-fold increase in velocity that was independent of bead size and by hollowing out of the comets. The actin filament-bundling proteins fimbrin and fascin did not appear to play a role in this transformation. Together with the idea that VASP enhances filament detachment and with the presence of pulling forces at the rear of the bead, a mesoscopic analysis of movement provides a possible explanation for our results.     

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.     

Chlamydia trachomatis Tarp cooperates with the Arp2/3 complex to increase the rate of actin polymerization

Actin polymerization is required for Chlamydia trachomatis entry into nonphagocytic host cells. Host and chlamydial actin nucleators are essential for internalization of chlamydiae by eukaryotic cells. The host cell Arp2/3 complex and the chlamydial translocated actin recruiting phosphoprotein (Tarp) are both required for entry. Tarp and the Arp2/3 complex exhibit unique actin polymerization kinetics individually, but the molecular details of how these two actin nucleators cooperate to promote bacterial entry is not understood. In this study we provide biochemical evidence that the two actin nucleators act synergistically by co-opting the unique attributes of each to enhance the dynamics of actin filament formation. This process is independent of Tarp phosphorylation. We further demonstrate that Tarp colocalization with actin filaments is independent of the Tarp phosphorylation domain. The results are consistent with a model in which chlamydial and host cell actin nucleators cooperate to increase the rate of actin filament formation.     

Dynamin2 and cortactin regulate actin assembly and filament organization

The GTPase dynamin is required for endocytic vesicle formation. Dynamin has also been implicated in regulating the actin cytoskeleton, but the mechanism by which it does so is unclear. Through interactions via its proline-rich domain (PRD), dynamin binds several proteins, including cortactin, profilin, syndapin, and murine Abp1, that regulate the actin cytoskeleton. We investigated the interaction of dynamin2 and cortactin in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreased actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decreased actin dynamics. Dynamin2 influenced actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhanced actin nucleation by Arp2/3 complex and cortactin, and high concentrations were inhibitory. Dynamin2 promoted the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis altered the organization of the filaments and the lipid vesicles. We conclude that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes.     

Interactions of ADF/cofilin, Arp2/3 complex, capping protein and profilin in remodeling of branched actin filament networks

BACKGROUND: Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Recently, using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, we showed how these proteins co-operate in the assembly of branched actin filament networks. Here, we investigated how they work together to disassemble the networks. RESULTS: Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduced the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevented annealing of short filaments after debranching and, with profilin, allowed filaments to depolymerize at the pointed ends. CONCLUSIONS: The low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends.     

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.     

Actin polymerization or myosin contraction: two ways to build up cortical tension for symmetry breaking

Cells use complex biochemical pathways to drive shape changes for polarization and movement. One of these pathways is the self-assembly of actin filaments and myosin motors that together produce the forces and tensions that drive cell shape changes. Whereas the role of actin and myosin motors in cell polarization is clear, the exact mechanism of how the cortex, a thin shell of actin that is underneath the plasma membrane, can drive cell shape changes is still an open question. Here, we address this issue using biomimetic systems: the actin cortex is reconstituted on liposome membranes, in an ‘outside geometry’. The actin shell is either grown from an activator of actin polymerization immobilized at the membrane by a biotin–streptavidin link, or built by simple adsorption of biotinylated actin filaments to the membrane, in the presence or absence of myosin motors. We show that tension in the actin network can be induced either by active actin polymerization on the membrane via the Arp2/3 complex or by myosin II filament pulling activity. Symmetry breaking and spontaneous polarization occur above a critical tension that opens up a crack in the actin shell. We show that this critical tension is reached by growing branched networks, nucleated by the Arp2/3 complex, in a concentration window of capping protein that limits actin filament growth and by a sufficient number of motors that pull on actin filaments. Our study provides the groundwork to understanding the physical mechanisms at work during polarization prior to cell shape modifications.     

A subset of dynamic actin rearrangements in Drosophila requires the Arp2/3 complex

The Arp2/3 complex has been shown to dramatically increase the slow spontaneous rate of actin filament nucleation in vitro, and it is known to be important for remodeling the actin cytoskeleton in vivo. We isolated and characterized loss of function mutations in genes encoding two subunits of the Drosophila Arp2/3 complex: Arpc1, which encodes the homologue of the p40 subunit, and Arp3, encoding one of the two actin-related proteins. We used these mutations to study how the Arp2/3 complex contributes to well-characterized actin structures in the ovary and the pupal epithelium. We found that the Arp2/3 complex is required for ring canal expansion during oogenesis but not for the formation of parallel actin bundles in nurse cell cytoplasm and bristle shaft cells. The requirement for Arp2/3 in ring canals indicates that the polymerization of actin filaments at the ring canal plasma membrane is important for driving ring canal growth.     

Pathway of actin filament branch formation by Arp2/3 complex

A spectroscopic assay using pyrene-labeled fission yeast Arp2/3 complex revealed that the complex binds to and dissociates from actin filaments extremely slowly with or without the nucleation-promoting factor fission yeast Wsp1-VCA. Wsp1-VCA binds both Arp2/3 complex and actin monomers with high affinity. These two ligands have only modest impacts on the interaction of the other ligand with VCA. Simulations of a mathematical model based on the kinetic parameters determined in this study and elsewhere account for the full time course of actin polymerization in the presence of Arp2/3 complex and Wsp1-VCA and show that an activation step, postulated to follow binding of a ternary complex of Arp2/3 complex, a bound nucleation-promoting factor, and an actin monomer to an actin filament, has a rate constant at least 0.15 s(-1). Kinetic parameters determined in this study constrain the process of actin filament branch formation during cellular motility to one main pathway.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

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.