ABSTRACT
Arp2/3 complex nucleates branched actin filaments that drive membrane invagination during endocytosis and leading-edge protrusion in lamellipodia. Arp2/3 complex is maximally activated in vitro by binding of a WASP family protein to two sites-one on the Arp3 subunit and one spanning Arp2 and ARPC1-but the importance of each site in the regulation of force-producing actin networks is unclear. Here, we identify mutations in budding yeast Arp2/3 complex that decrease or block engagement of Las17, the budding yeast WASP, at each site. As in the mammalian system, both sites are required for maximal activation in vitro. Dimerization of Las17 partially restores activity of mutations at both CA-binding sites. Arp2/3 complexes defective at either site assemble force-producing actin networks in a bead motility assay, but their reduced activity hinders motility by decreasing actin assembly near the bead surface and by failing to suppress actin filament bundling within the networks. While even the most defective Las17-binding site mutants assembled actin filaments at endocytic sites, they showed significant internalization defects, potentially because they lack the proper architecture to drive plasma membrane remodeling. Together, our data indicate that both Las17-binding sites are important to assemble functional endocytic actin networks in budding yeast, but Arp2/3 complex retains some activity in vitro and in vivo even with a severe defect at either Las17-binding site.
Subject(s)
Actin-Related Protein 2-3 Complex , Actins , Saccharomyces cerevisiae Proteins , Wiskott-Aldrich Syndrome Protein , Animals , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Binding Sites , Mammals/metabolism , Protein Binding , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Wiskott-Aldrich Syndrome Protein/metabolismABSTRACT
Actin-related protein 2/3 complex (Arp2/3 complex) catalyzes the nucleation of branched actin filaments that push against membranes in processes like cellular motility and endocytosis. During activation by WASP proteins, the complex must bind WASP and engage the side of a pre-existing (mother) filament before a branched filament is nucleated. Recent high-resolution structures of activated Arp2/3 complex revealed two major sets of activating conformational changes. How these activating conformational changes are triggered by interactions of Arp2/3 complex with actin filaments and WASP remains unclear. Here we use a recent high-resolution structure of Arp2/3 complex at a branch junction to design all-atom molecular dynamics simulations that elucidate the pathway between the active and inactive states. We ran a total of â¼4.6 microseconds of both unbiased and steered all-atom molecular dynamics simulations starting from three different binding states, including Arp2/3 complex within a branch junction, bound only to a mother filament, and alone in solution. These simulations indicate that the contacts with the mother filament are mostly insensitive to the massive rigid body motion that moves Arp2 and Arp3 into a short pitch helical (filament-like) arrangement, suggesting actin filaments alone do not stimulate the short pitch conformational change. In contrast, contacts with the mother filament stabilize subunit flattening in Arp3, an intrasubunit change that converts Arp3 from a conformation that mimics an actin monomer to one that mimics a filamentous actin subunit. Our results support a multistep activation pathway that has important implications for understanding how WASP-mediated activation allows Arp2/3 complex to assemble force-producing actin networks.
Subject(s)
Actin Cytoskeleton , Actin-Related Protein 2-3 Complex , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Molecular Dynamics Simulation , Protein Structure, Quaternary , Animals , CattleABSTRACT
Dendrite morphogenesis is essential for neural circuit formation, yet the molecular mechanisms underlying complex dendrite branching remain elusive. Previous studies on the highly branched Caenorhabditis elegans PVD sensory neuron identified a membrane co-receptor complex that links extracellular signals to intracellular actin remodeling machinery, promoting high-order dendrite branching. In this complex, the claudin-like transmembrane protein HPO-30 recruits the WAVE regulatory complex (WRC) to dendrite branching sites, stimulating the Arp2/3 complex to polymerize actin. We report here our biochemical and structural analysis of this interaction, revealing that the intracellular domain (ICD) of HPO-30 is intrinsically disordered and employs two distinct mechanisms to regulate the actin cytoskeleton. First, HPO-30 ICD binding to the WRC requires dimerization and involves the entire ICD sequence, rather than a short linear peptide motif. This interaction enhances WRC activation by the GTPase Rac1. Second, HPO-30 ICD directly binds to the sides and barbed end of actin filaments. Binding to the barbed end requires ICD dimerization and inhibits both actin polymerization and depolymerization, resembling the actin capping protein CapZ. These dual functions provide an intriguing model of how membrane proteins can integrate distinct mechanisms to fine-tune local actin dynamics.
Subject(s)
Actin Cytoskeleton , Actins , Animals , Actins/metabolism , Actin Cytoskeleton/metabolism , Carrier Proteins/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Membrane Proteins/metabolism , Caenorhabditis elegans/metabolism , Dendrites/metabolismABSTRACT
Fragmentation ion spectral analysis of chemically cross-linked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify cross-linked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable cross-linkers, and identification of cross-links between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of cross-links improve the sensitivity and accuracy of correct cross-link identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multiplatform.
Subject(s)
Proteomics , Tandem Mass Spectrometry , Proteomics/methods , Tandem Mass Spectrometry/methods , Peptides/analysis , Proteins/chemistry , Software , Cross-Linking Reagents/chemistryABSTRACT
Arp2/3 complex nucleates branched actin filaments that provide pushing forces to drive cellular processes such as lamellipodial protrusion and endocytosis. Arp2/3 complex is intrinsically inactive, and multiple classes of nucleation promoting factors (NPFs) stimulate its nucleation activity. When activated by WASP family NPFs, the complex must bind to the side of a preexisting (mother) filament of actin to complete the nucleation process, ensuring that WASP-mediated activation creates branched rather than linear actin filaments. How actin filaments contribute to activation is currently not understood, largely due to the lack of high-resolution structures of activated Arp2/3 complex bound to the side of a filament. Here, we present the 3.9-Å cryo-electron microscopy structure of the Arp2/3 complex at a branch junction. The structure reveals contacts between Arp2/3 complex and the side of the mother actin filament that likely stimulate subunit flattening, a conformational change that allows the actin-related protein subunits in the complex (Arp2 and Arp3) to mimic filamentous actin subunits. In contrast, limited contact between the bottom half of the complex and the mother filament suggests that clamp twisting, a second major conformational change observed in the active state, is not stimulated by actin filaments, potentially explaining why actin filaments are required but insufficient to trigger nucleation during WASP-mediated activation. Along with biochemical and live-cell imaging data and molecular dynamics simulations, the structure reveals features critical for the interaction of Arp2/3 complex with actin filaments and regulated assembly of branched actin filament networks in cells.
Subject(s)
Actin Cytoskeleton , Actin-Related Protein 2-3 Complex , Actin Cytoskeleton/chemistry , Actin-Related Protein 2-3 Complex/chemistry , Actin-Related Protein 2-3 Complex/metabolism , Cryoelectron Microscopy , Cytoskeleton/metabolism , Molecular Dynamics Simulation , Protein Conformation , Wiskott-Aldrich Syndrome Protein/metabolismABSTRACT
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.
Subject(s)
Actin Cytoskeleton , Actin-Related Protein 2-3 Complex , Schizosaccharomyces pombe Proteins , Schizosaccharomyces , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Pseudopodia/metabolism , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Arp2/3 complex nucleates branched actin filaments important for processes such as DNA repair, endocytosis, and cellular motility. Multiple factors are required to activate branching nucleation by Arp2/3 complex, including a WASP family protein and a pre-existing actin filament. Activation is achieved through two major conformational changes-subunit flattening and movement into the short pitch conformation-that allow the actin-related proteins (Arps) within the complex (Arp2 and Arp3) to mimic filamentous actin subunits, thereby templating new filament assembly. Some models suggest that these changes are concerted and stimulated cooperatively by WASP and actin filaments, but how Arp2/3 complex integrates signals from multiple factors to drive switch-like activation of branching nucleation has been unknown. Here, we use biochemical assays to show that instead of a concerted mechanism, signal integration by Arp2/3 complex occurs via distinct and unconcerted conformational changes; WASP stimulates the short pitch arrangement of Arp2 and Arp3, while actin filaments trigger a different activation step. An engineered Arp2/3 complex that bypasses the need for WASP but not actin filaments in activation potently assembles isotropic actin networks but fails to assemble sustained force-producing actin networks in bead motility assays. The engineered complex, which is crosslinked into the short pitch conformation, fails to target nucleation to the surface of the bead, creating unproductive branching events that deplete unpolymerized actin and halt assembly. Together, our data demonstrate the requirement for multifactor signal integration by Arp2/3 complex and highlight the importance of both the WASP- and actin filament-mediated activation steps in the assembly of functional actin networks.
Subject(s)
Actin-Related Protein 2-3 Complex , Actins , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Cytoskeleton/metabolism , Wiskott-Aldrich Syndrome Protein/metabolismABSTRACT
The actin filament nucleator Arp2/3 complex is activated at cortical sites in Schizosaccharomyces pombe to assemble branched actin networks that drive endocytosis. Arp2/3 complex activators Wsp1 and Dip1 are required for proper actin assembly at endocytic sites, but how they coordinately control Arp2/3-mediated actin assembly is unknown. Alone, Dip1 activates Arp2/3 complex without preexisting actin filaments to nucleate 'seed' filaments that activate Wsp1-bound Arp2/3 complex, thereby initiating branched actin network assembly. In contrast, because Wsp1 requires preexisting filaments to activate, it has been assumed to function exclusively in propagating actin networks by stimulating branching from preexisting filaments. Here we show that Wsp1 is important not only for propagation but also for initiation of endocytic actin networks. Using single molecule total internal reflection fluorescence microscopy we show that Wsp1 synergizes with Dip1 to co-activate Arp2/3 complex. Synergistic co-activation does not require preexisting actin filaments, explaining how Wsp1 contributes to actin network initiation in cells.
Subject(s)
Actins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Gene Expression Regulation, Fungal/physiology , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
Arp2/3 complex, a crucial actin filament nucleator, undergoes structural rearrangements during activation by nucleation-promoting factors (NPFs). However, the conformational pathway leading to the nucleation-competent state is unclear due to lack of high-resolution structures of the activated state. Here we report a ~3.9 Å resolution cryo-EM structure of activated Schizosaccharomyces pombe Arp2/3 complex bound to the S. pombe NPF Dip1 and attached to the end of the nucleated actin filament. The structure reveals global and local conformational changes that allow the two actin-related proteins in Arp2/3 complex to mimic a filamentous actin dimer and template nucleation. Activation occurs through a clamp-twisting mechanism, in which Dip1 forces two core subunits in Arp2/3 complex to pivot around one another, shifting half of the complex into a new activated position. By showing how Dip1 stimulates activation, the structure reveals how NPFs can activate Arp2/3 complex in diverse cellular processes.
Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Schizosaccharomyces/metabolism , Actin Cytoskeleton/chemistry , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/ultrastructure , Actin-Related Protein 2-3 Complex/chemistry , Actin-Related Protein 2-3 Complex/ultrastructure , Cryoelectron Microscopy , Models, Molecular , Protein Binding , Protein Conformation , Protein Multimerization , Schizosaccharomyces/chemistry , Schizosaccharomyces pombe Proteins/chemistry , Schizosaccharomyces pombe Proteins/ultrastructureABSTRACT
Arp2/3 complex nucleates branched actin filaments important for cellular motility, endocytosis, meiosis, and cellular differentiation [1-4]. Wiskott-Aldrich syndrome proteins (WASPs), the prototypical Arp2/3 complex activators, activate Arp2/3 complex only once it is bound to the side of an actin filament [5, 6]. This ensures WASP-activated Arp2/3 complex only nucleates branched actin filaments but means branched actin networks must be seeded with an initial preformed filament. Dip1 and other WISH/DIP/SPIN90 family proteins activate Arp2/3 complex without preformed filaments [7], creating seed filaments that activate WASP-bound Arp2/3 complex [8]. Importantly, Dip1-mediated activation of Arp2/3 complex creates linear filaments instead of branches [7]. Cells may therefore need to limit Dip1 activity relative to WASP to preserve the dendritic nature of actin networks, although it is unclear whether such regulatory mechanisms exist. Here, we use total internal reflection fluorescence (TIRF) microscopy to show that Dip1 causes actin assembled with WASP and Arp2/3 complex to form disconnected networks with many linear filaments rather than highly branched arrays. We discover a key biochemical difference between Dip1 and WASP that may limit linear filament nucleation in cells; although WASP must be released for nucleation, Dip1 stays associated with Arp2/3 complex on the pointed ends of nucleated actin filaments, so Dip1 is consumed in the reaction. Using live-cell imaging of fission yeast, we provide evidence that Dip1 is a single-turnover activator of Arp2/3 complex in vivo, revealing a mechanism by which Dip1 can initiate branched actin networks at endocytic sites without disrupting their branched architectures.
Subject(s)
Actin Cytoskeleton/chemistry , Actin-Related Protein 2-3 Complex/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/physiology , Actin-Related Protein 2-3 Complex/metabolism , Protein Binding , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
When activated by Wiskott-Aldrich syndrome proteins (WASP), Arp2/3 complex nucleates branched actin filaments important for processes like cellular motility and endocytosis [1]. WASP-mediated activation of Arp2/3 complex requires a preformed actin filament, ensuring that activation by WASP creates branched instead of linear filaments. However, this biochemical requirement also means that assembly of branched actin networks must be primed with an initial seed filament [2-4]. We recently described a class of activators called WISH/DIP/SPIN90 (WDS) proteins, which, unlike WASP, activate Arp2/3 complex without a preformed filament [4]. Although this property may allow WDS proteins to serve as seed filament generators, it is unknown whether actin filaments nucleated by WDS-activated Arp2/3 complex can activate WASP-bound Arp2/3 complex. Further, despite their potential importance as branched actin network initiators, little is known about how WDS proteins turn on Arp2/3 complex. Here, we use two-color single-molecule total internal reflection fluorescence (TIRF) microscopy to show that Dip1, the S. pombe WDS protein [5], co-opts features of branching nucleation to activate Arp2/3 complex. Specifically, it activates Arp2/3 complex to nucleate linear filaments analogous to the branch created by WASP-mediated activation. The barbed ends of Dip1-Arp2/3 nucleated filaments are free to elongate, and their pointed ends remain anchored to Dip1-bound Arp2/3 complex. The linear filaments nucleated by Dip1-bound Arp2/3 complex activate WASP-bound Arp2/3 complex as potently as spontaneously nucleated or branched actin filaments. These observations provide important insights into the regulation of Arp2/3 complex by its activators and the molecular basis for initiation of branched actin networks.
Subject(s)
Actin-Related Protein 2-3 Complex/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Actin-Related Protein 2-3 Complex/metabolism , Cytoskeleton/metabolism , Protein Binding , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolismABSTRACT
Unlike the WASP family of Arp2/3 complex activators, WISH/DIP/SPIN90 (WDS) family proteins activate actin filament nucleation by the Arp2/3 complex without the need for a preformed actin filament. This allows WDS proteins to initiate branched actin network assembly by providing seed filaments that activate WASP-bound Arp2/3 complex. Despite their important role in actin network initiation, it is unclear how WDS proteins drive the activating steps that require both WASP and pre-existing actin filaments during WASP-mediated nucleation. Here, we show that SPIN90 folds into an armadillo repeat domain that binds a surface of Arp2/3 complex distinct from the two WASP sites, straddling a hinge point that may stimulate movement of the Arp2 subunit into the activated short-pitch conformation. SPIN90 binds a surface on Arp2/3 complex that overlaps with actin filament binding, explaining how it could stimulate the same structural rearrangements in the complex as pre-existing actin filaments. By revealing how WDS proteins activate the Arp2/3 complex, these data provide a molecular foundation to understand initiation of dendritic actin networks and regulation of Arp2/3 complex by its activators.
Subject(s)
Actin Cytoskeleton/chemistry , Actin-Related Protein 2-3 Complex/chemistry , Adaptor Proteins, Signal Transducing/chemistry , Muscle Proteins/chemistry , Actin Cytoskeleton/genetics , Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 2-3 Complex/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Cattle , Humans , Muscle Proteins/genetics , Muscle Proteins/metabolism , Protein Domains , Protein Structure, Quaternary , Structure-Activity RelationshipABSTRACT
Arp2/3 complex nucleates branched actin filaments important for cellular motility and endocytosis. WASP family proteins are Arp2/3 complex activators that play multiple roles in branching nucleation, but little is known about the structural bases of these WASP functions, owing to an incomplete understanding of how WASP binds Arp2/3 complex. Recent data show WASP binds two sites, and biochemical and structural studies led to models in which the WASP C segment engages the barbed ends of the Arp3 and Arp2 subunits while the WASP A segment binds the back side of the complex on Arp3. However, electron microscopy reconstructions showed density for WASP inconsistent with these models on the opposite (front) side of Arp2/3 complex. Here we use chemical cross-linking and mass spectrometry (XL-MS) along with computational docking and structure-based mutational analysis to map the two WASP binding sites on the complex. Our data corroborate the barbed end and back side binding models and show one WASP binding site on Arp3, on the back side of the complex, and a second site on the bottom of the complex, spanning Arp2 and ARPC1. The XL-MS-identified cross-links rule out the front side binding model and show that the A segment of WASP binds along the bottom side of the ARPC1 subunit, instead of at the Arp2/ARPC1 interface, as suggested by FRET experiments. The identified binding sites support the Arp3 tail release model to explain WASP-mediated activating conformational changes in Arp2/3 complex and provide insight into the roles of WASP in branching nucleation.
Subject(s)
Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Protein Conformation , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Wiskott-Aldrich Syndrome Protein/metabolism , Actin Cytoskeleton/chemistry , Actin-Related Protein 2-3 Complex/chemistry , Amino Acid Sequence , Binding Sites , Protein Binding , Protein Interaction Mapping , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/chemistry , Sequence Homology , Wiskott-Aldrich Syndrome Protein/chemistryABSTRACT
Nucleation of branched actin filaments by Arp2/3 complex is tightly regulated to control actin assembly in cells. Arp2/3 complex activation involves conformational changes brought about by ATP, Nucleation Promoting Factor (NPF) proteins, actin filaments and NPF-recruited actin monomers. To understand how these factors promote activation, we must first understand how the complex is held inactive in their absence. Here we demonstrate that the Arp3 C-terminal tail is a structural switch that prevents Arp2/3 complex from adopting an active conformation. The interaction between the tail and a hydrophobic groove in Arp3 blocks movement of Arp2 and Arp3 into an activated filament-like (short pitch) conformation. Our data indicate ATP binding destabilizes this interaction via an allosteric link between the Arp3 nucleotide cleft and the hydrophobic groove, thereby promoting the short-pitch conformation. Our results help explain how Arp2/3 complex is locked in an inactive state without activators and how autoinhibition is relieved.
Subject(s)
Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Actin-Related Protein 2/metabolism , Adenosine Triphosphate/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Actin-Related Protein 2/genetics , Actin-Related Protein 2-3 Complex/chemistry , Actin-Related Protein 2-3 Complex/genetics , Actin-Related Protein 3/genetics , Actin-Related Protein 3/metabolism , Amino Acid Sequence , Conserved Sequence , Protein Conformation , Protein Multimerization , Protein Stability , Saccharomyces cerevisiae Proteins/genetics , Schizosaccharomyces/genetics , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/geneticsABSTRACT
The Arp2/3 (Actin-related proteins 2/3) complex is activated by WASP (Wiskott-Aldrich syndrome protein) family proteins to nucleate branched actin filaments that are important for cellular motility. WASP recruits actin monomers to the complex and stimulates movement of Arp2 and Arp3 into a "short-pitch" conformation that mimics the arrangement of actin subunits within filaments. The relative contribution of these functions in Arp2/3 complex activation and the mechanism by which WASP stimulates the conformational change have been unknown. We purified budding yeast Arp2/3 complex held in or near the short-pitch conformation by an engineered covalent cross-link to determine if the WASP-induced conformational change is sufficient for activity. Remarkably, cross-linked Arp2/3 complex bypasses the need for WASP in activation and is more active than WASP-activated Arp2/3 complex. These data indicate that stimulation of the short-pitch conformation is the critical activating function of WASP and that monomer delivery is not a fundamental requirement for nucleation but is a specific requirement for WASP-mediated activation. During activation, WASP limits nucleation rates by releasing slowly from nascent branches. The cross-linked complex is inhibited by WASP's CA region, even though CA potently stimulates cross-linking, suggesting that slow WASP detachment masks the activating potential of the short-pitch conformational switch. We use structure-based mutations and WASP-Arp fusion chimeras to determine how WASP stimulates movement toward the short-pitch conformation. Our data indicate that WASP displaces the autoinhibitory Arp3 C-terminal tail from a hydrophobic groove at Arp3's barbed end to destabilize the inactive state, providing a mechanism by which WASP stimulates the short-pitch conformation and activates Arp2/3 complex.
Subject(s)
Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/metabolism , Wiskott-Aldrich Syndrome Protein/metabolism , Amino Acid Sequence , Cross-Linking Reagents , Molecular Conformation , Molecular Sequence Data , Saccharomyces cerevisiae , SchizosaccharomycesABSTRACT
Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.
Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Actins/metabolism , Cortactin/metabolism , Wiskott-Aldrich Syndrome Protein Family/metabolism , Amino Acid Sequence , Animals , Binding, Competitive , Cattle , DNA Mutational Analysis , Microscopy, Fluorescence , Molecular Sequence Data , Muscle, Skeletal/metabolism , Protein Binding , Pseudopodia/metabolism , Pyrenes/chemistry , Rabbits , Sequence Homology, Amino AcidABSTRACT
BACKGROUND: Arp2/3 complex is a key actin cytoskeletal regulator that creates branched actin filament networks in response to cellular signals. WASP-activated Arp2/3 complex assembles branched actin networks by nucleating new filaments from the sides of pre-existing ones. WASP-mediated activation requires seed filaments, to which the WASP-bound Arp2/3 complex can bind to form branches, but the source of the first substrate filaments for branching is unknown. RESULTS: Here we show that Dip1, a member of the WISH/DIP/SPIN90 family of actin regulators, potently activates Arp2/3 complex without preformed filaments. Unlike other Arp2/3 complex activators, Dip1 does not bind actin monomers or filaments, and it interacts with the complex using a non-WASP-like binding mode. In addition, Dip1-activated Arp2/3 complex creates linear instead of branched actin filament networks. CONCLUSIONS: Our data show the mechanism by which Dip1 and other WISH/DIP/SPIN90 proteins can provide seed filaments to Arp2/3 complex to serve as master switches in initiating branched actin assembly. This mechanism is distinct from other known activators of Arp2/3 complex.
Subject(s)
Actin Cytoskeleton/metabolism , Actin-Related Protein 2-3 Complex/genetics , Schizosaccharomyces pombe Proteins/genetics , Schizosaccharomyces/genetics , Actin-Related Protein 2-3 Complex/metabolism , Amino Acid Sequence , Electrophoresis, Polyacrylamide Gel , Immunoblotting , Microscopy, Fluorescence , Molecular Sequence Data , Schizosaccharomyces/metabolism , Schizosaccharomyces pombe Proteins/metabolism , Sequence AlignmentABSTRACT
Nucleation promoting factors (NPFs) initiate branched actin network assembly by activating Arp2/3 complex, a branched actin filament nucleator. Cellular actin networks contain multiple NPFs, but how they coordinately regulate Arp2/3 complex is unclear. Cortactin is an NPF that activates Arp2/3 complex weakly on its own, but with WASP/N-WASP, another class of NPFs, potently activates. We dissect the mechanism of synergy and propose a model in which cortactin displaces N-WASP from nascent branches as a prerequisite for nucleation. Single-molecule imaging revealed that unlike WASP/N-WASP, cortactin remains bound to junctions during nucleation, and specifically targets junctions with a â¼160-fold increased on rate over filament sides. N-WASP must be dimerized for potent synergy, and targeted mutations indicate release of dimeric N-WASP from nascent branches limits nucleation. Mathematical modeling shows cortactin-mediated displacement but not N-WASP recycling or filament recruitment models can explain synergy. Our results provide a molecular basis for coordinate Arp2/3 complex regulation. DOI:http://dx.doi.org/10.7554/eLife.00884.001.
Subject(s)
Actin-Related Protein 2-3 Complex/metabolism , Cortactin/metabolism , Wiskott-Aldrich Syndrome Protein, Neuronal/metabolism , PolymerizationABSTRACT
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.
Subject(s)
Actin-Related Protein 2-3 Complex/chemistry , Actin-Related Protein 2-3 Complex/metabolism , Glia Maturation Factor/chemistry , Glia Maturation Factor/metabolism , Actin-Related Protein 2-3 Complex/genetics , Actins/chemistry , Actins/metabolism , Amino Acid Sequence , Animals , Cattle , Crystallography, X-Ray , Glia Maturation Factor/genetics , Mice , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Interaction Domains and Motifs , Protein Subunits , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Wiskott-Aldrich Syndrome Protein Family/chemistry , Wiskott-Aldrich Syndrome Protein Family/metabolismABSTRACT
Actin-related protein 2/3 (Arp2/3) complex is a seven-subunit assembly that nucleates branched actin filaments. Small molecule inhibitors CK-666 and CK-869 bind to Arp2/3 complex and inhibit nucleation, but their modes of action are unknown. Here, we use biochemical and structural methods to determine the mechanism of each inhibitor. Our data indicate that CK-666 stabilizes the inactive state of the complex, blocking movement of the Arp2 and Arp3 subunits into the activated filament-like (short pitch) conformation, while CK-869 binds to a serendipitous pocket on Arp3 and allosterically destabilizes the short pitch Arp3-Arp2 interface. These results provide key insights into the relationship between conformation and activity in Arp2/3 complex and will be critical for interpreting the influence of the inhibitors on actin filament networks in vivo.