[Show abstract][Hide abstract] ABSTRACT: Arp2/3 complex initiates the growth of branched actin-filament networks by inducing actin polymerization from the sides of pre-existing filaments. Nucleation promoting factors (NPFs) are essential for the branching reaction through interactions with the Arp2/3 complex prior to branch formation. The modes by which NPFs bind Arp2/3 complex and associated conformational changes have remained elusive. Here, we used electron microscopy to determine three-dimensional structures at ~2 nm resolution of Arp2/3 complex with three different bound NPFs: N-WASp, Scar-VCA and cortactin. All of these structures adopt a conformation with the two actin-related proteins in an actin-filament-like dimer and the NPF bound to the pointed end. Distance constraints derived by fluorescence resonance energy transfer independently verified the NPF location. Furthermore, all bound NPFs partially occlude the actin-filament binding site, suggesting that additional local structural rearrangements are required in the pathway of Arp2/3 complex activation to allow branch formation.
The EMBO Journal 09/2011; 31(1):236-47. · 9.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The actin-related protein 2/3 (Arp2/3) complex mediates the formation of branched actin filaments at the leading edge of motile cells and in the comet tails moving certain intracellular pathogens. Crystal structures of the Arp2/3 complex are available, but the architecture of the junction formed by the Arp2/3 complex at the base of the branch was not known. In this study, we use electron tomography to reconstruct the branch junction with sufficient resolution to show how the Arp2/3 complex interacts with the mother filament. Our analysis reveals conformational changes in both the mother filament and Arp2/3 complex upon branch formation. The Arp2 and Arp3 subunits reorganize into a dimer, providing a short-pitch template for elongation of the daughter filament. Two subunits of the mother filament undergo conformational changes that increase stability of the branch. These data provide a rationale for why branch formation requires cooperative interactions among the Arp2/3 complex, nucleation-promoting factors, an actin monomer, and the mother filament.
The Journal of Cell Biology 04/2008; 180(5):887-95. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Biological Chemistry 02/2007; 282(1):426-35. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Subversion of the host cell cytoskeleton is the hallmark of enterohaemorrhagic Escherichia coli (EHEC) infection. EHEC translocates the trans-membrane receptor protein Tir (translocated intimin receptor), which links the extracellular bacterium to the eukaryotic cell actin cytoskeleton, triggering formation of actin-rich pedestals beneath adherent bacteria. Tir-mediated actin accretion by EHEC requires TccP (Tir cytoskeleton coupling protein), a recently discovered type III secretion system effector protein which, following translocation, binds and activates Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the actin-related protein 2/3 complex leading to localized polymerization of actin. In this study, truncated N-WASP and TccP derivatives were generated and tested in in vitro actin polymerization and epithelial cell infection assays. The C-terminal amino acids 253-276 of the GTPase binding domain (GBD) of N-WASP were identified as essential, although not sufficient, for TccP:N-WASP protein:protein interaction, TccP-mediated N-WASP activation and induction of actin polymerization. TccP from EHEC O157:H7 strain EDL933 consists of a unique N-terminal domain and six proline-rich repeats. Progressive deletions within the N-terminus of TccP revealed that residues 1-21 are necessary and sufficient for its translocation, while amino acids 1-181, encompassing the N-terminal translocation signal and two proline-rich repeats, are sufficient for triggering actin polymerization in EHEC-infected epithelial cells and in in vitro actin polymerization assays. This study defines the modular domain structure of TccP and the molecular basis of TccP-mediated N-WASP activation and EHEC-induced remodelling of the host actin cytoskeleton.
[Show abstract][Hide abstract] ABSTRACT: N-WASP is a member of the WASP family of proteins, which play essential roles in actin dynamics during cell adhesion and migration. hnRNPK is a member of the heterogeneous nuclear ribonucleoprotein complex, which has also been implicated in the regulation of cell spreading. Here, we identify a direct interaction between N-WASP and hnRNPK. We show that this interaction is mediated by the N-terminal WH1 domain of N-WASP and the segment of hnRNPK containing its K interaction (KI) domain. Furthermore, these two proteins are co-localized at the cell periphery in the spreading initiation center during the early stage of cell spreading. We found that co-expression of hnRNPK with N-WASP reverses the stimulation of cell spreading by N-WASP, and this effect is correlated with hnRNPK binding to N-WASP. Expression of hnRNPK does not affect subcellular localization of N-WASP protein. However, co-expression of hnRNPK with N-WASP reduced filopodia formation stimulated by N-WASP in spreading cells. Together, these results identify hnRNPK as a new negative regulator of N-WASP and suggest that hnRNPK may regulate the initial stage of cell spreading by direct association with N-WASP in the spreading initiation center.
Journal of Biological Chemistry 07/2006; 281(22):15352-60. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The VCA domain of the neuronal Wiskott-Aldrich syndrome protein (N-WASP) is a potent activator of the Arp2/3 complex, a 240 kDa heteroheptameric actin-nucleating assembly. We used site-directed spin labeling of N-WASP peptides in conjunction with methyl-TROSY spectra of the intact, selectively labeled Arp2/3 complex to identify regions of the VCA that are proximal to the ARPC3 subunit of the assembly. We also cross-linked CA peptides to the Arp3, Arp2, ARPC1, and ARPC3 subunits. The combined data suggest that the extreme C-terminus of the A region and the C-terminus of the C region of N-WASP are proximal to ARPC3. These results have implications for the mechanism of Arp2/3 complex activation by VCA peptides. This study also demonstrates the utility of NMR spectroscopy for studying ligand binding events in large, asymmetric, macromolecular assemblies.
[Show abstract][Hide abstract] ABSTRACT: Actin branch junctions are conserved cytoskeletal elements critical for the generation of protrusive force during actin polymerization-driven cellular motility. Assembly of actin branch junctions requires the Arp2/3 complex, upon activation, to initiate a new actin (daughter) filament branch from the side of an existing (mother) filament, leading to the formation of a dendritic actin network with the fast growing (barbed) ends facing the direction of movement. Using genetic labeling and electron microscopy, we have determined the structural organization of actin branch junctions assembled in vitro with 1-nm precision. We show here that the activators of the Arp2/3 complex, except cortactin, dissociate after branch formation. The Arp2/3 complex associates with the mother filament through a comprehensive network of interactions, with the long axis of the complex aligned nearly perpendicular to the mother filament. The actin-related proteins, Arp2 and Arp3, are positioned with their barbed ends facing the direction of daughter filament growth. This subunit map brings direct structural insights into the mechanism of assembly and mechanical stability of actin branch junctions.
[Show abstract][Hide abstract] ABSTRACT: Cortactin is an actin-associated scaffolding protein that regulates cell migration. Amplification of the human gene, EMS1, has been detected in breast, head and neck tumors, where it correlates with increased invasiveness. Cortactin can regulate actin dynamics directly via its N-terminal half, which can bind and activate the Arp2/3 complex. The C-terminal portion of cortactin, however, is thought to have limited function in its regulation of the actin polymerization machinery. In this report, we identify a role for the cortactin C-terminus in regulating cell migration and, more specifically, actin dynamics. Overexpression of either full-length cortactin or cortactin C-terminus is sufficient to enhance migration of mammary epithelial cells. In vitro, cortactin binds to and activates, via its SH3 domain, a regulator of the Arp2/3 complex, neural Wiskott Aldrich Syndrome protein (N-WASP). This in vitro activation of N-WASP is likely to be important in vivo, as cortactin-enhanced migration is dependent upon N-WASP. Thus, our results suggest that cortactin has multiple mechanisms by which it can recruit and modulate the actin machinery and ultimately regulate cell migration.
[Show abstract][Hide abstract] ABSTRACT: The actin-related protein 2 and 3 (Arp2/3) complex is a seven-subunit protein complex that nucleates actin filaments at the cell cortex. Despite extensive cross-linking, crystallography, genetic and biochemical studies, the contribution of each subunit to the activity of the complex remains largely unclear. In this study we characterized the function of the 40-kDa subunit, ARPC1/Arc40, of the yeast Arp2/3 complex. We showed that this subunit is indeed a stable component of the Arp2/3 complex, but its highly unusual electrophoretic mobility eluded detection in previous studies. Recombinant Arc40 bound the VCA domain of Wiskott-Aldrich syndrome protein family activators at a K(d) of 0.45 mum, close to that of the full complex with VCA (0.30 microm), and this interaction was dependent on the conserved tryptophan at the COOH terminus of VCA. Using a newly constructed Delta arc40 yeast strain, we showed that loss of Arc40 severely reduced the binding affinity of the Arp2/3 complex with VCA as well as the nucleation activity of the complex, suggesting that Arc40 contains an important contact site of the Arp2/3 complex with VCA. The Delta arc40 cells exhibited reduced growth rate, loss of actin patches, and accumulation of cables like actin aggregates, phenotypes typical of other subunit nulls, suggesting that Arc40 functions exclusively within the Arp2/3 complex.
Journal of Biological Chemistry 01/2005; 279(52):54629-36. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Arp2/3 complex, a highly conserved nucleator of F-actin polymerization, plays a key role in the regulation of actin dynamics eukaryotic cells. In animal cells and yeasts, Wiskott-Aldrich Syndrome protein (WASP)/suppressor of cAMP receptor (Scar)/WASP family verprolin homologous (WAVE) family proteins activate the Arp2/3 complex in response to localized cues. Like other eukaryotes, plants have an Arp2/3 complex, which has recently been shown to play an important role in F-actin organization and cell morphogenesis. However, no activators of the Arp2/3 complex have been identified in plants, which lack obvious homologs of WASP/Scar/WAVE family proteins. Here, we identify a family of Scar/WAVE-related plant Arp2/3 activators. Like Scar/WAVE proteins, four proteins identified in Arabidopsis thaliana (AtSCAR1 to AtSCAR4) and one in maize (ZmSCAR1) have a C-terminal WASP homology 2 (WH2)/acidic (WA)-verprolin homology/cofilin homology/acidic (VCA)-like domain, which we show can activate the bovine Arp2/3 complex. At their N termini, AtSCAR1 to ATSCAR4, along with a fifth protein lacking a VCA/WA-like domain at its C terminus (At4g18600), are related to the N-terminal Scar homology domains of Scar/WAVE family proteins. Analysis of gene expression patterns suggests functional redundancy among members of the AtSCAR family. Full-length AtSCAR1 and ATSCAR3 proteins and their Scar homology domains bind in vitro to AtBRICK 1 (AtBRK1), the Arabidopsis homolog of HSPC300, a WAVE-binding protein recently identified as a component of a complex implicated in the regulation of Scar/WAVE activity. Thus, AtSCAR proteins are likely to function in association with AtBRK1, and perhaps other Arabidopsis homologs of WAVE complex components, to regulate activation of the Arp2,3 complex in vivo.
Proceedings of the National Academy of Sciences 12/2004; 101(46):16379-84. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Motile and morphogenetic cellular processes are driven by site-directed assembly of actin filaments. Formins, proteins characterized by formin homology domains FH1 and FH2, are initiators of actin assembly. How formins simply bind to filament barbed ends in rapid equilibrium or find free energy to become a processive motor of filament assembly remains enigmatic. Here we demonstrate that the FH1-FH2 domain accelerates hydrolysis of ATP coupled to profilin-actin polymerization and uses the derived free energy for processive polymerization, increasing 15-fold the rate constant for profilin-actin association to barbed ends. Profilin is required for and takes part in the processive function. Single filaments grow at least 10 microm long from formin bound beads without detaching. Transitory formin-associated processes are generated by poisoning of the processive cycle by barbed-end capping proteins. We successfully reconstitute formin-induced motility in vitro, demonstrating that this mechanism accounts for the puzzlingly rapid formin-induced actin processes observed in vivo.
[Show abstract][Hide abstract] ABSTRACT: Actin polymerization, the main driving force for cell locomotion, is also used by the bacteria Listeria and Shigella and vaccinia virus for intracellular and intercellular movements. Seminal studies have shown the key function of the Arp2/3 complex in nucleating actin and generating a branched array of actin filaments during membrane extension and pathogen movement. Arp2/3 requires activation by proteins such as the WASP-family proteins or ActA of Listeria. We previously reported that actin tails of Rickettsia conorii, another intracellular bacterium, unlike those of Listeria, Shigella or vaccinia, are made of long unbranched actin filaments apparently devoid of Arp2/3 (ref. 4). Here we identify a R. conorii surface protein, RickA, that activates Arp2/3 in vitro, although less efficiently than ActA. In infected cells, Arp2/3 is detected on the rickettsial surface but not in actin tails. When expressed in mammalian cells and targeted to the membrane, RickA induces filopodia. Thus RickA-induced actin polymerization, by generating long actin filaments reminiscent of those present in filopodia, has potential as a tool for studying filopodia formation.
[Show abstract][Hide abstract] ABSTRACT: Large macromolecular machines are among the most important and challenging targets for structural and mechanistic analyses. Consequently, there is great interest in development of NMR methods for the study of multicomponent systems in the 50-500 kDa range. Biochemical methods also must be developed in concert to produce such systems in selectively labeled form. Here, we present (1)H/(13)C-HSQC spectra of protonated methyl groups in a model system that mimics molecular weights up to approximately 560 kDa. Signals from side chain methyl groups of Ile, Leu, and Val residues are clearly detectable at correlation times up to approximately 330 ns. We have also developed a biochemical procedure to produce the 240 kDa, heteroheptameric Arp2/3 actin nucleation complex selectively labeled at one subunit and obtained (1)H/(13)C-HSQC spectra of this assembly. Sensitivity in spectra of both the Arp2/3 complex and the model system indicate that methyl groups will be useful sources of information in nonsymmetric systems with molecular weights greater than 600 kDa at concentrations less than 100 microM. Methyl analyses will complement TROSY and CRINEPT analyses of amides in NMR studies of structure and molecular interactions of extremely large macromolecules and assemblies.
[Show abstract][Hide abstract] ABSTRACT: The seven-subunit Arp2/3 complex choreographs the formation of branched actin networks at the leading edge of migrating cells.
When activated by Wiskott-Aldrich Syndrome protein (WASp), the Arp2/3 complex initiates actin filament branches from the sides
of existing filaments. Electron cryomicroscopy and three-dimensional reconstruction of Acanthamoeba castellanii andSaccharomyces cerevisiae Arp2/3 complexes bound to the WASp carboxy-terminal domain reveal asymmetric, oblate ellipsoids. Image analysis of actin
branches indicates that the complex binds the side of the mother filament, and Arp2 and Arp3 (for actin-related protein) are
the first two subunits of the daughter filament. Comparison to the actin-free, WASp-activated complexes suggests that branch
initiation involves large-scale structural rearrangements within Arp2/3.
[Show abstract][Hide abstract] ABSTRACT: Cortactin, a filamentous actin (F-actin)-associated protein and prominent substrate of Src, is implicated in progression of breast tumours through gene amplification at chromosome 11q13. However, the function of cortactin remains obscure. Here we show that cortactin co-localizes with the Arp2/3 complex, a de novo actin nucleator, at dynamic particulate structures enriched with actin filaments. Cortactin binds directly to the Arp2/3 complex and activates it to promote nucleation of actin filaments. The interaction of cortactin with the Arp2/3 complex occurs at an amino-terminal domain that is rich in acidic amino acids. Mutations in a conserved amino-acid sequence of DDW abolish both the interaction with the Arp2/3 complex and complex activation. The N-terminal domain is not only essential but also sufficient to target cortactin to actin-enriched patches within cells. Interestingly, the ability of cortactin to activate the Arp2/3 complex depends on an activity for F-actin binding, which is almost 20-fold higher than that of the Arp2/3 complex. Our data indicate a new mechanism for activation of actin polymerization involving an enhanced interaction between the Arp2/3 complex and actin filaments.