The Conserved Tarp Actin Binding Domain Is Important for Chlamydial Invasion

The translocated actin recruiting phosphoprotein (Tarp) is conserved among all pathogenic chlamydial species. Previous reports identified single C. trachomatis Tarp actin binding and proline rich domains required for Tarp mediated actin nucleation. A peptide antiserum specific for the Tarp actin binding domain was generated and inhibited actin polymerization in vitro and C. trachomatis entry in vivo, indicating an essential role for Tarp in chlamydial pathogenesis. Sequence analysis of Tarp orthologs from additional chlamydial species and C. trachomatis serovars indicated multiple putative actin binding sites. In order to determine whether the identified actin binding domains are functionally conserved, GST-Tarp fusions from multiple chlamydial species were examined for their ability to bind and nucleate actin. Chlamydial Tarps harbored variable numbers of actin binding sites and promoted actin nucleation as determined by in vitro polymerization assays. Our findings indicate that Tarp mediated actin binding and nucleation is a conserved feature among diverse chlamydial species and this function plays a critical role in bacterial invasion of host cells.     

Interaction between Microtubules and the Drosophila Formin Cappuccino and Its Effect on Actin Assembly

Formin family actin nucleators are potential coordinators of the actin and microtubule cytoskeletons, as they can both nucleate actin filaments and bind microtubules in vitro. To gain a more detailed mechanistic understanding of formin-microtubule interactions and formin-mediated actin-microtubule cross-talk, we studied microtubule binding by Cappuccino (Capu), a formin involved in regulating actin and microtubule organization during Drosophila oogenesis. We found that two distinct domains within Capu, FH2 and tail, work together to promote high-affinity microtubule binding. The tail domain appears to bind microtubules through nonspecific charge-based interactions. In contrast, distinct residues within the FH2 domain are important for microtubule binding. We also report the first visualization of a formin polymerizing actin filaments in the presence of microtubules. Interestingly, microtubules are potent inhibitors of the actin nucleation activity of Capu but appear to have little effect on Capu once it is bound to the barbed end of an elongating filament. Because Capu does not simultaneously bind microtubules and assemble actin filaments in vitro, its actin assembly and microtubule binding activities likely require spatial and/or temporal regulation within the Drosophila oocyte.     

Rickettsia Sca2 Recruits Two Actin Subunits for Nucleation but Lacks WH2 Domains

The Rickettsia ～1800-amino-acid autotransporter protein surface cell antigen 2 (Sca2) promotes actin polymerization on the surface of the bacterium to drive its movement using an actin comet-tail mechanism. Sca2 mimics eukaryotic formins in that it promotes both actin filament nucleation and elongation and competes with capping protein to generate filaments that are long and unbranched. However, despite these functional similarities, Sca2 is structurally unrelated to eukaryotic formins and achieves these functions through an entirely different mechanism. Thus, while formins are dimeric, Sca2 functions as a monomer. However, Sca2 displays intramolecular interactions and functional cooperativity between its N- and C-terminal domains that are crucial for actin nucleation and elongation. Here, we map the interaction of N- and C- terminal fragments of Sca2 and their contribution to actin binding and nucleation. We find that both the N- and C-terminal regions of Sca2 interact with actin monomers but only weakly, whereas the full-length protein binds two actin monomers with high affinity. Moreover, deletions at both ends of the N- and C-terminal regions disrupt their ability to interact with each other, suggesting that they form a contiguous ring-like structure that wraps around two actin subunits, analogous to the formin homology-2 domain. The discovery of Sca2 as an actin nucleator followed the identification of what appeared to be a repeat of three Wiskott-Aldrich syndrome homology 2 (WH2) domains in the middle of the molecule, consistent with the presence of WH2 domains in most actin nucleators. However, we show here that contrary to previous assumptions, Sca2 does not contain WH2 domains. Instead, our analysis indicates that the region containing the putative WH2 domains is folded as a globular domain that cooperates with other parts of the Sca2 molecule for actin binding and nucleation.     

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.     

Structure of a Longitudinal Actin Dimer Assembled by Tandem W Domains: Implications for Actin Filament Nucleation

Actin filament nucleators initiate polymerization in cells in a regulated manner. A common architecture among these molecules consists of tandem WASP homology 2 domains (W domains) that recruit three to four actin subunits to form a polymerization nucleus. We describe a low-resolution crystal structure of an actin dimer assembled by tandem W domains, where the first W domain is cross-linked to Cys374 of the actin subunit bound to it, whereas the last W domain is followed by the C-terminal pointed end-capping helix of thymosin β4. While the arrangement of actin subunits in the dimer resembles that of a long-pitch helix of the actin filament, important differences are observed. These differences result from steric hindrance of the W domain with intersubunit contacts in the actin filament. We also determined the structure of the first W domain of Vibrio parahaemolyticus VopL cross-linked to actin Cys374 and show it to be nearly identical with non-cross-linked W-Actin structures. This result validates the use of cross-linking as a tool for the study of actin nucleation complexes, whose natural tendency to polymerize interferes with most structural methods. Combined with a biochemical analysis of nucleation, the structures may explain why nucleators based on tandem W domains with short inter-W linkers have relatively weak activity, cannot stay bound to filaments after nucleation, and are unlikely to influence filament elongation. The findings may also explain why nucleation-promoting factors of the Arp2/3 complex, which are related to tandem-W-domain nucleators, are ejected from branch junctions after nucleation. We finally show that the simple addition of the C-terminal pointed end-capping helix of thymosin β4 to tandem W domains can change their activity from actin filament nucleation to monomer sequestration.     

Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases

The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.     

The Role of Formin Tails in Actin Nucleation,Processive Elongation,and Filament Bundling

Formins are multidomain proteins that assemble actin in a wide variety of biological processes. They both nucleate and remain processively associated with growing filaments, in some cases accelerating filament growth. The well conserved formin homology 1 and 2 domains were originally thought to be solely responsible for these activities. Recently a role in nucleation was identified for the Diaphanous autoinhibitory domain (DAD), which is C-terminal to the formin homology 2 domain. The C-terminal tail of the Drosophila formin Cappuccino (Capu) is conserved among FMN formins but distinct from other formins. It does not have a DAD domain. Nevertheless, we find that Capu-tail plays a role in filament nucleation similar to that described for mDia1 and other formins. Building on this, replacement of Capu-tail with DADs from other formins tunes nucleation activity. Capu-tail has low-affinity interactions with both actin monomers and filaments. Removal of the tail reduces actin filament binding and bundling. Furthermore, when the tail is removed, we find that processivity is compromised. Despite decreased processivity, the elongation rate of filaments is unchanged. Again, replacement of Capu-tail with DADs from other formins tunes the processive association with the barbed end, indicating that this is a general role for formin tails. Our data show a role for the Capu-tail domain in assembling the actin cytoskeleton, largely mediated by electrostatic interactions. Because of its multifunctionality, the formin tail is a candidate for regulation by other proteins during cytoskeletal rearrangements.     

Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.     

Toxoplasma gondii Actin Depolymerizing Factor Acts Primarily to Sequester G-actin

Toxoplasma gondii is a protozoan parasite belonging to the phylum Apicomplexa. Parasites in this phylum utilize a unique process of motility termed gliding, which is dependent on parasite actin filaments. Surprisingly, 98% of parasite actin is maintained as G-actin, suggesting that filaments are rapidly assembled and turned over. Little is known about the regulated disassembly of filaments in the Apicomplexa. In higher eukaryotes, the related actin depolymerizing factor (ADF) and cofilin proteins are essential regulators of actin filament turnover. ADF is one of the few actin-binding proteins conserved in apicomplexan parasites. In this study we examined the mechanism by which T. gondii ADF (TgADF) regulates actin filament turnover. Unlike other members of the ADF/cofilin (AC) family, apicomplexan ADFs lack key F-actin binding sites. Surprisingly, this promotes their enhanced disassembly of actin filaments. Restoration of the C-terminal F-actin binding site to TgADF stabilized its interaction with filaments but reduced its net filament disassembly activity. Analysis of severing activity revealed that TgADF is a weak severing protein, requiring much higher concentrations than typical AC proteins. Investigation of TgADF interaction with T. gondii actin (TgACT) revealed that TgADF disassembled short TgACT oligomers. Kinetic and steady-state polymerization assays demonstrated that TgADF has strong monomer-sequestering activity, inhibiting TgACT polymerization at very low concentrations. Collectively these data indicate that TgADF promoted the efficient turnover of actin filaments via weak severing of filaments and strong sequestering of monomers. This suggests a dual role for TgADF in maintaining high G-actin concentrations and effecting rapid filament turnover.     

Actin Re-Organization Induced by Chlamydia trachomatis Serovar D - Evidence for a Critical Role of the Effector Protein CT166 Targeting Rac

The intracellular bacterium Chlamydia trachomatis causes infections of urogenital tract, eyes or lungs. Alignment reveals homology of CT166, a putative effector protein of urogenital C. trachomatis serovars, with the N-terminal glucosyltransferase domain of clostridial glucosylating toxins (CGTs). CGTs contain an essential DXD-motif and mono-glucosylate GTP-binding proteins of the Rho/Ras families, the master regulators of the actin cytoskeleton. CT166 is preformed in elementary bodies of C. trachomatis D and is detected in the host-cell shortly after infection. Infection with high MOI of C. trachomatis serovar D containing the CT166 ORF induces actin re-organization resulting in cell rounding and a decreased cell diameter. A comparable phenotype was observed in HeLa cells treated with the Rho-GTPase-glucosylating Toxin B from Clostridium difficile (TcdB) or HeLa cells ectopically expressing CT166. CT166 with a mutated DXD-motif (CT166-mut) exhibited almost unchanged actin dynamics, suggesting that CT166-induced actin re-organization depends on the glucosyltransferase motif of CT166. The cytotoxic necrotizing factor 1 (CNF1) from E. coli deamidates and thereby activates Rho-GTPases and transiently protects them against TcdB-induced glucosylation. CNF1-treated cells were found to be protected from TcdB- and CT166-induced actin re-organization. CNF1 treatment as well as ectopic expression of non-glucosylable Rac1-G12V, but not RhoA-G14A, reverted CT166-induced actin re-organization, suggesting that CT166-induced actin re-organization depends on the glucosylation of Rac1. In accordance, over-expression of CT166-mut diminished TcdB induced cell rounding, suggesting shared substrates. Cell rounding induced by high MOI infection with C. trachomatis D was reduced in cells expressing CT166-mut or Rac1-G12V, and in CNF1 treated cells. These observations indicate that the cytopathic effect of C. trachomatis D is mediated by CT166 induced Rac1 glucosylation. Finally, chlamydial uptake was impaired in CT166 over-expressing cells. Our data strongly suggest CT166''s participation as an effector protein during host-cell entry, ensuring a balanced uptake into host-cells by interfering with Rac-dependent cytoskeletal changes.     

MARCKS-Related Protein Binds to Actin without Significantly Affecting Actin Polymerization or Network Structure

Actinis a 42-kDa protein which, due to its ability to polymerize into filaments (F-actin), is one of the major constituents of the cytoskeleton. It has been proposed that MARCKS (an acronym for myristoylated alanine-rich C kinase substrate) proteins play an important role in regulating the structure and mechanical properties of the actin cytoskeleton by cross-linking actin filaments. We have recently reported that peptides corresponding to the effector domain of MARCKS proteins promote actin polymerization and cause massive bundling of actin filaments. We now investigate the effect of MARCKS-related protein, a 20-kDa member of the MARCKS family, on both filament structure and the kinetics of actin polymerization in vitro. Our experiments document that MRP binds to F-actin with micromolar affinity and that the myristoyl chain at the N-terminus of MRP is not required for this interaction. In marked contrast to the effector peptide, binding of MRP is not accompanied by an acceleration of actin polymerization kinetics, and we also could not reliably observe an actin cross-linking activity of MRP.     

Myosin Motors Drive Long Range Alignment of Actin Filaments

The bulk alignment of actin filament sliding movement, powered by randomly oriented myosin molecules, has been observed and studied using an in vitro motility assay. The well established, actin filament gliding assay is a minimal experimental system for studying actomyosin motility. Here, we show that when the assay is performed at densities of actin filaments approaching those found in living cells, filament gliding takes up a preferred orientation. The oriented patterns of movement that we have observed extend over a length scale of 10–100 μm, similar to the size of a mammalian cell. We studied the process of filament alignment and found that it depends critically upon filament length and density. We developed a simple quantitative measure of filament sliding orientation and this enabled us to follow the time course of alignment and the formation and disappearance of oriented domains. Domains of oriented filaments formed spontaneously and were separated by distinct boundaries. The pattern of the domain structures changed on the time scale of several seconds and the collision of neighboring domains led to emergence of new patterns. Our results indicate that actin filament crowding may play an important role in structuring the leading edge of migrating cells. Filament alignment due to near-neighbor mechanical interactions can propagate over a length scale of several microns; much greater than the size of individual filaments and analogous to a log drive. Self-alignment of actin filaments may make an important contribution to cell polarity and provide a mechanism by which cell migration direction responds to chemical cues.     

A Highlights from MBoC Selection: Mathematical Modeling of Endocytic Actin Patch Kinetics in Fission Yeast: Disassembly Requires Release of Actin Filament Fragments

We used the dendritic nucleation hypothesis to formulate a mathematical model of the assembly and disassembly of actin filaments at sites of clathrin-mediated endocytosis in fission yeast. We used the wave of active WASp recruitment at the site of the patch formation to drive assembly reactions after activation of Arp2/3 complex. Capping terminated actin filament elongation. Aging of the filaments by ATP hydrolysis and γ-phosphate dissociation allowed actin filament severing by cofilin. The model could simulate the assembly and disassembly of actin and other actin patch proteins using measured cytoplasmic concentrations of the proteins. However, to account quantitatively for the numbers of proteins measured over time in the accompanying article (Sirotkin et al., 2010 , MBoC 21: 2792–2802), two reactions must be faster in cells than in vitro. Conditions inside the cell allow capping protein to bind to the barbed ends of actin filaments and Arp2/3 complex to bind to the sides of filaments faster than the purified proteins in vitro. Simulations also show that depolymerization from pointed ends cannot account for rapid loss of actin filaments from patches in 10 s. An alternative mechanism consistent with the data is that severing produces short fragments that diffuse away from the patch.     

Structure and Function of Palladin's Actin Binding Domain

Here, we report the NMR structure of the actin-binding domain contained in the cell adhesion protein palladin. Previously, we demonstrated that one of the immunoglobulin domains of palladin (Ig3) is both necessary and sufficient for direct filamentous actin binding in vitro. In this study, we identify two basic patches on opposite faces of Ig3 that are critical for actin binding and cross-linking. Sedimentation equilibrium assays indicate that the Ig3 domain of palladin does not self-associate. These combined data are consistent with an actin cross-linking mechanism that involves concurrent attachment of two actin filaments by a single palladin molecule by an electrostatic mechanism. Palladin mutations that disrupt actin binding show altered cellular distributions and morphology of actin in cells, revealing a functional requirement for the interaction between palladin and actin in vivo.     

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.     

The Interaction of Mip-90 with Microtubules and Actin Filaments in Human Fibroblasts

The novel microtubule-interacting protein Mip-90 was originally isolated from HeLa cells by using affinity columns of agarose derivatized with peptides from the C-terminal regulatory domain on β-tubulin. Biochemical and immunocytochemical data have suggested that the association of Mip-90 with the microtubule system contributes to its cellular organization. Here we report the interaction patterns of Mip-90 with microtubules and actin filaments in interphase human fibroblasts. A polyclonal monospecific antibody against Mip-90 was used for immunofluorescence microscopy analysis to compare the distribution patterns of this protein with tubulin and actin. A detailed observation of fibroblasts revealed the colocalization of Mip-90 with microtubules and actin filaments. These studies were complemented with experiments using cytoskeleton-disrupting drugs which showed that colocalization patterns of Mip-90 with microtubules and actin filaments requires the integrity of these cytoskeletal components. Interestingly, a colocalization of Mip-90 with actin at the leading edge of fibroblasts grown under subconfluency was observed, suggesting that Mip-90 could play a role in actin organization, particularly at this cellular domain. Mip-90 interaction with actin polymers was further supportedin vitroby cosedimentation and immunoprecipitation experiments. The cosedimentation analysis indicated that Mip-90 bound to actin filaments with an association constantK_a= 1 × 10⁶M⁻¹, while an stoichiometry Mip-90/actin of 1:12 mol/mol was calculated. Western blots of the immunoprecipitates revealed that Mip-90 associated to both actin and tubulin in fibroblasts extracts. These studies indicate that Mip-90, described as a microtubule-interacting protein, also bears the capacity to interact with the microfilament network, suggesting that it may play a role in modulating the interactions between these cytoskeletal filaments in nonneuronal cells.     

Arabidopsis CROLIN1, a Novel Plant Actin-binding Protein,Functions in Cross-linking and Stabilizing Actin Filaments

Higher order actin filament structures are necessary for cytoplasmic streaming, organelle movement, and other physiological processes. However, the mechanism by which the higher order cytoskeleton is formed in plants remains unknown. In this study, we identified a novel actin-cross-linking protein family (named CROLIN) that is well conserved only in the plant kingdom. There are six isovariants of CROLIN in the Arabidopsis genome, with CROLIN1 specifically expressed in pollen. In vitro biochemical analyses showed that CROLIN1 is a novel actin-cross-linking protein with binding and stabilizing activities. Remarkably, CROLIN1 can cross-link actin bundles into actin networks. CROLIN1 loss of function induces pollen germination and pollen tube growth hypersensitive to latrunculin B. All of these results demonstrate that CROLIN1 may play an important role in stabilizing and remodeling actin filaments by binding to and cross-linking actin filaments.     

Actin hydrophobic loop 262-274 and filament nucleation and elongation

The importance of actin hydrophobic loop 262-274 dynamics to actin polymerization and filament stability has been shown recently with the use of the yeast mutant actin L180C/L269C/C374A, in which the hydrophobic loop could be locked in a “parked” conformation by a disulfide bond between C180 and C269. Such a cross-linked globular actin monomer does not form filaments, suggesting nucleation and/or elongation inhibition. To determine the role of loop dynamics in filament nucleation and/or elongation, we studied the polymerization of the cross-linked actin in the presence of cofilin, to assist with actin nucleation, and with phalloidin, to stabilize the elongating filament segments. We demonstrate here that together, but not individually, phalloidin and cofilin co-rescue the polymerization of cross-linked actin. The polymerization was also rescued by filament seeds added together with phalloidin but not with cofilin. Thus, loop immobilization via cross-linking inhibits both filament nucleation and elongation. Nevertheless, the conformational changes needed to catalyze ATP hydrolysis by actin occur in the cross-linked actin. When actin filaments are fully decorated by cofilin, the helical twist of filamentous actin (F-actin) changes by ∼ 5° per subunit. Electron microscopic analysis of filaments rescued by cofilin and phalloidin revealed a dense contact between opposite strands in F-actin and a change of twist by ∼ 1° per subunit, indicating either partial or disordered attachment of cofilin to F-actin and/or competition between cofilin and phalloidin to alter F-actin symmetry. Our findings show an importance of the hydrophobic loop conformational dynamics in both actin nucleation and elongation and reveal that the inhibition of these two steps in the cross-linked actin can be relieved by appropriate factors.     

Dynamin2 GTPase and Cortactin Remodel Actin Filaments

The large GTPase dynamin, best known for its activities that remodel membranes during endocytosis, also regulates F-actin-rich structures, including podosomes, phagocytic cups, actin comet tails, subcortical ruffles, and stress fibers. The mechanisms by which dynamin regulates actin filaments are not known, but an emerging view is that dynamin influences F-actin via its interactions with proteins that interact directly or indirectly with actin filaments. We show here that dynamin2 GTPase activity remodels actin filaments in vitro via a mechanism that depends on the binding partner and F-actin-binding protein, cortactin. Tightly associated actin filaments cross-linked by dynamin2 and cortactin became loosely associated after GTP addition when viewed by transmission electron microscopy. Actin filaments were dynamically unraveled and fragmented after GTP addition when viewed in real time using total internal reflection fluorescence microscopy. Cortactin stimulated the intrinsic GTPase activity of dynamin2 and maintained a stable link between actin filaments and dynamin2, even in the presence of GTP. Filaments remodeled by dynamin2 GTPase in vitro exhibit enhanced sensitivity to severing by the actin depolymerizing factor, cofilin, suggesting that GTPase-dependent remodeling influences the interactions of actin regulatory proteins and F-actin. The global organization of the actomyosin cytoskeleton was perturbed in U2-OS cells depleted of dynamin2, implicating dynamin2 in remodeling actin filaments that comprise supramolecular F-actin arrays in vivo. We conclude that dynamin2 GTPase remodels actin filaments and plays a role in orchestrating the global actomyosin cytoskeleton.Controlled assembly and disassembly of actin filaments underlies movement, shape, division, trafficking of lipids and proteins of the cell and pathogenesis by infectious bacteria and viruses. Several proteins and signaling circuits modulate actin filament dynamics, including proteins that nucleate formation of new filaments, filament cross-linking proteins that stabilize branched and bundled filament arrays, and depolymerizing factors that promote filament disassembly (1). Studies with reconstituted systems show that a single actin nucleating factor, such as the Arp2/3 complex together with a nucleation-promoting factor, a barbed end capping protein to preserve the actin monomer pool and promote nucleation, and a filament disassembly factor, such as ADF/cofilin, are sufficient to establish a dynamic dendritic actin network in vitro that mimics many properties of actin networks at the leading edge of migrating cells (2–4). However, the mechanisms for coordinating the organization and dynamics of actin filaments associated with higher-order cellular structures such as the subcortical F-actin network, F-actin at focal adhesions, and actomyosin arrays are not as well understood.Considerable evidence indicates that the large GTPase dynamin, a key mediator of membrane remodeling and fission, also influences actin filaments (reviewed in Refs. 5–7). Although the mechanisms are unknown, dynamin could influence actin filaments via its interactions with a number of proteins that directly or indirectly regulate actin filament assembly, filament stability, or filament organization. For example, several protein scaffolds biochemically link dynamin and the Arp2/3 complex activating factor, N-WASP, suggesting that the machinery for de novo actin assembly may be targeted or activated by dynamin (6, 8, 9). Dynamin2 is associated with several dynamic F-actin-containing structures in vivo, including podosomes, F-actin comet tails, phagocytic cups, dynamic cortical ruffles, and pedestal structures elaborated by enteropathogenic Escherichia coli (10–20). Cortactin, which directly binds both dynamin and actin filaments, is associated with many of the same dynamic actin structures as dynamin (5, 7) and is required for both clathrin-dependent and -independent endocytosis (21, 22). Thus, dynamin-cortactin interaction may be an important link between actin filaments and dynamin during formation or turnover of F-actin-rich structures.Considerable evidence supports the notion that GTP hydrolysis by dynamin catalyzes membrane fission activity via GTPase-dependent changes in conformation (23, 24) or via GTPase-dependent cycles of assembly and disassembly (25, 26). We hypothesize that GTPase-dependent changes in dynamin linked via its interacting proteins to actin filaments or actin regulators could similarly influence actin filaments. Overexpressed, dominant negative dynamin mutant proteins impaired in binding or hydrolyzing GTP (most often the dynamin-K44A mutation) perturb a variety of F-actin-rich cellular structures, including stress fibers and focal adhesions (27, 28), dendritic spines of neurons (29), podosomes (12, 30), actin comet tails (13, 14), phagocytic cups and bacteria-induced pedestal structures (16, 19), and dynamic cortical ruffles (15, 17). In addition, F-actin of stress fibers and overall cell morphology were perturbed in Clone9 cells expressing a mutant dynamin2 protein lacking the C-terminal proline-rich domain, the domain through which dynamin2 interacts with actin regulatory factors (11). Whereas existing data indicates that the specific effects of dynamin GTPase activity on F-actin structures are cell type- and structure-specific, a general conclusion is that dynamin GTPase activity influences the organization or turnover of a subset of actin filaments.To determine the mechanisms by which dynamin2 GTPase activity influences actin filaments, we developed biochemical and microscopic approaches to quantitatively assess and observe GTPase-dependent effects on actin filaments formed in vitro with Arp2/3 complex, cortactin, and dynamin2. The activities of dynamin2 on actin filaments in vivo were examined in cells with disrupted dynamin2 function using siRNA²-mediated suppression or pharmacologic inhibition. We report that dynamin2 GTPase, together with cortactin, functions as a dynamic actin filament remodeling complex that influences the global organization of the actomyosin cytoskeleton.