A Highlights from MBoC Selection: Cell type–dependent mechanisms for formin-mediated assembly of filopodia

Filopodia are finger-like protrusions from the plasma membrane and are of fundamental importance to cellular physiology, but the mechanisms governing their assembly are still in question. One model, called convergent elongation, proposes that filopodia arise from Arp2/3 complex–nucleated dendritic actin networks, with factors such as formins elongating these filaments into filopodia. We test this model using constitutively active constructs of two formins, FMNL3 and mDia2. Surprisingly, filopodial assembly requirements differ between suspension and adherent cells. In suspension cells, Arp2/3 complex is required for filopodial assembly through either formin. In contrast, a subset of filopodia remains after Arp2/3 complex inhibition in adherent cells. In adherent cells only, mDia1 and VASP also contribute to filopodial assembly, and filopodia are disproportionately associated with focal adhesions. We propose an extension of the existing models for filopodial assembly in which any cluster of actin filament barbed ends in proximity to the plasma membrane, either Arp2/3 complex dependent or independent, can initiate filopodial assembly by specific formins.     

Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end

Understanding how a particular cell type expresses the lamellipodial or filopodial form of the actin machinery is essential to understanding a cell's functional interactions. To determine how a cell "chooses" among these alternative modes of "molecular hardware," we tested the role of key proteins that affect actin filament barbed ends. Depletion of capping protein (CP) by short hairpin RNA (shRNA) caused loss of lamellipodia and explosive formation of filopodia. The knockdown phenotype was rescued by a CP mutant refractory to shRNA, but not by another barbed-end capper, gelsolin, demonstrating that the phenotype was specific for CP. In Ena/VASP deficient cells, CP depletion resulted in ruffling instead of filopodia. We propose a model for selection of lamellipodial versus filopodial organization in which CP is a negative regulator of filopodia formation and Ena/VASP has recruiting/activating functions downstream of actin filament elongation in addition to its previously suggested anticapping and antibranching activities.     

Filopodia formation in the absence of functional WAVE- and Arp2/3-complexes

Cell migration is initiated by plasma membrane protrusions, in the form of lamellipodia and filopodia. The latter rod-like projections may exert sensory functions and are found in organisms as distant in evolution as mammals and amoeba such as Dictyostelium discoideum. In mammals, lamellipodia protrusion downstream of the small GTPase Rac1 requires a multimeric protein assembly, the WAVE-complex, which activates Arp2/3-mediated actin filament nucleation and actin network assembly. A current model of filopodia formation postulates that these structures arise from a dendritic network of lamellipodial actin filaments by selective elongation and bundling. Here, we have analyzed filopodia formation in mammalian cells abrogated in expression of essential components of the lamellipodial actin polymerization machinery. Cells depleted of the WAVE-complex component Nck-associated protein 1 (Nap1), and, in consequence, of lamellipodia, exhibited normal filopodia protrusion. Likewise, the Arp2/3-complex, which is essential for lamellipodia protrusion, is dispensable for filopodia formation. Moreover, genetic disruption of nap1 or the WAVE-orthologue suppressor of cAMP receptor (scar) in Dictyostelium was also ineffective in preventing filopodia protrusion. These data suggest that the molecular mechanism of filopodia formation is conserved throughout evolution from Dictyostelium to mammals and show that lamellipodia and filopodia formation are functionally separable.     

Formation of filopodia-like bundles in vitro from a dendritic network

We report the development and characterization of an in vitro system for the formation of filopodia-like bundles. Beads coated with actin-related protein 2/3 (Arp2/3)-activating proteins can induce two distinct types of actin organization in cytoplasmic extracts: (1) comet tails or clouds displaying a dendritic array of actin filaments and (2) stars with filament bundles radiating from the bead. Actin filaments in these bundles, like those in filopodia, are long, unbranched, aligned, uniformly polar, and grow at the barbed end. Like filopodia, star bundles are enriched in fascin and lack Arp2/3 complex and capping protein. Transition from dendritic to bundled organization was induced by depletion of capping protein, and add-back of this protein restored the dendritic mode. Depletion experiments demonstrated that star formation is dependent on Arp2/3 complex. This poses the paradox of how Arp2/3 complex can be involved in the formation of both branched (lamellipodia-like) and unbranched (filopodia-like) actin structures. Using purified proteins, we showed that a small number of components are sufficient for the assembly of filopodia-like bundles: Wiskott-Aldrich syndrome protein (WASP)-coated beads, actin, Arp2/3 complex, and fascin. We propose a model for filopodial formation in which actin filaments of a preexisting dendritic network are elongated by inhibition of capping and subsequently cross-linked into bundles by fascin.     

Dia-interacting protein modulates formin-mediated actin assembly at the cell cortex

BACKGROUND: Mammalian Diaphanous (mDia)-related formins and the N-WASP-activated Arp2/3 complex initiate the assembly of filamentous actin. Dia-interacting protein (DIP) binds via its amino-terminal SH3 domain to the proline-rich formin homology 1 (FH1) domain of mDia1 and mDia2 and to the N-WASp proline-rich region. RESULTS: Here, we investigated an interaction between a conserved leucine-rich region (LRR) in DIP and the mDia FH2 domain that nucleates, processively elongates, and bundles actin filaments. DIP binding to mDia2 was regulated by the same Rho-GTPase-controlled autoinhibitory mechanism modulating formin-mediated actin assembly. DIP was previously shown to interact with and stimulate N-WASp-dependent branched filament assembly via Arp2/3. Despite direct binding to both mDia1 and mDia2 FH2 domains, DIP LRR inhibited only mDia2-dependent filament assembly and bundling in vitro. DIP expression interfered with filopodia formation, consistent with a role for mDia2 in assembly of these structures. After filopodia retraction into the cell body, DIP expression induced excessive nonapoptotic membrane blebbing, a physiological process involved in both cytokinesis and amoeboid cell movement. DIP-induced blebbing was dependent on mDia2 but did not require the activities of either mDia1 or Arp2/3. CONCLUSIONS: These observations point to a pivotal role for DIP in the control of nonbranched and branched actin-filament assembly that is mediated by Diaphanous-related formins and activators of Arp2/3, respectively. The ability of DIP to trigger blebbing also suggests a role for mDia2 in the assembly of cortical actin necessary for maintaining plasma-membrane integrity.     

Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation

Cortactin is a c-src substrate associated with sites of dynamic actin assembly at the leading edge of migrating cells. We previously showed that cortactin binds to Arp2/3 complex, the essential molecular machine for nucleating actin filament assembly. In this study, we demonstrate that cortactin activates Arp2/3 complex based on direct visualization of filament networks and pyrene actin assays. Strikingly, cortactin potently inhibited the debranching of filament networks. When cortactin was added in combination with the active VCA fragment of N-WASp, they synergistically enhanced Arp2/3-induced actin filament branching. The N-terminal acidic and F-actin binding domains of cortactin were both necessary to activate Arp2/3 complex. These results support a model in which cortactin modulates actin filament dendritic nucleation by two mechanisms, (1) direct activation of Arp2/3 complex and (2) stabilization of newly generated filament branch points. By these mechanisms, cortactin may promote the formation and stabilization of the actin network that drives protrusion at the leading edge of migrating cells.     

Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins

Ena/VASP proteins influence the organization of actin filament networks within lamellipodia and filopodia of migrating cells and in actin comet tails. The molecular mechanisms by which Ena/VASP proteins control actin dynamics are unknown. We investigated how Ena/VASP proteins regulate actin polymerization at actin filament barbed ends in vitro in the presence and absence of barbed end capping proteins. Recombinant His-tagged VASP increased the rate of actin polymerization in the presence of the barbed end cappers, heterodimeric capping protein (CP), CapG, and gelsolin-actin complex. Profilin enhanced the ability of VASP to protect barbed ends from capping by CP, and this required interactions of profilin with G-actin and VASP. The VASP EVH2 domain was sufficient to protect barbed ends from capping, and the F-actin and G-actin binding motifs within EVH2 were required. Phosphorylation by protein kinase A at sites within the VASP EVH2 domain regulated anti-capping and F-actin bundling by VASP. We propose that Ena/VASP proteins associate at or near actin filament barbed ends, promote actin assembly, and restrict the access of barbed end capping proteins.     

The interaction of capping protein with the barbed end of the actin filament

The interaction of capping protein (CP) with actin filaments is an essential element of actin assembly and actin-based motility in nearly all eukaryotes. The dendritic nucleation model for Arp2/3-based lamellipodial assembly features capping of barbed ends by CP, and the formation of filopodia is proposed to involve inhibition of capping by formins and other proteins. To understand the molecular basis for how CP binds the barbed end of the actin filament, we have used a combination of computational and experimental approaches, primarily involving molecular docking and site-directed mutagenesis. We arrive at a model that supports all of our biochemical data and agrees very well with a cryo-electron microscopy structure of the capped filament. CP interacts with both actin protomers at the barbed end of the filament, and the amphipathic helix at the C-terminus of the β-subunit binds to the hydrophobic cleft on actin, in a manner similar to that of WH2 domains. These studies provide us with new molecular insight into how CP binds to the actin filament.     

Cdc42 induces filopodia by promoting the formation of an IRSp53:Mena complex.

BACKGROUND: The Rho GTPases Rho, Rac, and Cdc42 regulate the organization of the actin cytoskeleton by interacting with multiple, distinct downstream effector proteins. Cdc42 controls the formation of actin bundle-containing filopodia at the cellular periphery. The molecular mechanism for this remains as yet unclear. RESULTS: We report here that Cdc42 interacts with IRSp53/BAP2 alpha, an SH3 domain-containing scaffold protein, at a partial CRIB motif and that an N-terminal fragment of IRSp53 binds, via an intramolecular interaction, to the CRIB motif-containing central region. Overexpression of IRSp53 in fibroblasts leads to the formation of filopodia, and both this and Cdc42-induced filopodia are inhibited by expression of the N-terminal IRSp53 fragment. Using affinity chromatography, we have identified Mena, an Ena/VASP family member, as interacting with the SH3 domain of IRSp53. Mena and IRSp53 act synergistically to promote filopodia formation. CONCLUSION: We conclude that the interaction of Cdc42 with the partial CRIB motif of IRSp53 relieves an intramolecular, autoinhibitory interaction with the N terminus, allowing the recruitment of Mena to the IRSp53 SH3 domain. This IRSp53:Mena complex initiates actin filament assembly into filopodia.     

The making of filopodia

Filopodia are rod-like cell surface projections filled with bundles of parallel actin filaments. They are found on a variety of cell types and have been ascribed sensory or exploratory functions. Filopodium formation is frequently associated with protrusion of sheet-like actin filament arrays called lamellipodia and membrane ruffles, but, in comparison to these structures, the molecular details underpinning the initiation and maintenance of filopodia are only just beginning to emerge. Recent advances have improved our understanding of the molecular requirements for filopodium protrusion and have yielded insights into the inter-relationships between lamellipodia and filopodia, the two 'sub-compartments' of the protrusive actin cytoskeleton.     

Defining mechanisms of actin polymerization and depolymerization during dendritic spine morphogenesis

Dendritic spines are small protrusions along dendrites where the postsynaptic components of most excitatory synapses reside in the mature brain. Morphological changes in these actin-rich structures are associated with learning and memory formation. Despite the pivotal role of the actin cytoskeleton in spine morphogenesis, little is known about the mechanisms regulating actin filament polymerization and depolymerization in dendritic spines. We show that the filopodia-like precursors of dendritic spines elongate through actin polymerization at both the filopodia tip and root. The small GTPase Rif and its effector mDia2 formin play a central role in regulating actin dynamics during filopodia elongation. Actin filament nucleation through the Arp2/3 complex subsequently promotes spine head expansion, and ADF/cofilin-induced actin filament disassembly is required to maintain proper spine length and morphology. Finally, we show that perturbation of these key steps in actin dynamics results in altered synaptic transmission.     

Cdc42 regulates arsenic-induced NADPH oxidase activation and cell migration through actin filament reorganization

Although arsenic is a human carcinogen, the molecular mechanisms of its action remain to be understood. The present study reports that exposure to arsenic induced actin filament reorganization, resulting in lamellipodia and filopodia structures through the activation of Cdc42 in SVEC4-10 endothelial cells. It was also found that arsenic induced the formation of the superoxide anion (O2*) in SVEC4-10 cells. Immunoprecipitation and Western blotting analysis demonstrated that arsenic stimulation induced serine phosphorylation of p47phox, a key component of NADPH oxidase, indicating that arsenic induces O2* formation through NADPH oxidase activation. Inhibition of arsenic-induced actin filament reorganization by either overexpression of a dominant negative Cdc42 or pretreatment of an actin filament stabilizing regent, jasplakinolide, abrogated arsenic-induced NADPH oxidase activation, showing that the activation of NADPH oxidase was regulated by Cdc42-mediated actin filament reorganization. This study also showed that overexpression of a dominant negative Rac1 was sufficient to abolish arsenic-induced O2*- production, implying that Rac1 activities are required for Cdc42-mediated NADPH oxidase activation in response to arsenic stimulation. Furthermore, arsenic stimulation induced cell migration, which can be inhibited by the inactivation of either Cdc42 or NADPH oxidase. Taken together, the results indicate that arsenic is able to activate NADPH oxidase through Cdc42-mediated actin filament reorganization, leading to the induction of an increase in cell migration in SVEC4-10 endothelial cells.     

Screening and target identification of bioactive compounds that modulate cell migration and autophagy

Cell migration is a fundamental step for embryonic development, wound repair, immune responses, and tumor cell invasion and metastasis. It is well known that protrusive structures, namely filopodia and lamellipodia, can be observed at the leading edge of migrating cells. The formation of these structures is necessary for cell migration; however, the molecular mechanisms behind the formation of these structures remain largely unclear. Therefore, bioactive compounds that modulate protrusive structures are extremely powerful tools for studying the mechanisms behind the formation of these structures and subsequent cell migration. Therefore, we have screened for bioactive compounds that inhibit the formation of filopodia, lamellipodia, or cell migration from natural products, and attempted to identify the target molecules of our isolated compounds. Additionally, autophagy is a bulk, non-specific protein degradation system that is involved in the pathogenesis of cancer and neurodegenerative disorders. Recent extensive studies have revealed the molecular mechanisms of autophagy, however, they also remain largely unclear. Thus, we also have screened for bioactive compounds that modulate autophagy, and identified the target molecules. In the present article, we introduce the phenotypic screening system and target identification of four bioactive compounds.     

The Effect of Tropomyosin Mutations on Actin-Tropomyosin Binding: In Search of Lost Time

The initial binding of tropomyosin onto actin filaments and then its polymerization into continuous cables on the filament surface must be precisely tuned to overall thin-filament structure, function, and performance. Low-affinity interaction of tropomyosin with actin has to be sufficiently strong to localize the tropomyosin on actin, yet not so tight that regulatory movement on filaments is curtailed. Likewise, head-to-tail association of tropomyosin molecules must be favorable enough to promote tropomyosin cable formation but not so tenacious that polymerization precedes filament binding. Arguably, little molecular detail on early tropomyosin binding steps has been revealed since Wegner’s seminal studies on filament assembly almost 40 years ago. Thus, interpretation of mutation-based actin-tropomyosin binding anomalies leading to cardiomyopathies cannot be described fully. In vitro, tropomyosin binding is masked by explosive tropomyosin polymerization once cable formation is initiated on actin filaments. In contrast, in silico analysis, characterizing molecular dynamics simulations of single wild-type and mutant tropomyosin molecules on F-actin, is not complicated by tropomyosin polymerization at all. In fact, molecular dynamics performed here demonstrates that a midpiece tropomyosin domain is essential for normal actin-tropomyosin interaction and that this interaction is strictly conserved in a number of tropomyosin mutant species. Elsewhere along these mutant molecules, twisting and bending corrupts the tropomyosin superhelices as they “lose their grip” on F-actin. We propose that residual interactions displayed by these mutant tropomyosin structures with actin mimic ones that occur in early stages of thin-filament generation, as if the mutants are recapitulating the assembly process but in reverse. We conclude therefore that an initial binding step in tropomyosin assembly onto actin involves interaction of the essential centrally located domain.     

Integrating the actin and vimentin cytoskeletons. adhesion-dependent formation of fimbrin-vimentin complexes in macrophages.

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH(2)-terminal domain of vimentin and the vimentin binding site at residues 143-188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin-vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.     

The activation of ezrin-radixin-moesin proteins is regulated by netrin-1 through Src kinase and RhoA/Rho kinase activities and mediates netrin-1-induced axon outgrowth

The receptor Deleted in Colorectal Cancer (DCC) mediates the attractive response of axons to the guidance cue netrin-1 during development. On netrin-1 stimulation, DCC is phosphorylated and induces the assembly of signaling complexes within the growth cone, leading to activation of cytoskeleton regulators, namely the GTPases Rac1 and Cdc42. The molecular mechanisms that link netrin-1/DCC to the actin machinery remain unclear. In this study we seek to demonstrate that the actin-binding proteins ezrin-radixin-moesin (ERM) are effectors of netrin-1/DCC signaling in embryonic cortical neurons. We show that ezrin associates with DCC in a netrin-1-dependent manner. We demonstrate that netrin-1/DCC induces ERM phosphorylation and activation and that the phosphorylation of DCC is required in that context. Moreover, Src kinases and RhoA/Rho kinase activities mediate netrin-1-induced ERM phosphorylation in neurons. We also observed that phosphorylated ERM proteins accumulate in growth cone filopodia, where they colocalize with DCC upon netrin-1 stimulation. Finally, we show that loss of ezrin expression in cortical neurons significantly decreases axon outgrowth induced by netrin-1. Together, our findings demonstrate that netrin-1 induces the formation of an activated ERM/DCC complex in growth cone filopodia, which is required for netrin-1-dependent cortical axon outgrowth.     

Molecular Architecture of Synaptic Actin Cytoskeleton in Hippocampal Neurons Reveals a Mechanism of Dendritic Spine Morphogenesis

Excitatory synapses in the brain play key roles in learning and memory. The formation and functions of postsynaptic mushroom-shaped structures, dendritic spines, and possibly of presynaptic terminals, rely on actin cytoskeleton remodeling. However, the cytoskeletal architecture of synapses remains unknown hindering the understanding of synapse morphogenesis. Using platinum replica electron microscopy, we characterized the cytoskeletal organization and molecular composition of dendritic spines, their precursors, dendritic filopodia, and presynaptic boutons. A branched actin filament network containing Arp2/3 complex and capping protein was a dominant feature of spine heads and presynaptic boutons. Surprisingly, the spine necks and bases, as well as dendritic filopodia, also contained a network, rather than a bundle, of branched and linear actin filaments that was immunopositive for Arp2/3 complex, capping protein, and myosin II, but not fascin. Thus, a tight actin filament bundle is not necessary for structural support of elongated filopodia-like protrusions. Dynamically, dendritic filopodia emerged from densities in the dendritic shaft, which by electron microscopy contained branched actin network associated with dendritic microtubules. We propose that dendritic spine morphogenesis begins from an actin patch elongating into a dendritic filopodium, which tip subsequently expands via Arp2/3 complex-dependent nucleation and which length is modulated by myosin II-dependent contractility.     

Common regulatory control of CTP synthase enzyme activity and filament formation

The ability of enzymes to assemble into visible supramolecular complexes is a widespread phenomenon. Such complexes have been hypothesized to play a number of roles; however, little is known about how the regulation of enzyme activity is coupled to the assembly/disassembly of these cellular structures. CTP synthase is an ideal model system for addressing this question because its activity is regulated via multiple mechanisms and its filament-forming ability is evolutionarily conserved. Our structure–function studies of CTP synthase in Saccharomyces cerevisiae reveal that destabilization of the active tetrameric form of the enzyme increases filament formation, suggesting that the filaments comprise inactive CTP synthase dimers. Furthermore, the sites responsible for feedback inhibition and allosteric activation control filament length, implying that multiple regions of the enzyme can influence filament structure. In contrast, blocking catalysis without disrupting the regulatory sites of the enzyme does not affect filament formation or length. Together our results argue that the regulatory sites that control CTP synthase function, but not enzymatic activity per se, are critical for controlling filament assembly. We predict that the ability of enzymes to form supramolecular structures in general is closely coupled to the mechanisms that regulate their activity.     

The Eps8/IRSp53/VASP network differentially controls actin capping and bundling in filopodia formation

There is a body of literature that describes the geometry and the physics of filopodia using either stochastic models or partial differential equations and elasticity and coarse-grained theory. Comparatively, there is a paucity of models focusing on the regulation of the network of proteins that control the formation of different actin structures. Using a combination of in-vivo and in-vitro experiments together with a system of ordinary differential equations, we focused on a small number of well-characterized, interacting molecules involved in actin-dependent filopodia formation: the actin remodeler Eps8, whose capping and bundling activities are a function of its ligands, Abi-1 and IRSp53, respectively; VASP and Capping Protein (CP), which exert antagonistic functions in controlling filament elongation. The model emphasizes the essential role of complexes that contain the membrane deforming protein IRSp53, in the process of filopodia initiation. This model accurately accounted for all observations, including a seemingly paradoxical result whereby genetic removal of Eps8 reduced filopodia in HeLa, but increased them in hippocampal neurons, and generated quantitative predictions, which were experimentally verified. The model further permitted us to explain how filopodia are generated in different cellular contexts, depending on the dynamic interaction established by Eps8, IRSp53 and VASP with actin filaments, thus revealing an unexpected plasticity of the signaling network that governs the multifunctional activities of its components in the formation of filopodia.     

Dissecting regulatory networks of filopodia formation in a Drosophila growth cone model

F-actin networks are important structural determinants of cell shape and morphogenesis. They are regulated through a number of actin-binding proteins. The function of many of these proteins is well understood, but very little is known about how they cooperate and integrate their activities in cellular contexts. Here, we have focussed on the cellular roles of actin regulators in controlling filopodial dynamics. Filopodia are needle-shaped, actin-driven cell protrusions with characteristic features that are well conserved amongst vertebrates and invertebrates. However, existing models of filopodia formation are still incomplete and controversial, pieced together from a wide range of different organisms and cell types. Therefore, we used embryonic Drosophila primary neurons as one consistent cellular model to study filopodia regulation. Our data for loss-of-function of capping proteins, enabled, different Arp2/3 complex components, the formin DAAM and profilin reveal characteristic changes in filopodia number and length, providing a promising starting point to study their functional relationships in the cellular context. Furthermore, the results are consistent with effects reported for the respective vertebrate homologues, demonstrating the conserved nature of our Drosophila model system. Using combinatorial genetics, we demonstrate that different classes of nucleators cooperate in filopodia formation. In the absence of Arp2/3 or DAAM filopodia numbers are reduced, in their combined absence filopodia are eliminated, and in genetic assays they display strong functional interactions with regard to filopodia formation. The two nucleators also genetically interact with enabled, but not with profilin. In contrast, enabled shows strong genetic interaction with profilin, although loss of profilin alone does not affect filopodia numbers. Our genetic data support a model in which Arp2/3 and DAAM cooperate in a common mechanism of filopodia formation that essentially depends on enabled, and is regulated through profilin activity at different steps.