New mechanisms and functions of actin nucleation

In cells the de novo nucleation of actin filaments from monomers requires actin-nucleating proteins. These fall into three main families--the Arp2/3 complex and its nucleation promoting factors (NPFs), formins, and tandem-monomer-binding nucleators. In this review, we highlight recent advances in understanding the molecular mechanism of actin nucleation by both well-characterized and newly identified nucleators, and explore current insights into their cellular functions in membrane trafficking, cell migration and division. The mechanisms and functions of actin nucleators are proving to be more complex than previously considered, with extensive cooperation and overlap in their cellular roles.     

Actin filament nucleation and elongation factors – structure–function relationships

The spontaneous and unregulated polymerization of actin filaments is inhibited in cells by actin monomer-binding proteins such as profilin and Tβ4. Eukaryotic cells and certain pathogens use filament nucleators to stabilize actin polymerization nuclei, whose formation is rate-limiting. Known filament nucleators include the Arp2/3 complex and its large family of nucleation promoting factors (NPFs), formins, Spire, Cobl, VopL/VopF, TARP and Lmod. These molecules control the time and location for polymerization, and additionally influence the structures of the actin networks that they generate. Filament nucleators are generally unrelated, but with the exception of formins they all use the WASP-Homology 2 domain (WH2 or W), a small and versatile actin-binding motif, for interaction with actin. A common architecture, found in Spire, Cobl and VopL/VopF, consists of tandem W domains that bind three to four actin subunits to form a nucleus. Structural considerations suggest that NPFs–Arp2/3 complex can also be viewed as a specialized form of tandem W-based nucleator. Formins are unique in that they use the formin-homology 2 (FH2) domain for interaction with actin and promote not only nucleation, but also processive barbed end elongation. In contrast, the elongation function among W-based nucleators has been “outsourced” to a dedicated family of proteins, Eva/VASP, which are related to WASP-family NPFs.     

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.     

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

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

The formin DAD domain plays dual roles in autoinhibition and actin nucleation

Formins are a large family of actin assembly-promoting proteins with many important biological roles. However, it has remained unclear how formins nucleate actin polymerization. All other nucleators are known to recruit actin monomers as a central part of their mechanisms. However, the actin-nucleating FH2 domain of formins lacks appreciable affinity for monomeric actin. Here, we found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we observed that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia1, mapped nucleation activity to conserved residues in the DAD, and demonstrated that DAD roles in nucleation and autoinhibition are separable. Furthermore, DAD enhancement of nucleation was independent of contributions from the FH1 domain to nucleation. Together, our data show that (1) the DAD has dual functions in autoinhibition and nucleation; (2) the FH1, FH2, and DAD form a tripartite nucleation machine; and (3) formins nucleate by recruiting actin monomers and therefore are more similar to other nucleators than previously thought.     

Controlling actin cytoskeletal organization and dynamics during neuronal morphogenesis

Coordinated functions of the actin cytoskeleton and microtubules, which need to be carefully controlled in time and space, are required for the drastic alterations of neuronal morphology during neuromorphogenesis and neuronal network formation. A key process in neuronal actin dynamics is filament formation by actin nucleators, such as the Arp2/3 complex, formins and the brain-enriched, novel WH2 domain-based nucleators Spire and cordon-bleu (Cobl). We here discuss in detail the currently available data on the roles of these actin nucleators during neuromorphogenesis and highlight how their required control at the plasma membrane may be brought about. The Arp2/3 complex was found to be especially important for proper growth cone translocation and axon development. The underlying molecular mechanisms for Arp2/3 complex activation at the neuronal plasma membrane include a recruitment and an activation of N-WASP by lipid- and F-actin-binding adaptor proteins, Cdc42 and phosphatidyl-inositol-(4,5)-bisphosphate (PIP(2)). Together, these components upstream of N-WASP and the Arp2/3 complex ensure fine-control of N-WASP-mediated Arp2/3 complex activation and control distinct functions during axon development. They are counteracted by Arp2/3 complex inhibitors, such as PICK, which likewise play an important role in neuromorphogenesis. In contrast to the crucial role of the Arp2/3 complex in proper axon development, dendrite formation and dendritic arborization was revealed to critically involve the newly identified actin nucleator Cobl. Cobl is a brain-enriched protein and uses three Wiskott-Aldrich syndrome protein homology 2 (WH2) domains for actin binding and for promoting the formation of non-bundled, unbranched filaments. Thus, cells use different actin nucleators to steer the complex remodeling processes underlying cell morphogenesis, the formation of cellular networks and the development of complex body plans.     

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.     

Actin nucleation: spire - actin nucleator in a class of its own

The rate limiting step for actin filament polymerisation is nucleation, and two types of nucleator have been described: the Arp2/3 complex and the formins. A recent study has now identified in Spire a third class of actin nucleator. The four short WH2 repeats within Spire bind four consecutive actin monomers to form a novel single strand nucleus for 'barbed end' actin filament elongation.     

Arp2/3 complex is important for filopodia formation, growth cone motility, and neuritogenesis in neuronal cells

A role of Arp2/3 complex in lamellipodia is well established, whereas its roles in filopodia formation remain obscure. We addressed this question in neuronal cells, in which motility is heavily based on filopodia, and we found that Arp2/3 complex is involved in generation of both lamellipodia and filopodia in growth cones, and in neuritogenesis, the processes thought to occur largely in Arp2/3 complex-independent manner. Depletion of Arp2/3 complex in primary neurons and neuroblastoma cells by small interfering RNA significantly decreased the F-actin contents and inhibited lamellipodial protrusion and retrograde flow in growth cones, but also initiation and dynamics of filopodia. Using electron microscopy, immunochemistry, and gene expression, we demonstrated the presence of the Arp2/3 complex-dependent dendritic network of actin filaments in growth cones, and we showed that individual actin filaments in filopodia originated at Arp2/3 complex-dependent branch points in lamellipodia, thus providing a mechanistic explanation of Arp2/3 complex functions during filopodia formation. Additionally, Arp2/3 complex depletion led to formation of multiple neurites, erratic pattern of neurite extension, and excessive formation of stress fibers and focal adhesions. Consistent with this phenotype, RhoA activity was increased in Arp2/3 complex-depleted cells, indicating that besides nucleating actin filaments, Arp2/3 complex may influence cell motility by altering Rho GTPase signaling.     

A malaria parasite formin regulates actin polymerization and localizes to the parasite-erythrocyte moving junction during invasion

Malaria parasites invade host cells using actin-based motility, a process requiring parasite actin filament nucleation and polymerization. Malaria and other apicomplexan parasites lack Arp2/3 complex, an actin nucleator widely conserved across eukaryotes, but do express formins, another type of actin nucleator. Here, we demonstrate that one of two malaria parasite formins, Plasmodium falciparum formin 1 (PfFormin 1), and its ortholog in the related parasite Toxoplasma gondii, follows the moving tight junction between the invading parasite and the host cell, which is the predicted site of the actomyosin motor that powers motility. Furthermore, in vitro, the PfFormin1 actin-binding formin homology 2 domain is a potent nucleator, stimulating actin polymerization and, like other formins, localizing to the barbed end during filament elongation. These findings support a conserved molecular mechanism underlying apicomplexan parasite motility and, given the essential role that actin plays in cell invasion, highlight formins as important determinants of malaria parasite pathogenicity.     

An actin nucleation mechanism mediated by Bni1 and profilin

Formins are required for cell polarization and cytokinesis, but do not have a defined biochemical activity. In Saccharomyces cerevisiae, formins and the actin-monomer-binding protein profilin are specifically required to assemble linear actin structures called 'actin cables'. These structures seem to be assembled independently of the Arp2/3 complex, the only well characterized cellular mediator of actin nucleation. Here, an activated yeast formin was purified and found to promote the nucleation of actin filaments in vitro. Formin-dependent actin nucleation was stimulated by profilin. Thus, formin and profilin mediate actin nucleation by an Arp2/3-independent mechanism. These findings suggest that distinct actin nucleation mechanisms may underlie the assembly of different actin cytoskeletal structures.     

Novel roles of formin mDia2 in lamellipodia and filopodia formation in motile cells

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (ΔGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of ΔGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that ΔGBD-mDia2–induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.     

Protein complexes regulating Arp2/3-mediated actin assembly

Key steps in regulating actin dynamics are the de novo nucleation and elongation of actin filaments, which can be catalysed by a limited number of proteins and protein complexes. Among these, Arp2/3 complex and formins are the best studied. Arp2/3-complex activity is controlled through signalling-dependent association with nucleation-promoting factors, such as the WASP/WAVE family proteins. A common theme for these molecules, which is well established for WAVEs but is only just beginning to emerge for WASPs, is that they act as coincident detectors of a variety of signalling pathways through the formation of large multi-molecular complexes.     

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.     

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.     

The actin nucleating Arp2/3 complex contributes to the formation of axonal filopodia and branches through the regulation of actin patch precursors to filopodia

The emergence of axonal filopodia is the first step in the formation of axon collateral branches. In vitro, axonal filopodia emerge from precursor cytoskeletal structures termed actin patches. However, nothing is known about the cytoskeletal dynamics of the axon leading to the formation of filopodia in the relevant tissue environment. In this study we investigated the role of the actin nucleating Arp2/3 complex in the formation of sensory axon actin patches, filopodia, and branches. By combining in ovo chicken embryo electroporation mediated gene delivery with a novel acute ex vivo spinal cord preparation, we demonstrate that actin patches form along sensory axons and give rise to filopodia in situ. Inhibition of Arp2/3 complex function in vitro and in vivo decreases the number of axonal filopodia. In vitro, Arp2/3 complex subunits and upstream regulators localize to actin patches. Analysis of the organization of actin filaments in actin patches using platinum replica electron microscopy reveals that patches consist of networks of actin filaments, and filaments in axonal filopodia exhibit an organization consistent with the Arp2/3-based convergent elongation mechanism. Nerve growth factor (NGF) promotes formation of axonal filopodia and branches through phosphoinositide 3-kinase (PI3K). Inhibition of the Arp2/3 complex impairs NGF/PI3K-induced formation of axonal actin patches, filopodia, and the formation of collateral branches. Collectively, these data reveal that the Arp2/3 complex contributes to the formation of axon collateral branches through its involvement in the formation of actin patches leading to the emergence of axonal filopodia.     

From solution to surface to filament: actin flux into branched networks

The actin cytoskeleton comprises a set of filament networks that perform essential functions in eukaryotic cells. The idea that actin filaments incorporate monomers directly from solution forms both the “textbook picture” of filament elongation and a conventional starting point for quantitative modeling of cellular actin dynamics. Recent work, however, reveals that filaments created by two major regulators, the formins and the Arp2/3 complex, incorporate monomers delivered by nearby proteins. Specifically, actin enters Arp2/3-generated networks via binding sites on nucleation-promoting factors clustered on membrane surfaces. Here, we describe three functions of this surface-associated actin monomer pool: (1) regulating network density via product inhibition of the Arp2/3 complex, (2) accelerating filament elongation as a distributive polymerase, and (3) converting profilin-actin into a substrate for the Arp2/3 complex. These linked functions control the architecture of branched networks and explain how capping protein enhances their growth.     

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.     

Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility

Diverse intracellular pathogens subvert the host actin-polymerization machinery to drive movement within and between cells during infection. Rickettsia in the spotted fever group (SFG) are Gram-negative, obligate intracellular bacterial pathogens that undergo actin-based motility and assemble distinctive 'comet tails' that consist of long, unbranched actin filaments. Despite this distinct organization, it was proposed that actin in Rickettsia comet tails is nucleated by the host Arp2/3 complex and the bacterial protein RickA, which assemble branched actin networks. However, a second bacterial gene, sca2, was recently implicated in actin-tail formation by R. rickettsii. Here, we demonstrate that Sca2 is a bacterial actin-assembly factor that functionally mimics eukaryotic formin proteins. Sca2 nucleates unbranched actin filaments, processively associates with growing barbed ends, requires profilin for efficient elongation, and inhibits the activity of capping protein, all properties shared with formins. Sca2 localizes to the Rickettsia surface and is sufficient to promote the assembly of actin filaments in cytoplasmic extract. These results suggest that Sca2 mimics formins to determine the unique organization of actin filaments in Rickettsia tails and drive bacterial motility, independently of host nucleators.     

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.