Actin Filament Elongation Research Articles

Twinfilin is a non-processive depolymerase which synergizes with formin to dramatically accelerate actin filament uncapping by 300-fold.

Cellular actin networks assemble by actin filament elongation at barbed ends and are thought to disassemble primarily by depolymerization at filament pointed ends. Contrary to this conventional understanding of actin dynamics, twinfilin was recently shown to promote barbed-end depolymerization. Twinfilin has additionally been suggested to sequester monomers and cap as well as uncap filament barbed ends. As a result, the exact mechanisms by which twinfilin affects barbed-end dynamics remain controversial. Using multicolor single-molecule microscopy, we show that both mouse and yeast twinfilin are non-processive depolymerases that interact only transiently with barbed ends (~0.2-0.5 s). Each twinfilin binding event, on average, results in the removal of one or two actin subunits. At CP-capped barbed ends, twinfilin synergizes with formin to accelerate uncapping by up to ~320-fold. We find that uncapping by twinfilin, alone and together with formin, depends on the nucleotide state of the filament, with the two proteins causing a much more modest enhancement of uncapping of newly assembled filaments. Our study thus establishes twinfilin as a multifunctional barbed-end binding protein capable of non-processively depolymerizing, transiently capping, and synergizing with formin to rapidly uncap actin filament barbed ends.

Mechanisms of actin filament severing and elongation by formins.

Humans express 15 formins that play crucial roles in actin-based processes, including cytokinesis, cell motility and mechanotransduction1,2. However, the lack of structures bound to the actin filament (F-actin) has been a major impediment to understanding formin function. Whereas formins are known for their ability to nucleate and elongate F-actin3-7, some formins can additionally depolymerize, sever or bundle F-actin. Two mammalian formins, inverted formin 2 (INF2) and diaphanous 1 (DIA1, encoded by DIAPH1), exemplify this diversity. INF2 shows potent severing activity but elongates weakly8-11 whereas DIA1 has potent elongation activity but does not sever4,8. Using cryo-electron microscopy (cryo-EM)we show five structural states of INF2 and two of DIA1 bound to the middle and barbed end of F-actin. INF2 and DIA1 bind differently to these sites, consistent with their distinct activities. The formin-homology 2 and Wiskott-Aldrich syndrome protein-homology 2 (FH2 andWH2, respectively) domains of INF2 are positioned to sever F-actin, whereas DIA1 appears unsuited for severing. These structures also show how profilin-actin is delivered to the fast-growing barbed end, and how this is followed by a transition of the incoming monomer into the F-actin conformation and the release of profilin. Combined, the seven structures presented here provide step-by-step visualization of the mechanisms of F-actin severing and elongation by formins.

Molecular mechanism of actin filament elongation by formins.

Formins control the assembly of actin filaments (F-actin) that drive cell morphogenesis and motility in eukaryotes. However, their molecular interaction with F-actin and their mechanism of action remain unclear. In this work, we present high-resolution cryo-electron microscopy structures of F-actin barbed ends bound by three distinct formins, revealing a common asymmetric formin conformation imposed by the filament. Formation of new intersubunit contacts during actin polymerization sterically displaces formin and triggers its translocation. This "undock-and-lock" mechanism explains how actin-filament growth is coordinated with formin movement. Filament elongation speeds are controlled by the positioning and stability of actin-formin interfaces, which distinguish fast and slow formins. Furthermore, we provide a structure of the actin-formin-profilin ring complex, which resolves how profilin is rapidly released from the barbed end during filament elongation.

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins.

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser-Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin β4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.

The non-muscle actinopathy-associated mutation E334Q in cytoskeletal γ-actin perturbs interaction of actin filaments with myosin and ADF/cofilin family proteins

Various heterozygous cytoskeletal γ-actin mutations have been shown to cause Baraitser–Winter cerebrofrontofacial syndrome, non-syndromic hearing loss, or isolated eye coloboma. Here, we report the biochemical characterization of human cytoskeletal γ-actin carrying mutation E334Q, a mutation that leads to a hitherto unspecified non-muscle actinopathy. Following expression, purification, and removal of linker and thymosin β4 tag sequences, the p.E334Q monomers show normal integration into linear and branched actin filaments. The mutation does not affect thermal stability, actin filament nucleation, elongation, and turnover. Model building and normal mode analysis predict significant differences in the interaction of p.E334Q filaments with myosin motors and members of the ADF/cofilin family of actin-binding proteins. Assays probing the interactions of p.E334Q filaments with human class 2 and class 5 myosin motor constructs show significant reductions in sliding velocity and actin affinity. E334Q differentially affects cofilin-mediated actin dynamics by increasing the rate of cofilin-mediated de novo nucleation of actin filaments and decreasing the efficiency of cofilin-mediated filament severing. Thus, it is likely that p.E334Q-mediated changes in myosin motor activity, as well as filament turnover, contribute to the observed disease phenotype.

Multi-monoubiquitylation controls VASP-mediated actin dynamics.

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics.

Role of actin-binding proteins in prostate cancer.

The molecular mechanisms driving the onset and metastasis of prostate cancer remain poorly understood. Actin, under the control of actin-binding proteins (ABPs), plays a crucial role in shaping the cellular cytoskeleton, which in turn supports the morphological alterations in normal cells, as well as the invasive spread of tumor cells. Previous research indicates that ABPs of various types serve distinct functions, and any disruptions in their activities could predispose individuals to prostate cancer. These ABPs are intricately implicated in the initiation and advancement of prostate cancer through a complex array of intracellular processes, such as severing, linking, nucleating, inducing branching, assembling, facilitating actin filament elongation, terminating elongation, and promoting actin molecule aggregation. As such, this review synthesizes existing literature on several ABPs linked to prostate cancer, including cofilin, filamin A, and fascin, with the aim of shedding light on the molecular mechanisms through which ABPs influence prostate cancer development and identifying potential therapeutic targets. Ultimately, this comprehensive examination seeks to contribute to the understanding and management of prostate diseases.

Probing the ligand binding specificity of FNBP4 WW domains and interaction with FH1 domain of FMN1

Formins are a group of actin-binding proteins that mediate nascent actin filament polymerization, filament elongation, and barbed end-capping function, thereby regulating different cellular and developmental processes. Developmental processes like vertebrate gastrulation, neural growth cone dynamics, and limb development require formins functioning in a regulated manner. Formin-binding proteins like Rho GTPase regulate the activation of auto-inhibited conformation of diaphanous formins. Unlike other diaphanous formins, Formin1 (FMN1) a non-diaphanous formin is not regulated by Rho GTPase. FMN1 acts as an antagonist of the Bone Morphogenetic Protein (BMP) signaling pathway during limb development. Several previous reports demonstrated that WW domain-containing proteins can interact with poly-proline-rich amino acid stretches of formins and play a crucial role in developmental processes. In contrast, WW domain-containing Formin-binding Protein 4 (FNBP4) protein plays an essential role in limb development. It has been hypothesized that the interaction between FNBP4 and FMN1 can further attribute to the role in limb development through the BMP signaling pathway. In this study, we have elucidated the binding kinetics of FNBP4 and FMN1 using surface plasmon resonance (SPR) and enzyme-linked immunosorbent assays (ELISA). Our findings confirm that the FNBP4 exhibits interaction with the poly-proline-rich formin homology 1 (FH1) domain of FMN1. Furthermore, only the first WW1 domains are involved in the interaction between the two domains. Thus, this study sheds light on the binding potentialities of WW domains of FNBP4 that might contribute to the regulation of FMN1 function.

Engineered muscle from micro-channeled PEG scaffold with magnetic Fe3O4 fixation towards accelerating esophageal muscle repair

Engineered scaffolds are used for repairing damaged esophagus to allow the precise alignment and movement of smooth muscle for peristalsis. However, most of these scaffolds focus solely on inducing cell alignment through directional apparatus, often overlooking the promotion of muscle tissue formation and causing reduced esophageal muscle repair effectiveness. To address this issue, we first introduced aligned nano-ferroferric oxide (Fe3O4) assemblies on a micropatterned poly(ethylene glycol) (PEG) hydrogel to form micro-/nano-stripes. Further modification using a gold coating was found to enhance cellular adhesion, orientation and organization within these micro-/nano-stripes, which consequently prevented excessive adhesion of smooth muscle cells (SMCs) to the thin PEG ridges, thereby effectively confining the cells to the Fe3O4-laid channels. This architectural design promotes the alignment of the cytoskeleton and elongation of actin filaments, leading to the organized formation of muscle bundles and a tendency for SMCs to adopt synthetic phenotypes. Muscle patches are harvested from the micro-/nano-stripes and transplanted into a rat esophageal defect model. In vivo experiments demonstrate the exceptional viability of these muscle patches and their ability to accelerate the regeneration of esophageal tissue. Overall, this study presents an efficient strategy for constructing muscle patches with directional alignment and muscle bundle formation of SMCs, holding significant promise for muscle tissue regeneration.

Reconstitution of the transition from a lamellipodia- to filopodia-like actin network with purified proteins

How cells utilize complex mixtures of actin binding proteins to assemble and maintain functionally diverse actin filament networks with distinct architectures and dynamics within a common cytoplasm is a longstanding question in cell biology. A compelling example of complex and specialized actin structures in cells are filopodia which sense extracellular chemical and mechanical signals to help steer motile cells. Filopodia have distinct actin architecture, composed of long, parallel actin filaments bundled by fascin, which form finger-like membrane protrusions. Elongation of the parallel actin filaments in filopodia can be mediated by two processive actin filament elongation factors, formin and Ena/VASP, which localize to the tips of filopodia. There remains debate as to how the architecture of filopodia are generated, with one hypothesis proposing that filopodia are generated from the lamellipodia, which consists of densely packed, branched actin filaments nucleated by Arp2/3 complex and kept short by capping protein. It remains unclear if different actin filament elongation factors are necessary and sufficient to facilitate the emergence of filopodia with diverse characteristics from a highly dense network of short-branched capped filaments. To address this question, we combined bead motility and micropatterning biomimetic assays with multi-color Total Internal Reflection Fluorescence microscopy imaging, to successfully reconstitute the formation of filopodia-like networks (FLN) from densely-branched lamellipodia-like networks (LLN) with eight purified proteins (actin, profilin, Arp2/3 complex, Wasp pWA, fascin, capping protein, VASP and formin mDia2). Saturating capping protein concentrations inhibit FLN assembly, but the addition of either formin or Ena/VASP differentially rescues the formation of FLN from LLN. Specifically, we found that formin/mDia2-generated FLNs are relatively long and lack capping protein, whereas VASP-generated FLNs are comparatively short and contain capping protein, indicating that the actin elongation factor can affect the architecture and composition of FLN emerging from LLN. Our biomimetic reconstitution systems reveal that formin or VASP are necessary and sufficient to induce the transition from a LLN to a FLN, and establish robust in vitro platforms to investigate FLN assembly mechanisms.

Chiral growth of adherent filopodia

Adherent filopodia are elongated finger-like membrane protrusions, extending from the edges of diverse cell types and participating in cell adhesion, spreading, migration, and environmental sensing. The formation and elongation of filopodia are driven by the polymerization of parallel actin filaments, comprising the filopodia cytoskeletal core. Here, we report that adherent filopodia, formed during the spreading of cultured cells on galectin-8-coated substrates, tend to change the direction of their extension in a chiral fashion, acquiring a left-bent shape. Cryoelectron tomography examination indicated that turning of the filopodia tip to the left is accompanied by the displacement of the actin core bundle to the right of the filopodia midline. Reduction of the adhesion to galectin-8 by treatment with thiodigalactoside abolished this filopodia chirality. By modulating the expression of a variety of actin-associated filopodia proteins, we identified myosin-X and formin DAAM1 as major filopodia chirality promoting factors. Formin mDia1, actin filament elongation factor VASP, and actin filament cross-linker fascin were also shown to be involved. Thus, the simple actin cytoskeleton of filopodia, together with a small number of associated proteins are sufficient to drive a complex navigation process, manifested by the development of left-right asymmetry in these cellular protrusions.

Regulation of processive actin filament elongation mediated by formin.

The formin family of proteins directs the assembly of unbranched actin filaments that are incorporated into a diverse set of higher-order cytoskeletal structures, including cytokinetic rings, filopodia and stress fibers. Formins mediate polymerization by binding the barbed end with their dimeric formin homology 2 (FH2) domains, which processively step onto incoming actin monomers to incorporate them into the filament. By remaining bound to filaments over thousands of cycles of subunit addition, formins specify changes in filament lengths while protecting the barbed ends from capping. Formin isoforms have been shown to possess a range of processive properties, which are readily modulated by changes in ionic strength, the concentrations of actin and the actin-binding protein profilin, and the application of force. However, despite its critical importance to filament assembly, the relationship between a formin's polymerization activity and its processivity remains poorly understood. To address this question, we investigated the mechanism of processive elongation mediated by the budding yeast formin Bni1p. Using in vitro assays to reconstitute polymerization under a range of conditions, we found that processive association of Bni1p with filament ends is regulated by both the rate and the mechanism by which actin monomers are added to the barbed end. As a result, the rate at which Bni1p dissociates from barbed ends and the lengths of the filaments it polymerizes are regulated differently by the polymerization conditions. This suggests that formin-mediated elongation and processivity can be independently tuned to generate networks of actin filaments with defined lengths.

The proline-rich domain of fission yeast WASp (Wsp1p) interacts with actin filaments and inhibits actin polymerization.

Members of the Wiskott-Aldrich Syndrome protein (WASp) family activate Arp2/3 complex (actin-related proteins 2 and 3 complex) to form actin filament branches. The proline-rich domain (PRD) of WASp contributes to branching nucleation, and the PRD of budding yeast Las17 binds actin filaments [Urbanek AN etal. (2013) Curr Biol 23, 196-203]. Biochemical assays showed the recombinant PRD of fission yeast Schizosaccharomyces pombe Wsp1p binds actin filaments with micromolar affinity. Recombinant PRDs of both Wsp1p and Las17p slowed the elongation of actin filaments by Mg-ATP-actin monomers by half and slowed the spontaneous polymerization of Mg-ATP-actin monomers modestly. The affinity of PRDs of WASp-family proteins for actin filaments is high enough to contribute to the reported stimulation of actin filament branching by Arp2/3 complex.

A structural model of the profilin–formin pacemaker system for actin filament elongation

The formins constitute a large class of multi-domain polymerases that catalyze the localization and growth of unbranched actin filaments in cells from yeast to mammals. The conserved FH2 domains form dimers that bind actin at the barbed end of growing filaments and remain attached as new subunits are added. Profilin–actin is recruited and delivered to the barbed end by formin FH1 domains via the binding of profilin to interspersed tracts of poly-l-proline. We present a structural model showing that profilin–actin can bind the FH2 dimer at the barbed end stabilizing a state where profilin prevents its associated actin subunit from directly joining the barbed end. It is only with the dissociation of profilin from the polymerase that an actin subunit rotates and docks into its helical position, consistent with observations that under physiological conditions optimal elongation rates depend on the dissociation rate of profilin, independently of cellular concentrations of actin subunits.

Pointed-end processive elongation of actin filaments by Vibrio effectors VopF and VopL.

According to the cellular actin dynamics paradigm, filaments grow at their barbed ends and depolymerize predominantly from their pointed ends to form polar structures and do productive work. We show that actin can elongate at the pointed end when assisted by Vibrio VopF/L toxins, which act as processive polymerases. In cells, processively moving VopF/L speckles are inhibited by factors blocking the pointed but not barbed ends. Multispectral single-molecule imaging confirmed that VopF molecules associate with the pointed end, actively promoting its elongation even in the presence of profilin. Consequently, VopF/L can break the actin cytoskeleton's polarity by compromising actin-based cellular processes. Therefore, actin filament design allows processive growth at both ends, which suggests unforeseen possibilities for cellular actin organization, particularly in specialized cells and compartments.

Actin assembly requirements of the formin Fus1 to build the fusion focus.

In formin-family proteins, actin filament nucleation and elongation activities reside in the formin homology 1 (FH1) and FH2 domains, with reaction rates that vary by at least 20-fold between formins. Each cell expresses distinct formins that assemble one or several actin structures, raising the question of what confers each formin its specificity. Here, using the formin Fus1 in Schizosaccharomyces pombe, we systematically probed the importance of formin nucleation and elongation rates in vivo. Fus1 assembles the actin fusion focus, necessary for gamete fusion to form the zygote during sexual reproduction. By constructing chimeric formins with combinations of FH1 and FH2 domains previously characterized in vitro, we establish that changes in formin nucleation and elongation rates have direct consequences on fusion focus architecture, and that Fus1 native high nucleation and low elongation rates are optimal for fusion focus assembly. We further describe a point mutant in Fus1 FH2 that preserves native nucleation and elongation rates in vitro but alters function in vivo, indicating an additional FH2 domain property. Thus, rates of actin assembly are tailored for assembly of specific actin structures.

Rapid assembly of a polar network architecture drives efficient actomyosin contractility.

SUMMARYActin network architecture and dynamics play a central role in cell contractility and tissue morphogenesis. RhoA-driven pulsed contractions are a generic mode of actomyosin contractility, but the mechanisms underlying how their specific architecture emerges and how this architecture supports the contractile function of the network remain unclear. Here we show that, during pulsed contractions, the actin network is assembled by two subpopulations of formins: a functionally inactive population (recruited) and formins actively participating in actin filament elongation (elongating). We then show that elongating formins assemble a polar actin network, with barbed ends pointing out of the pulse. Numerical simulations demonstrate that this geometry favors rapid network contraction. Our results show that formins convert a local RhoA activity gradient into a polar network architecture, causing efficient network contractility, underlying the key function of kinetic controls in the assembly and mechanics of cortical network architectures.

Ena/VASP proteins in cell edge protrusion, migration and adhesion.

The tightly coordinated, spatiotemporal control of actin filament remodeling provides the basis of fundamental cellular processes, such as cell migration and adhesion. Specific protein assemblies, composed of various actin-binding proteins, are thought to operate in these processes to nucleate and elongate new filaments, arrange them into complex three-dimensional (3D) arrays and recycle them to replenish the actin monomer pool. Actin filament assembly is not only necessary to generate pushing forces against the leading edge membrane or to propel pathogens through the cytoplasm, but also coincides with the generation of stress fibers (SFs) and focal adhesions (FAs) that generate, transmit and sense mechanical tension. The only protein families known to date that directly enhance the elongation of actin filaments are formins and the family of Ena/VASP proteins. Their mechanisms of action, however, in enhancing processive filament elongation are distinct. The aim of this Review is to summarize our current knowledge on the molecular mechanisms of Ena/VASP-mediated actin filament assembly, and to discuss recent insights into the cell biological functions of Ena/VASP proteins in cell edge protrusion, migration and adhesion.

Carmil proteins regulate capping protein to control branched actin network architecture

The general function of the actin cytoskeleton is to shape, support, and move membranes. Actin regulatory proteins guide the self-organization of actin into functional structures like stereocilia, lamellipodia, filopodia, and ultimately cell motility. Among these regulatory proteins is Capping Protein. Capping Protein is a heterodimer that terminates individual actin filament elongation; yet it promotes branched network assembly via Arp2/3, competes with Nucleation Promoting Factors (NPF) to nucleate barbed ends, and is essential for the growth of polarized, force-generating actin networks.

VASP boosts protrusive activity of macroendocytic cups and drives phagosome rocketing after internalization

Ena/VASP proteins are powerful actin polymerases that drive the processive elongation of actin filaments. Members of this protein family have been implicated in a variety of important cellular processes including axon guidance, cell migration and adhesion. However, the specific function of these proteins in macroendocytosis, comprising macropinocytosis and phagocytosis remain rather poorly understood. Here, we used the professional phagocyte Dictyostelium discoideum to address the function and dynamics of its only family member VASP in macroendocytosis. Confocal time-lapse imaging revealed that VASP localized prominently in a circumferential narrow band at the advancing rim of the phagocytic cup followed by its aperture-like convergence upon particle internalization. Loss of VASP resulted in substantial defects in both, macropinocytosis of bulk fluid and phagocytosis of yeast particles. Consistently, VASP-deficiency coincided with diminished speed of the protruding rim and an impaired internalization rate. Most intriguingly, after cup closure, VASP condensed at the distal side of internalized phagosomes and initiated localized de-novo actin assembly to propel the phagosome by an actin-rich comet deeper into the cell, resembling intracellular movement of rocketing Listeria cells. In line with these findings, travelled distance and speed of rocketing phagosomes in VASP-deficient cells were markedly impaired.