Models for actin polymerization motors

Actin polymerization drives cell membrane protrusions and the propulsion of intracellular pathogens. The molecular mechanisms driving actin polymerization are not yet fully understood. Various mathematical models have been proposed to explain how cells convert chemical energy released upon actin polymerization into a pushing force on a surface. These models have attempted to explain puzzling properties of actin-based motility, including persistent attachment of the network to the membrane during propulsion and the interesting trajectories of propelled particles. These models fall generally into two classes: those requiring filament (+)-ends to fluctuate freely from the membrane to add subunits, and those where filaments elongate with their (+)-ends persistently associated with surface through filament end-tracking proteins ("actoclampin" models). This review compares and contrasts the key predictions of these two classes of models with regard to force-velocity profiles, and evaluates them with respect to experiments with biomimetic particles, and the experimental evidence on the role of end-tracking proteins such as formins and nucleation-promoting factors in actin-based motility.     

Structure of MFP2 and its function in enhancing MSP polymerization in Ascaris sperm amoeboid motility

The simplicity and specialization of the cell motility machinery of Ascaris sperm provides a powerful system in which to probe the basic molecular mechanism of amoeboid cell motility. Although Ascaris sperm locomotion closely resembles that seen in many other types of crawling cell, movement is generated by modulation of a cytoskeleton based on the major sperm protein (MSP) rather than the actin present in other cell types. The Ascaris motility machinery can be studied conveniently in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibres constructed from bundles of MSP filaments. In addition to ATP, MSP and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins to orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. One of these proteins, MFP2, accelerates the rate of movement in this assay. Here, we describe crystal structures of two isoforms of MFP2 and show that both are constructed from two domains that have the same fold based on a novel, compact beta sheet arrangement. Patterns of conservation observed in a structure-based analysis of MFP2 sequences from different nematode species identified regions that may be putative functional interfaces involved both in interactions between MFP2 domains and also with other components of the sperm motility machinery. Analysis of the growth of fibres in vitro in the presence of added MFP2 indicated that MFP2 increases the rate of locomotion by enhancing the effective rate of MSP filament polymerization. This observation, together with the structural data, suggests that MFP2 may function in a manner analogous to formins in actin-based motility.     

Diffusion rate limitations in actin-based propulsion of hard and deformable particles

The mechanism by which actin polymerization propels intracellular vesicles and invasive microorganisms remains an open question. Several recent quantitative studies have examined propulsion of biomimetic particles such as polystyrene microspheres, phospholipid vesicles, and oil droplets. In addition to allowing quantitative measurement of parameters such as the dependence of particle speed on its size, these systems have also revealed characteristic behaviors such a saltatory motion of hard particles and oscillatory deformation of soft particles. Such measurements and observations provide tests for proposed mechanisms of actin-based motility. In the actoclampin filament end-tracking motor model, particle-surface-bound filament end-tracking proteins are involved in load-insensitive processive insertion of actin subunits onto elongating filament plus-ends that are persistently tethered to the surface. In contrast, the tethered-ratchet model assumes working filaments are untethered and the free-ended filaments grow as thermal ratchets in a load-sensitive manner. This article presents a model for the diffusion and consumption of actin monomers during actin-based particle propulsion to predict the monomer concentration field around motile particles. The results suggest that the various behaviors of biomimetic particles, including dynamic saltatory motion of hard particles and oscillatory vesicle deformations, can be quantitatively and self-consistently explained by load-insensitive, diffusion-limited elongation of (+)-end-tethered actin filaments, consistent with predictions of the actoclampin filament-end tracking mechanism.     

Actin binding proteins that change extent and rate of actin monomer-polymer distribution by different mechanisms

Actin binding proteins control actin assembly and disassembly by altering the critical concentration and by changing the kinetics of polymerization. All of these control mechanisms in some way or the other make use of the energy of hydrolysis of actin-bound ATP. Capping of barbed filament ends increases the critical concentration as long as ATP hydrolysis maintains a difference in the actin monomer binding constants of the two ends. A further increase in the critical concentration on adding a second cap, tropomodulin, to the other, pointed filament end also requires ATP hydrolysis as described by the model presented here. Changes in the critical concentration are amplified into much larger changes of the monomer pool by actin sequestering proteins, provided their actin binding equilibrium constants fall within a relatively narrow range around the values for the two critical concentrations of actin. Cofilin greatly speeds up treadmilling, which requires ATP hydroysis, by increasing the rate constant of depolymerization. Profilin increases the rate of elongation at the barbed filament end, coupled to a lowering of the critical concentration, only if ATP hydrolysis makes profilin binding to the barbed end independent of its binding constant for actin monomers.     

Nonlinear increase of elongation rate of actin filaments with actin monomer concentration

The nonlinear increase of the elongation rate of actin filaments above the critical monomer concentration was investigated by nucleated polymerization of actin. Significant deviations from linearity were observed when actin was polymerized in the presence of magnesium ions. When magnesium ions were replaced by potassium or calcium ions, no deviations from linearity could be detected. The nonlinearity was analyzed by two simple assembly mechanisms. In the first model, if the ATP hydrolysis by polymeric actin is approximately as fast as the incorporation of monomers into filaments, terminal subunits of lengthening filaments are expected to carry to some extent ADP. As ADP-containing subunits dissociate from the ends of actin filaments faster than ATP-containing subunits, the rate of elongation of actin filaments would be nonlinearly correlated with the monomer concentration. In the second model (conformational change model), actin monomers and filament subunits were assumed to occur in two conformations. The association and dissociation rates of actin molecules in the two conformations were thought to be different. The equilibrium distribution between the two conformations was assumed to be different for monomers and filament subunits. The ATP hydrolysis was thought to lag behind polymerization and conformational change. As under the experimental conditions the rate of ATP hydrolysis by polymeric actin was independent of the concentration of filament ends, the observed nonlinear increase of the rate of elongation with the monomer concentration above the critical monomer concentration was unlikely to be caused by ATP hydrolysis at the terminal subunits. The conformational change model turned out to be the simplest assembly mechanism by which all available experimental data could be explained.     

Actin Depolymerizing Factor (ADF/Cofilin) Enhances the Rate of Filament Turnover: Implication in Actin-based Motility

Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes. ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family. The function of ADF in actin dynamics has been examined using a combination of physical–chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8. ADF binds the ADPbound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi– bound forms. A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia. ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails. These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament. The kinetic effects of ADF are end specific and cannot be accounted for by filament severing. The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged. This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes. In conclusion, the function of ADF is not to sequester G-actin. ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.     

Force generation by cytoskeletal filament end-tracking proteins

Force generation in several types of cell motility is driven by rapidly elongating cytoskeletal filaments that are persistently tethered at their polymerizing ends to propelled objects. These properties are not easily explained by force-generation models that require free (i.e., untethered) filament ends to fluctuate away from the surface for addition of new monomers. In contrast, filament end-tracking proteins that processively advance on filament ends can facilitate rapid elongation and substantial force generation by persistently tethered filaments. Such processive end-tracking proteins, termed here filament end-tracking motors, maintain possession of filament ends and, like other biomolecular motors, advance by means of 5'-nucleoside triphosphate (NTP) hydrolysis-driven affinity-modulated interactions. On-filament NTP hydrolysis/phosphate release yields substantially more energy than that required for driving steady-state assembly/disassembly of free filament ends (i.e., filament treadmilling), as revealed by an energy inventory on the treadmilling cycle. The kinetic and thermodynamic properties of two simple end-tracking mechanisms (an end-tracking stepping motor and a direct-transfer end-tracking motor) are analyzed to illustrate the advantages of an end-tracking motor over free filament-end elongation, and over passive end-trackers that operate without the benefit of NTP hydrolysis, in terms of generating force, facilitating rapid monomer addition, and maintaining tight possession of the filament ends. We describe an additional cofactor-assisted end-tracking motor to account for suggested roles of cofactors in the affinity-modulated interactions, such as profilin in actin-filament end-tracking motors and EB1 in microtubule end-tracking motors.     

Preparing to move: assembly of the MSP amoeboid motility apparatus during spermiogenesis in Ascaris

We exploited the rapid, inducible conversion of non-motile Ascaris spermatids into crawling spermatozoa to examine the pattern of assembly of the MSP motility apparatus that powers sperm locomotion. In live sperm, the first detectable motile activity is the extension of spikes and, later, blebs from the cell surface. However, examination of cells by EM revealed that the formation of surface protrusions is preceded by assembly of MSP filament tails on the membranous organelles in the peripheral cytoplasm. These organelle-associated filament meshworks assemble within 30 sec after induction of spermiogenesis and persist until the membranous organelles are sequestered into the cell body when the lamellipod extends. The filopodia-like spikes, which are packed with bundles of filaments, extend and retract rapidly but last only a few seconds before giving way to, or converting into, blebs. Coalescence of these blebs, each supported by a dense mesh of filaments, often initiates lamellipod extension, which culminates in the formation of the robust, dynamic MSP fiber complexes that generate sperm motility. The same membrane phosphoprotein that orchestrates assembly of the fiber complexes at the leading edge of the lamellipod of mature sperm is also found at all sites of filament assembly during spermiogenesis. The orderly progression of steps that leads to construction of a functional motility apparatus illustrates the precise spatio-temporal control of MSP filament assembly in the developing cell and highlights the remarkable similarity in organization and plasticity shared by the MSP cytoskeleton and the actin filament arrays in conventional crawling cells.     

Hydrostatic Pressure Shows That Lamellipodial Motility in Ascaris Sperm Requires Membrane-associated Major Sperm Protein Filament Nucleation and Elongation

Sperm from nematodes use a major sperm protein (MSP) cytoskeleton in place of an actin cytoskeleton to drive their ameboid locomotion. Motility is coupled to the assembly of MSP fibers near the leading edge of the pseudopod plasma membrane. This unique motility system has been reconstituted in vitro in cell-free extracts of sperm from Ascaris suum: inside-out vesicles derived from the plasma membrane trigger assembly of meshworks of MSP filaments, called fibers, that push the vesicle forward as they grow (Italiano, J.E., Jr., T.M. Roberts, M. Stewart, and C.A. Fontana. 1996. Cell. 84:105–114). We used changes in hydrostatic pressure within a microscope optical chamber to investigate the mechanism of assembly of the motile apparatus. The effects of pressure on the MSP cytoskeleton in vivo and in vitro were similar: pressures >50 atm slowed and >300 atm stopped fiber growth. We focused on the in vitro system to show that filament assembly occurs in the immediate vicinity of the vesicle. At 300 atm, fibers were stable, but vesicles often detached from the ends of fibers. When the pressure was dropped, normal fiber growth occurred from detached vesicles but the ends of fibers without vesicles did not grow. Below 300 atm, pressure modulates both the number of filaments assembled at the vesicle (proportional to fiber optical density and filament nucleation rate), and their rate of assembly (proportional to the rates of fiber growth and filament elongation). Thus, fiber growth is not simply because of the addition of subunits onto the ends of existing filaments, but rather is regulated by pressure-sensitive factors at or near the vesicle surface. Once a filament is incorporated into a fiber, its rates of addition and loss of subunits are very slow and disassembly occurs by pathways distinct from assembly. The effects of pressure on fiber assembly are sensitive to dilution of the extract but largely independent of MSP concentration, indicating that a cytosolic component other than MSP is required for vesicle-association filament nucleation and elongation. Based on these data we present a model for the mechanism of locomotion-associated MSP polymerization the principles of which may apply generally to the way cells assemble filaments locally to drive protrusion of the leading edge.     

Dissection of the Ascaris sperm motility machinery identifies key proteins involved in major sperm protein-based amoeboid locomotion

Although Ascaris sperm motility closely resembles that seen in many other types of crawling cells, the lamellipodial dynamics that drive movement result from modulation of a cytoskeleton based on the major sperm protein (MSP) rather than actin. The dynamics of the Ascaris sperm cytoskeleton can be studied in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibers constructed from bundles of MSP filaments. In addition to ATP, MSP, and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins that orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. Here, we identify a fraction of cytosol that is comprised of a small number of proteins but contains all of the soluble components required to assemble fibers. We have purified two of these proteins, designated MSP fiber proteins (MFPs) 1 and 2 and demonstrated by immunolabeling that both are located in the MSP cytoskeleton in cells and in fibers. These proteins had reciprocal effects on fiber assembly in vitro: MFP1 decreased the rate of fiber growth, whereas MFP2 increased the growth rate.     

Biomimetic systems for studying actin-based motility

Actin polymerization provides a major driving force for eukaryotic cell motility. Successive intercalation of monomeric actin subunits between the plasma membrane and the filamentous actin network results in protrusions of the membrane enabling the cell to move or to change shape. One of the challenges in understanding eukaryotic cell motility is to dissect the elementary biochemical and biophysical steps that link actin polymerization to mechanical force generation. Recently, significant progress was made using biomimetic, in vitro systems that are inspired by the actin-based motility of bacterial pathogens such as Listeria monocytogenes. Polystyrene microspheres and synthetic phospholipid vesicles coated with proteins that initiate actin polymerization display motile behavior similar to Listeria, mimicking the leading edge of lamellipodia and filopodia. A major advantage of these biomimetic systems is that both biochemical and physical parameters can be controlled precisely. These systems provide a test bed for validating theoretical models on force generation and polarity establishment resulting from actin polymerization. In this review, we discuss recent experimental progress using biomimetic systems propelled by actin polymerization and discuss these results in the light of recent theoretical models on actin-based motility.     

Formin is a processive motor that requires profilin to accelerate actin assembly and associated ATP hydrolysis

Motile and morphogenetic cellular processes are driven by site-directed assembly of actin filaments. Formins, proteins characterized by formin homology domains FH1 and FH2, are initiators of actin assembly. How formins simply bind to filament barbed ends in rapid equilibrium or find free energy to become a processive motor of filament assembly remains enigmatic. Here we demonstrate that the FH1-FH2 domain accelerates hydrolysis of ATP coupled to profilin-actin polymerization and uses the derived free energy for processive polymerization, increasing 15-fold the rate constant for profilin-actin association to barbed ends. Profilin is required for and takes part in the processive function. Single filaments grow at least 10 microm long from formin bound beads without detaching. Transitory formin-associated processes are generated by poisoning of the processive cycle by barbed-end capping proteins. We successfully reconstitute formin-induced motility in vitro, demonstrating that this mechanism accounts for the puzzlingly rapid formin-induced actin processes observed in vivo.     

Clamped-filament elongation model for actin-based motors.

Although actin-based motility drives cell crawling and intracellular locomotion of organelles and certain pathogens, the underlying mechanism of force generation remains a mystery. Recent experiments demonstrated that Listeria exhibit episodes of 5.4-nm stepwise motion corresponding to the periodicity of the actin filament subunits, and extremely small positional fluctuations during the intermittent pauses [S. C. Kuo and J. L. McGrath. 2000. Nature. 407:1026-1029]. These findings suggest that motile bacteria remain firmly bound to actin filament ends as they elongate, a behavior that appears to rule out previous models for actin-based motility. We propose and analyze a new mechanochemical model (called the "Lock, Load & Fire" mechanism) for force generation by means of affinity-modulated, clamped-filament elongation. During the locking step, the filament's terminal ATP-containing subunit binds tightly to a clamp situated on the surface of a motile object; in the loading step, actin.ATP monomer(s) bind to the filament end, an event that triggers the firing step, wherein ATP hydrolysis on the clamped subunit attenuates the filament's affinity for the clamp. This last step initiates translocation of the new ATP-containing terminus to the clamp, whereupon another cycle begins anew. This model explains how surface-tethered filaments can grow while exerting flexural or tensile force on the motile surface. Moreover, stochastic simulations of the model reproduce the signature motions of Listeria. This elongation motor, which we term actoclampin, exploits actin's intrinsic ATPase activity to provide a simple, high-fidelity enzymatic reaction cycle for force production that does not require elongating filaments to dissociate from the motile surface. This mechanism may operate whenever actin polymerization is called upon to generate the forces that drive cell crawling or intracellular organelle motility.     

A direct-transfer polymerization model explains how the multiple profilin-binding sites in the actoclampin motor promote rapid actin-based motility

The high actin-based motility rates observed in nonmuscle cells require the per-second addition of 400-500 monomers to the barbed ends of growing actin filaments. The chief polymerization-competent species is profilin.actin.ATP (present at 5-40 microM intracellular concentrations), whereas G-actin.ATP is much less abundant ( approximately 0.1-1 microM). While earlier studies unambiguously demonstrated that profilin.actin is highly concentrated within the polymerization zone, profilin-actin localization on the motile surface cannot increase the local solution-phase concentration of polymerizable actin. To explain these high rates of actin polymerization, we present and analyze a novel polymerization model in which monomers are directly transferred to growing filament ends in the actoclampin motor. This direct-transfer polymerization mechanism endows the polymerization zone with properties unavailable to bulk-phase actin monomers, and our model also indicates why profilin is the ideal mobile carrier for actin monomers.     

细胞运动、细胞迁移与细胞骨架研究进展

细胞定向运动与细胞骨架的动态循环密切相关。运动细胞在其伪足前沿依靠细胞骨架的不断聚合推动细胞膜的前进,在基部靠近细胞体部位通过细胞骨架的不断解聚收缩拖拉细胞体向前运动,细胞骨架的聚合与解聚通过伪足与支撑表面的吸附与解吸附而偶连。肌动蛋白组成的微丝骨架是大多数运动细胞的主要成分。外界刺激引起微丝细胞骨架动态变化的信号通路已逐步明了。线虫精子细胞的运动行为与阿米巴变形运动相似,但是在线虫精子细胞中没有肌动蛋白,而是以精子主要蛋白为基础形成细胞骨架驱动精子细胞的运动。与肌动蛋白不同,精子主要蛋白没有分子极性、ATP结合位点和马达蛋白。通过比较研究以上两种运动体系将有助于在分子水平上进一步阐明细胞运动的机理。     

Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles

Regulated actin filament assembly is critical for eukaryotic cell physiology. Actin filaments are polar structures, and those with free high affinity or barbed ends are crucial for actin dynamics and cell motility. Actin filament barbed-end-capping proteins inhibit filament elongation after binding, and their regulated disassociation is proposed to provide a source of free filament ends to drive processes dependent on actin polymerization. To examine whether dissociation of actin filament capping proteins occurs with the correct spatio-temporal constraints to contribute to regulated actin assembly in live cells, I measured the dissociation of an actin capping protein, gelsolin, from actin in cells using a variation of fluorescence resonance energy transfer (FRET). Uncapping was found to occur in cells at sites of active actin assembly, including protruding lamellae and rocketing vesicles, with the correct spatio-temporal properties to provide sites of actin filament polymerization during protrusion. These observations are consistent with models where uncapping of existing filaments provides sites of actin filament elongation.     

The beta-thymosin/WH2 domain; structural basis for the switch from inhibition to promotion of actin assembly

The widespread beta-thymosin/WH2 actin binding domain has versatile regulatory properties in actin dynamics and motility. beta-thymosins (isolated WH2 domain) maintain monomeric actin in a "sequestered" nonpolymerizable form. In contrast, when repeated in tandem or inserted in modular proteins, the beta-thymosin/WH2 domain promotes actin assembly at filament barbed ends, like profilin. The structural basis for these opposite functions is addressed using ciboulot, a three beta-thymosin repeat protein. Only the first repeat binds actin and possesses the function of ciboulot. The region that shows the strongest interaction with actin is an amphipathic N-terminal alpha helix, present in all beta-thymosin/WH2 domains, which recognizes the ATP bound actin structure and uses the shear motion of actin linked to ATP hydrolysis to control polymerization. Crystallographic ((1)H, (15)N), NMR, and mutagenetic data reveal that the weaker interaction of the C-terminal region of beta-thymosin/WH2 domain with actin accounts for the switch in function from inhibition to promotion of actin assembly.     

An integrative simulation model linking major biochemical reactions of actin-polymerization to structural properties of actin filaments

We report on an advanced universal Monte Carlo simulation model of actin polymerization processes offering a broad application panel. The model integrates major actin-related reactions, such as assembly of actin nuclei, association/dissociation of monomers to filament ends, ATP-hydrolysis via ADP-Pi formation and ADP-ATP exchange, filament branching, fragmentation and annealing or the effects of regulatory proteins. Importantly, these reactions are linked to information on the nucleotide state of actin subunits in filaments (ATP hydrolysis) and the distribution of actin filament lengths. The developed stochastic simulation modelling schemes were validated on: i) synthetic theoretical data generated by a deterministic model and ii) sets of our and published experimental data obtained from fluorescence pyrene-actin experiments. Build on an open-architecture principle, the designed model can be extended for predictive evaluation of the activities of other actin-interacting proteins and can be applied for the analysis of experimental pyrene actin-based or fluorescence microscopy data. We provide a user-friendly, free software package ActinSimChem that integrates the implemented simulation algorithms and that is made available to the scientific community for modelling in silico any specific actin-polymerization system.     

A Ser/Thr kinase required for membrane-associated assembly of the major sperm protein motility apparatus in the amoeboid sperm of Ascaris

Leading edge protrusion in the amoeboid sperm of Ascaris suum is driven by the localized assembly of the major sperm protein (MSP) cytoskeleton in the same way that actin assembly powers protrusion in other types of crawling cell. Reconstitution of this process in vitro led to the identification of two accessory proteins required for MSP polymerization: an integral membrane phosphoprotein, MSP polymerization-organizing protein (MPOP), and a cytosolic component, MSP fiber protein 2 (MFP2). Here, we identify and characterize a 34-kDa cytosolic protein, MSP polymerization-activating kinase (MPAK) that links the activities of MPOP and MFP2. Depletion/add-back assays of sperm extracts showed that MPAK, which is a member of the casein kinase 1 family of Ser/Thr protein kinases, is required for motility. MPOP and MPAK comigrated by native gel electrophoresis, coimmunoprecipitated, and colocalized by immunofluorescence, indicating that MPOP binds to and recruits MPAK to the membrane surface. MPAK, in turn, phosphorylated MFP2 on threonine residues, resulting in incorporation of MFP2 into the cytoskeleton. Beads coated with MPAK assembled a surrounding cloud of MSP filaments when incubated in MPAK-depleted sperm extract, but only when supplemented with detergent-solubilized MPOP. Our results suggest that interactions involving MPOP, MPAK, and MFP2 focus MSP polymerization to the plasma membrane at the leading edge of the cell thereby generating protrusion and minimizing nonproductive filament formation elsewhere.     

Signaling mechanisms that regulate actin-based motility processes in the nervous system

Actin-based motility is critical for nervous system development. Both the migration of neurons and the extension of neurites require organized actin polymerization to push the cell membrane forward. Numerous extracellular stimulants of motility and axon guidance cues regulate actin-based motility through the rho GTPases (rho, rac, and cdc42). The rho GTPases reorganize the actin cytoskeleton, leading to stress fiber, filopodium, or lamellipodium formation. The activity of the rho GTPases is regulated by a variety of proteins that either stimulate GTP uptake (activation) or hydrolysis (inactivation). These proteins potentially link extracellular signals to the activation state of rho GTPases. Effectors downstream of the rho GTPases that directly influence actin polymerization have been identified and are involved in neurite development. The Arp2/3 complex nucleates the formation of new actin branches that extend the membrane forward. Ena/VASP proteins can cause the formation of longer actin filaments, characteristic of growth cone actin morphology, by preventing the capping of barbed ends. Actin-depolymerizing factor (ADF)/cofilin depolymerizes and severs actin branches in older parts of the actin meshwork, freeing monomers to be re-incorporated into actively growing filaments. The signaling mechanisms by which extracellular cues that guide axons to their targets lead to direct effects on actin filament dynamics are becoming better understood.