Localization of PKA phosphorylation site, Ser(2030), in the three-dimensional structure of cardiac ryanodine receptor

Cofilin regulates actin filament dynamics by stimulating actin filament disassembly and plays a critical role in cytokinesis and chemotactic migration. Aip1 (actin-interacting protein 1), also called WDR1 (WD-repeat protein 1), is a highly conserved WD-repeat protein in eukaryotes and promotes cofilin-mediated actin filament disassembly in vitro; however, little is known about the mechanisms by which Aip1 functions in cytokinesis and cell migration in mammalian cells. In the present study, we investigated the roles of Aip1 in cytokinesis and chemotactic migration of human cells by silencing the expression of Aip1 using siRNA (small interfering RNA). Knockdown of Aip1 in HeLa cells increased the percentage of multinucleate cells; this effect was reversed by expression of an active form of cofilin. In Aip1-knockdown cells, the cleavage furrow ingressed normally from anaphase to early telophase; however, an excessive accumulation of actin filaments was observed on the contractile ring in late telophase. These results suggest that Aip1 plays a crucial role in the completion of cytokinesis by promoting cofilin-mediated actin filament disassembly in telophase. We have also shown that Aip1 knockdown significantly suppressed chemokine-induced chemotactic migration of Jurkat T-lymphoma cells, and this was blocked by expression of an active form of cofilin. Whereas control cells mostly formed a single lamellipodium in response to chemokine stimulation, Aip1 knockdown cells abnormally exhibited multiple protrusions around the cells before and after cell stimulation. This indicates that Aip1 plays an important role in directional cell migration by restricting the stimulus-induced membrane protrusion to one direction via promoting cofilin activity.     

Aip1p interacts with cofilin to disassemble actin filaments.

Actin interacting protein 1 (Aip1) is a conserved component of the actin cytoskeleton first identified in a two-hybrid screen against yeast actin. Here, we report that Aip1p also interacts with the ubiquitous actin depolymerizing factor cofilin. A two-hybrid-based approach using cofilin and actin mutants identified residues necessary for the interaction of actin, cofilin, and Aip1p in an apparent ternary complex. Deletion of the AIP1 gene is lethal in combination with cofilin mutants or act1-159, an actin mutation that slows the rate of actin filament disassembly in vivo. Aip1p localizes to cortical actin patches in yeast cells, and this localization is disrupted by specific actin and cofilin mutations. Further, Aip1p is required to restrict cofilin localization to cortical patches. Finally, biochemical analyses show that Aip1p causes net depolymerization of actin filaments only in the presence of cofilin and that cofilin enhances binding of Aip1p to actin filaments. We conclude that Aip1p is a cofilin-associated protein that enhances the filament disassembly activity of cofilin and restricts cofilin localization to cortical actin patches.     

Aip1 and cofilin promote rapid turnover of yeast actin patches and cables: a coordinated mechanism for severing and capping filaments

Rapid turnover of actin structures is required for dynamic remodeling of the cytoskeleton and cell morphogenesis, but the mechanisms driving actin disassembly are poorly defined. Cofilin plays a central role in promoting actin turnover by severing/depolymerizing filaments. Here, we analyze the in vivo function of a ubiquitous actin-interacting protein, Aip1, suggested to work with cofilin. We provide the first demonstration that Aip1 promotes actin turnover in living cells. Further, we reveal an unanticipated role for Aip1 and cofilin in promoting rapid turnover of yeast actin cables, dynamic structures that are decorated and stabilized by tropomyosin. Through systematic mutagenesis of Aip1 surfaces, we identify two well-separated F-actin-binding sites, one of which contributes to actin filament binding and disassembly specifically in the presence of cofilin. We also observe a close correlation between mutations disrupting capping of severed filaments in vitro and reducing rates of actin turnover in vivo. We propose a model for balanced regulation of actin cable turnover, in which Aip1 and cofilin function together to "prune" tropomyosin-decorated cables along their lengths. Consistent with this model, deletion of AIP1 rescues the temperature-sensitive growth and loss of actin cable defects of tpm1Delta mutants.     

Coordinated regulation of actin filament turnover by a high-molecular-weight Srv2/CAP complex, cofilin, profilin, and Aip1

BACKGROUND: Dynamic remodeling of the actin cytoskeleton requires rapid turnover of actin filaments, which is regulated in part by the actin filament severing/depolymerization factor cofilin/ADF. Two factors that cooperate with cofilin are Srv2/CAP and Aip1. Human CAP enhances cofilin-mediated actin turnover in vitro, but its biophysical properties have not been defined, and there has been no in vivo evidence reported for its role in turnover. Xenopus Aip1 forms a cofilin-dependent cap at filament barbed ends. It has been unclear how these diverse activities are coordinated in vivo. RESULTS: Purified native yeast Srv2/CAP forms a high molecular weight structure comprised solely of actin and Srv2. The complex is linked to actin filaments via the SH3 domain of Abp1. Srv2 complex catalytically accelerates cofilin-dependent actin turnover by releasing cofilin from ADP-actin monomers and enhances the ability of profilin to stimulate nucleotide exchange on ADP-actin. Yeast Aip1 forms a cofilin-dependent filament barbed end cap, disrupted by the cof1-19 mutant. Genetic analyses show that specific combinations of activities mediated by cofilin, Srv2, Aip1, and capping protein are required in vivo. CONCLUSIONS: We define two genetically and biochemically separable functions for cofilin in actin turnover. One is formation of an Aip1-cofilin cap at filament barbed ends. The other is cofilin-mediated severing/depolymerization of filaments, accelerated indirectly by Srv2 complex. We show that the Srv2 complex is a large multimeric structure and functions as an intermediate in actin monomer processing, converting cofilin bound ADP-actin monomers to profilin bound ATP-actin monomers and recycling cofilin for new rounds of filament depolymerization.     

Cofilin promotes stimulus-induced lamellipodium formation by generating an abundant supply of actin monomers

Cofilin stimulates actin filament disassembly and accelerates actin filament turnover. Cofilin is also involved in stimulus-induced actin filament assembly during lamellipodium formation. However, it is not clear whether this occurs by replenishing the actin monomer pool, through filament disassembly, or by creating free barbed ends, through its severing activity. Using photoactivatable Dronpa-actin, we show that cofilin is involved in producing more than half of all cytoplasmic actin monomers and that the rate of actin monomer incorporation into the tip of the lamellipodium is dependent on the size of this actin monomer pool. Finally, in cofilin-depleted cells, stimulus-induced actin monomer incorporation at the cell periphery is attenuated, but the incorporation of microinjected actin monomers is not. We propose that cofilin contributes to stimulus-induced actin filament assembly and lamellipodium extension by supplying an abundant pool of cytoplasmic actin monomers.     

Tropomyosin prevents depolymerization of actin filaments from the pointed end

Regulation of the pointed, or slow-growing, end of actin filaments is essential to the regulation of filament length. The purpose of this study is to investigate the role of skeletal muscle tropomyosin (TM) in regulating pointed end assembly and disassembly in vitro. The effects of TM upon assembly and disassembly of actin monomers from the pointed filament end were measured using pyrenyl-actin fluorescence assays in which the barbed ends were capped by villin. Tropomyosin did not affect pointed end elongation; however, filament disassembly from the pointed end stopped in the presence of TM under conditions where control filaments disassembled within minutes. The degree of protection against depolymerization was dependent upon free TM concentration and upon filament length. When filaments were diluted to a subcritical actin concentration in TM, up to 95% of the filamentous actin remained after 24 h and did not depolymerize further. Longer actin filaments (150 monomers average length) were more effectively protected from depolymerization than short filaments (50 monomers average length). Although filaments stopped depolymerizing in the presence of TM, they were not capped as shown by elongation assays. This study demonstrates that a protein, such as TM, which binds to the side of the actin filament can prevent dissociation of monomers from the end without capping the end to elongation. In skeletal muscle, tropomyosin could prevent thin filament disassembly from the pointed end and constitute a mechanism for regulating filament length.     

Synergies between Aip1p and capping protein subunits (Acp1p and Acp2p) in clathrin-mediated endocytosis and cell polarization in fission yeast

Aip1p cooperates with actin-depolymerizing factor (ADF)/cofilin to disassemble actin filaments in vitro and in vivo, and is proposed to cap actin filament barbed ends. We address the synergies between Aip1p and the capping protein heterodimer Acp1p/Acp2p during clathrin-mediated endocytosis in fission yeast. Using quantitative microscopy and new methods we have developed for data alignment and analysis, we show that heterodimeric capping protein can replace Aip1p, but Aip1p cannot replace capping protein in endocytic patches. Our quantitative analysis reveals that the actin meshwork is organized radially and is compacted by the cross-linker fimbrin before the endocytic vesicle is released from the plasma membrane. Capping protein and Aip1p help maintain the high density of actin filaments in meshwork by keeping actin filaments close enough for cross-linking. Our experiments also reveal new cellular functions for Acp1p and Acp2p independent of their capping activity. We identified two independent pathways that control polarization of endocytic sites, one depending on acp2⁺ and aip1⁺ during interphase and the other independent of acp1⁺, acp2⁺, and aip1⁺ during mitosis.     

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

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

Role of actin-filament disassembly in lamellipodium protrusion in motile cells revealed using the drug jasplakinolide.

BACKGROUND: In motile cells, protrusion of the lamellipodium (a type of cell margin) requires assembly of actin monomers into actin filaments at the tip of the lamellipodium. The importance of actin-filament disassembly in this process is less well understood, and is assessed here using the actin drug jasplakinolide, which has two known activities - inhibition of filament disassembly and induction of an increase in actin polymer. RESULTS: In cells the two activities of jasplakinolide were found to be separable; 1 microM jasplakinolide could permeate cells, bind cellular filamentous actin (F-actin) and inhibit filament disassembly within 3.5 minutes, but significant increase in actin polymer was not detected until 60 minutes of treatment. In live, permeabilised cells, jasplakinolide did not inhibit filament assembly from supplied, purified actin monomers. In migrating chick fibroblasts, lamellipodium protrusion was blocked within 1-5 minutes of treatment with 1 microM jasplakinolide, without any perturbation of actin organisation. In non-migrating chick fibroblasts, there was a delay in the onset of jasplakinolide-induced inhibition of lamellipodium protrusion, during which lamellipodium length increased linearly with no increase in protrusion rate. Motility of the bacterium Listeria in infected PtK2 cells was reduced 2.3-fold within 3 minutes of treatment with 1 microM jasplakinolide. CONCLUSIONS: Actin-filament disassembly is tightly coupled to lamellipodium protrusion in migrating chick fibroblasts and motility of Listeria in PtK2 cells. One simple interpretation of these data is a situation whereby ongoing actin-filament assembly uses free actin monomer derived from filament disassembly, in preference to stored monomer.     

A Highlights from MBoC Selection: Srv2/cyclase-associated protein forms hexameric shurikens that directly catalyze actin filament severing by cofilin

Actin filament severing is critical for the dynamic turnover of cellular actin networks. Cofilin severs filaments, but additional factors may be required to increase severing efficiency in vivo. Srv2/cyclase-associated protein (CAP) is a widely expressed protein with a role in binding and recycling actin monomers ascribed to domains in its C-terminus (C-Srv2). In this paper, we report a new biochemical and cellular function for Srv2/CAP in directly catalyzing cofilin-mediated severing of filaments. This function is mediated by its N-terminal half (N-Srv2), and is physically and genetically separable from C-Srv2 activities. Using dual-color total internal reflection fluorescence microscopy, we determined that N-Srv2 stimulates filament disassembly by increasing the frequency of cofilin-mediated severing without affecting cofilin binding to filaments. Structural analysis shows that N-Srv2 forms novel hexameric star-shaped structures, and disrupting oligomerization impairs N-Srv2 activities and in vivo function. Further, genetic analysis shows that the combined activities of N-Srv2 and Aip1 are essential in vivo. These observations define a novel mechanism by which the combined activities of cofilin and Srv2/CAP lead to enhanced filament severing and support an emerging view that actin disassembly is controlled not by cofilin alone, but by a more complex set of factors working in concert.     

Identification of Arabidopsis cyclase-associated protein 1 as the first nucleotide exchange factor for plant actin

The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP-actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP- and ATP-monomeric actin (Kd approximately 1.3 microM). Binding of AtCAP1 to ATP-actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of subunits through actin filament barbed ends. Collectively, these results and our understanding of other actin-binding proteins implicate CAP1 as a central player in regulating the pool of unpolymerized ATP-actin.     

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

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

Actin filament oxidation by MICAL1 suppresses protections from cofilin‐induced disassembly

Proteins of the ADF/cofilin family play a central role in the disassembly of actin filaments, and their activity must be tightly regulated in cells. Recently, the oxidation of actin filaments by the enzyme MICAL1 was found to amplify the severing action of cofilin through unclear mechanisms. Using single filament experiments in vitro, we found that actin filament oxidation by MICAL1 increases, by several orders of magnitude, both cofilin binding and severing rates, explaining the dramatic synergy between oxidation and cofilin for filament disassembly. Remarkably, we found that actin oxidation bypasses the need for cofilin activation by dephosphorylation. Indeed, non‐activated, phosphomimetic S3D‐cofilin binds and severs oxidized actin filaments rapidly, in conditions where non‐oxidized filaments are unaffected. Finally, tropomyosin Tpm1.8 loses its ability to protect filaments from cofilin severing activity when actin is oxidized by MICAL1. Together, our results show that MICAL1‐induced oxidation of actin filaments suppresses their physiological protection from the action of cofilin. We propose that, in cells, direct post‐translational modification of actin filaments by oxidation is a way to trigger their disassembly.     

Regulation of the Cortical Actin Cytoskeleton in Budding Yeast by Twinfilin,a Ubiquitous Actin Monomer-sequestering Protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)–twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

The Three Mouse Actin-depolymerizing Factor/Cofilins Evolved to Fulfill Cell-Type–specific Requirements for Actin Dynamics

Actin-depolymerizing factor (ADF)/cofilins are essential regulators of actin filament turnover. Several ADF/cofilin isoforms are found in multicellular organisms, but their biological differences have remained unclear. Herein, we show that three ADF/cofilins exist in mouse and most likely in all other mammalian species. Northern blot and in situ hybridization analyses demonstrate that cofilin-1 is expressed in most cell types of embryos and adult mice. Cofilin-2 is expressed in muscle cells and ADF is restricted to epithelia and endothelia. Although the three mouse ADF/cofilins do not show actin isoform specificity, they all depolymerize platelet actin filaments more efficiently than muscle actin. Furthermore, these ADF/cofilins are biochemically different. The epithelial-specific ADF is the most efficient in turning over actin filaments and promotes a stronger pH-dependent actin filament disassembly than the two other isoforms. The muscle-specific cofilin-2 has a weaker actin filament depolymerization activity and displays a 5-10-fold higher affinity for ATP-actin monomers than cofilin-1 and ADF. In steady-state assays, cofilin-2 also promotes filament assembly rather than disassembly. Taken together, these data suggest that the three biochemically distinct mammalian ADF/cofilin isoforms evolved to fulfill specific requirements for actin filament dynamics in different cell types.     

Direct demonstration of actin filament annealing in vitro

Direct electron microscopic examination confirms that short actin filaments rapidly anneal end-to-end in vitro, leading over time to an increase in filament length at steady state. During annealing of mixtures of native unlabeled filaments and glutaraldehyde-fixed filaments labeled with myosin subfragment-1, the structural polarity within heteropolymers is conserved absolutely. Annealing does not appear to require either ATP hydrolysis or the presence of exogenous actin monomers, suggesting that joining occurs through the direct association of filament ends. During recovery from sonication the initial rate of annealing is consistent with a second-order reaction involving the collision of two filament ends with an apparent annealing rate constant of 10(7) M-1s-1. This rapid phase lasts less than 10 s and is followed by a slow phase lasting minutes to hours. Annealing is calculated to contribute minimally to filament elongation during the initial stages of self-assembly. However, the rapid rate of annealing of sonicated fixed filaments observed in vitro suggests that it may be an efficient mechanism for repairing breaks in filaments and that annealing together with polymer-severing mechanisms may contribute significantly to the dynamics and function of actin filaments in vivo.     

Cyclase-associated Protein (CAP) Acts Directly on F-actin to Accelerate Cofilin-mediated Actin Severing across the Range of Physiological pH

Fast actin depolymerization is necessary for cells to rapidly reorganize actin filament networks. Utilizing a Listeria fluorescent actin comet tail assay to monitor actin disassembly rates, we observed that although a mixture of actin disassembly factors (cofilin, coronin, and actin-interacting protein 1 is sufficient to disassemble actin comet tails in the presence of physiological G-actin concentrations this mixture was insufficient to disassemble actin comet tails in the presence of physiological F-actin concentrations. Using biochemical complementation, we purified cyclase-associated protein (CAP) from thymus extracts as a factor that protects against the inhibition of excess F-actin. CAP has been shown to participate in actin dynamics but has been thought to act by liberating cofilin from ADP·G-actin monomers to restore cofilin activity. However, we found that CAP augments cofilin-mediated disassembly by accelerating the rate of cofilin-mediated severing. We also demonstrated that CAP acts directly on F-actin and severs actin filaments at acidic, but not neutral, pH. At the neutral pH characteristic of cytosol in most mammalian cells, we demonstrated that neither CAP nor cofilin are capable of severing actin filaments. However, the combination of CAP and cofilin rapidly severed actin at all pH values across the physiological range. Therefore, our results reveal a new function for CAP in accelerating cofilin-mediated actin filament severing and provide a mechanism through which cells can maintain high actin turnover rates without having to alkalinize cytosol, which would affect many biochemical reactions beyond actin depolymerization.     

WAVE binds Ena/VASP for enhanced Arp2/3 complex–based actin assembly

The WAVE complex is the main activator of the Arp2/3 complex for actin filament nucleation and assembly in the lamellipodia of moving cells. Other important players in lamellipodial protrusion are Ena/VASP proteins, which enhance actin filament elongation. Here we examine the molecular coordination between the nucleating activity of the Arp2/3 complex and the elongating activity of Ena/VASP proteins for the formation of actin networks. Using an in vitro bead motility assay, we show that WAVE directly binds VASP, resulting in an increase in Arp2/3 complex–based actin assembly. We show that this interaction is important in vivo as well, for the formation of lamellipodia during the ventral enclosure event of Caenorhabditis elegans embryogenesis. Ena/VASP''s ability to bind F-actin and profilin-complexed G-actin are important for its effect, whereas Ena/VASP tetramerization is not necessary. Our data are consistent with the idea that binding of Ena/VASP to WAVE potentiates Arp2/3 complex activity and lamellipodial actin assembly.     

Expression of Actin-interacting Protein 1 Suppresses Impaired Chemotaxis of Dictyostelium Cells Lacking the Na+-H+ Exchanger NHE1

Increased intracellular pH is an evolutionarily conserved signal necessary for directed cell migration. We reported previously that in Dictyostelium cells lacking H⁺ efflux by a Na⁺-H⁺ exchanger (NHE; Ddnhe1⁻), chemotaxis is impaired and the assembly of filamentous actin (F-actin) is attenuated. We now describe a modifier screen that reveals the C-terminal fragment of actin-interacting protein 1 (Aip1) enhances the chemotaxis defect of Ddnhe1⁻ cells but has no effect in wild-type Ax2 cells. However, expression of full-length Aip1 mostly suppresses chemotaxis defects of Ddnhe1⁻ cells and restores F-actin assembly. Aip1 functions to promote cofilin-dependent actin remodeling, and we found that although full-length Aip1 binds cofilin and F-actin, the C-terminal fragment binds cofilin but not F-actin. Because pH-dependent cofilin activity is attenuated in mammalian cells lacking H⁺ efflux by NHE1, our current data suggest that full-length Aip1 facilitates F-actin assembly when cofilin activity is limited. We predict the C-terminus of Aip1 enhances defective chemotaxis of Ddnhe1⁻ cells by sequestering the limited amount of active cofilin without promoting F-actin assembly. Our findings indicate a cooperative role of Aip1 and cofilin in pH-dependent cell migration, and they suggest defective chemotaxis in Ddnhe1⁻ cells is determined primarily by loss of cofilin-dependent actin dynamics.     

Quantitative analysis of actin turnover in Listeria comet tails: evidence for catastrophic filament turnover

Rapid assembly and disassembly (turnover) of actin filaments in cytoplasm drives cell motility and shape remodeling. While many biochemical processes that facilitate filament turnover are understood in isolation, it remains unclear how they work together to promote filament turnover in cells. Here, we studied cellular mechanisms of actin filament turnover by combining quantitative microscopy with mathematical modeling. Using live cell imaging, we found that actin polymer mass decay in Listeria comet tails is very well fit by a simple exponential. By analyzing candidate filament turnover pathways using stochastic modeling, we found that exponential polymer mass decay is consistent with either slow treadmilling, slow Arp2/3-dissociation, or catastrophic bursts of disassembly, but is inconsistent with acceleration of filament turnover by severing. Imaging of single filaments in Xenopus egg extract provided evidence that disassembly by bursting dominates isolated filament turnover in a cytoplasmic context. Taken together, our results point to a pathway where filaments grow transiently from barbed ends, rapidly terminate growth to enter a long-lived stable state, and then undergo a catastrophic burst of disassembly. By keeping filament lengths largely constant over time, such catastrophic filament turnover may enable cellular actin assemblies to maintain their mechanical integrity as they are turning over.