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.     

The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.     

Actin filament bundling by fimbrin is important for endocytosis, cytokinesis, and polarization in fission yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament "gatekeeper" and as a filament cross-linker.     

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.     

Roles of type II myosin and a tropomyosin isoform in retrograde actin flow in budding yeast

Retrograde flow of cortical actin networks and bundles is essential for cell motility and retrograde intracellular movement, and for the formation and maintenance of microvilli, stereocilia, and filopodia. Actin cables, which are F-actin bundles that serve as tracks for anterograde and retrograde cargo movement in budding yeast, undergo retrograde flow that is driven, in part, by actin polymerization and assembly. We find that the actin cable retrograde flow rate is reduced by deletion or delocalization of the type II myosin Myo1p, and by deletion or conditional mutation of the Myo1p motor domain. Deletion of the tropomyosin isoform Tpm2p, but not the Tpm1p isoform, increases the rate of actin cable retrograde flow. Pretreatment of F-actin with Tpm2p, but not Tpm1p, inhibits Myo1p binding to F-actin and Myo1p-dependent F-actin gliding. These data support novel, opposing roles of Myo1p and Tpm2 in regulating retrograde actin flow in budding yeast and an isoform-specific function of Tpm1p in promoting actin cable function in myosin-driven anterograde cargo transport.     

Actin cable distribution and dynamics arising from cross-linking,motor pulling,and filament turnover

The growth of fission yeast relies on the polymerization of actin filaments nucleated by formin For3p, which localizes at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. A big challenge is to develop a quantitative understanding of these cellular actin structures. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated individual actin filaments as semiflexible polymers in three dimensions composed of beads connected with springs. Polymerization out of For3p cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin are simulated as growth, cross-linking, pulling, and turnover of the semiflexible polymers. With the foregoing mechanisms, the model generates actin cable structures and dynamics similar to those observed in live-cell experiments. Our simulations reproduce the particular actin cable structures in myoVΔ cells and predict the effect of increased myosin V pulling. Increasing cross-linking parameters generates thicker actin cables. It also leads to antiparallel and parallel phases with straight or curved cables, consistent with observations of cells overexpressing α-actinin. Finally, the model predicts that clustering of formins at cell tips promotes actin cable formation.     

Tropomyosins Regulate the Severing Activity of Gelsolin in Isoform-Dependent and Independent Manners

The actin cytoskeleton fulfills numerous key cellular functions, which are tightly regulated in activity, localization, and temporal patterning by actin binding proteins. Tropomyosins and gelsolin are two such filament-regulating proteins. Here, we investigate how the effects of tropomyosins are coupled to the binding and activity of gelsolin. We show that the three investigated tropomyosin isoforms (Tpm1.1, Tpm1.12, and Tpm3.1) bind to gelsolin with micromolar or submicromolar affinities. Tropomyosin binding enhances the activity of gelsolin in actin polymerization and depolymerization assays. However, the effects of the three tropomyosin isoforms varied. The tropomyosin isoforms studied also differed in their ability to protect pre-existing actin filaments from severing by gelsolin. Based on the observed specificity of the interactions between tropomyosins, actin filaments, and gelsolin, we propose that tropomyosin isoforms specify which populations of actin filaments should be targeted by, or protected from, gelsolin-mediated depolymerization in living cells.     

Non-muscle tropomyosin (Tpm3) is crucial for asymmetric cell division and maintenance of cortical integrity in mouse oocytes

Tropomyosins are actin-binding cytoskeletal proteins that play a pivotal role in regulating the function of actin filaments in muscle and non-muscle cells; however, the roles of non-muscle tropomyosins in mouse oocytes are unknown. This study investigated the expression and functions of non-muscle tropomyosin (Tpm3) during meiotic maturation of mouse oocytes. Tpm3 mRNA was detected at all developmental stages in mouse oocytes. Tpm3 protein was localized at the cortex during the germinal vesicle and germinal vesicle breakdown stages. However, the overall fluorescence intensity of Tpm3 immunostaining was markedly decreased in metaphase II oocytes. Knockdown of Tpm3 impaired asymmetric division of oocytes and spindle migration, considerably reduced the amount of cortical actin, and caused membrane blebbing during cytokinesis. Expression of a constitutively active cofilin mutant and Tpm3 overexpression confirmed that Tpm3 protects cortical actin from depolymerization by cofilin. The data indicate that Tpm3 plays crucial roles in maintaining cortical actin integrity and asymmetric cell division during oocyte maturation, and that dynamic regulation of cortical actin by Tpm3 is critical to ensure proper polar body protrusion.     

Formins direct Arp2/3-independent actin filament assembly to polarize cell growth in yeast

Formins have been implicated in the regulation of cytoskeletal structure in animals and fungi. Here we show that the formins Bni1 and Bnr1 of budding yeast stimulate the assembly of actin filaments that function as precursors to tropomyosin-stabilized cables that direct polarized cell growth. With loss of formin function, cables disassemble,whereas increased formin activity causes the hyperaccumulation of cable-like filaments. Unlike the assembly of cortical actin patches, cable assembly requires profilin but not the Arp2/3 complex. Thus formins control a distinct pathway for assembling actin filaments that organize the overall polarity of the cell.     

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.     

Intracellular motility: myosin and tropomyosin in actin cable flow

A new study has found that retrograde flow of budding yeast actin cables is facilitated by myosin II but is inhibited by a specific tropomyosin isoform (Tpm2p). Budding yeast therefore contains a minimal component system for elucidating the mechanistic details of retrograde actin flow.     

Tropomyosin is essential for processive movement of a class v Myosin from budding yeast

Myosin V is an actin-based motor protein involved in intracellular cargo transport [1]. Given this physiological role, it was widely assumed that all class V myosins are processive, able to take multiple steps along actin filaments without dissociating. This notion was challenged when several class?V myosins were characterized as nonprocessive in?vitro, including Myo2p, the essential class V myosin from S.?cerevisiae [2-6]. Myo2p moves cargo including secretory vesicles and other organelles for several microns along actin cables in?vivo. This demonstrated cargo transporter must therefore either operate in small ensembles or?behave processively in the cellular context. Here we show?that Myo2p moves processively in?vitro as a single motor when it walks on an actin track that more closely resembles the actin cables found in?vivo. The key to processivity is tropomyosin: Myo2p is not processive on bare actin?but highly processive on actin-tropomyosin. The major yeast tropomyosin isoform, Tpm1p, supports the most robust processivity. Tropomyosin slows the rate of MgADP release, thus increasing the time the motor spends strongly attached to actin. This is the first example of tropomyosin switching a motor from nonprocessive to processive motion on actin.     

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.     

Functional effects of substitutions I92T and V95A in actin-binding period 3 of tropomyosin

Tropomyosin polymerizes along actin filaments and together with troponin regulates muscle contraction in a Ca-dependent manner. Actin-binding periods are homologous residues, which repeat along tropomyosin sequence, form tropomyosin-actin interface and determine regulatory functions. To learn how period 3 is involved in tropomyosin functions we examined effects of two mutations in Tpm1.1, I92T and V95A, which have been linked to dilated and hypertrophic cardiomyopathies characterized respectively by hyper- and hypocontractile phenotypes. In this work the functional consequences of both mutations were studied in vitro by using actin thin filaments reconstituted in the presence of mutant Tpm1.1 homodimers carrying the substitutions in both tropomyosin chains, Tpm1.1 heterodimers with substitution only in one Tpm1.1 chain, and Tpm1.1/Tpm2.2 heterodimers with substitution in Tpm1.1 chain and wild type Tpm2.2 in the second chain. The presence of the substitution I92T decreased the tropomyosin affinity for actin, abolished Ca²⁺-dependent activation of the actomyosin ATPase, decreased the sensitivity of the tropomyosin-troponin complex to subsaturating Ca²⁺ concentrations and reduced the cooperativity of the myosin-induced transition of the thin filament to a fully active state. The substitution V95A had opposite effects: increased actin affinity, increased the actomyosin ATPase activity above the level observed for wild type Tpm and increased cooperativity of myosin-induced activation of the thin filaments reconstructed with homo- and heterodimers of tropomyosin. Substitutions I92T and V95A were dominant, but the formation of heterodimers modified the effects observed for homodimers.     

Cofilin,but not profilin,is required for myosin-I-induced actin polymerization and the endocytic uptake in yeast

Mutations in the budding yeast myosins-I (MYO3 and MYO5) cause defects in the actin cytoskeleton and in the endocytic uptake. Robust evidence also indicates that these proteins induce Arp2/3-dependent actin polymerization. Consistently, we have recently demonstrated, using fluorescence microscopy, that Myo5p is able to induce cytosol-dependent actin polymerization on the surface of Sepharose beads. Strikingly, we now observed that, at short incubation times, Myo5p induced the formation of actin foci that resembled the yeast cortical actin patches, a plasma membrane-associated structure that might be involved in the endocytic uptake. Analysis of the machinery required for the formation of the Myo5p-induced actin patches in vitro demonstrated that the Arp2/3 complex was necessary but not sufficient in the assay. In addition, we found that cofilin was directly involved in the process. Strikingly though, the cofilin requirement seemed to be independent of its ability to disassemble actin filaments and profilin, a protein that closely cooperates with cofilin to maintain a rapid actin filament turnover, was not needed in the assay. In agreement with these observations, we found that like the Arp2/3 complex and the myosins-I, cofilin was essential for the endocytic uptake in vivo, whereas profilin was dispensable.     

Ultrastructure of the yeast actin cytoskeleton and its association with the plasma membrane

We characterized the yeast actin cytoskeleton at the ultrastructural level using immunoelectron microscopy. Anti-actin antibodies primarily labeled dense, patchlike cortical structures and cytoplasmic cables. This localization recapitulates results obtained with immunofluorescence light microscopy, but at much higher resolution. Immuno-EM double-labeling experiments were conducted with antibodies to actin together with antibodies to the actin binding proteins Abp1p and cofilin. As expected from immunofluorescence experiments, Abp1p, cofilin, and actin colocalized in immuno-EM to the dense patchlike structures but not to the cables. In this way, we can unambiguously identify the patches as the cortical actin cytoskeleton. The cortical actin patches were observed to be associated with the cell surface via an invagination of plasma membrane. This novel cortical cytoskeleton- plasma membrane interface appears to consist of a fingerlike invagination of plasma membrane around which actin filaments and actin binding proteins are organized. We propose a possible role for this unique cortical structure in wall growth and osmotic regulation.     

Dual roles of tropomyosin as an F-actin stabilizer and a regulator of muscle contraction in Caenorhabditis elegans body wall muscle

Tropomyosin is a well-characterized regulator of muscle contraction. It also stabilizes actin filaments in a variety of muscle and non-muscle cells. Although these two functions of tropomyosin could have different impacts on actin cytoskeletal organization, their functional relationship has not been studied in the same experimental system. Here, we investigated how tropomyosin stabilizes actin filaments and how this function is influenced by muscle contraction in Caenorhabditis elegans body wall muscle. We confirmed the antagonistic role of tropomyosin against UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament organization using multiple UNC-60B mutant alleles. Tropomyosin was also antagonistic to UNC-78 (AIP1) in vivo and protected actin filaments from disassembly by UNC-60B and UNC-78 in vitro, suggesting that tropomyosin protects actin filaments from the ADF/cofilin-AIP1 actin disassembly system in muscle cells. A mutation in the myosin heavy chain caused greater reduction in contractility than tropomyosin depletion. However, the myosin mutation showed much weaker suppression of the phenotypes of ADF/cofilin or AIP1 mutants than tropomyosin depletion. These results suggest that muscle contraction has only minor influence on the tropomyosin's protective role against ADF/cofilin and AIP1, and that the two functions of tropomyosin in actin stability and muscle contraction are independent of each other.     

A Highlights from MBoC Selection: Initial Polarized Bud Growth by Endocytic Recycling in the Absence of Actin Cable–dependent Vesicle Transport in Yeast

The assembly of filamentous actin is essential for polarized bud growth in budding yeast. Actin cables, which are assembled by the formins Bni1p and Bnr1p, are thought to be the only actin structures that are essential for budding. However, we found that formin or tropomyosin mutants, which lack actin cables, are still able to form a small bud. Additional mutations in components for cortical actin patches, which are assembled by the Arp2/3 complex to play a pivotal role in endocytic vesicle formation, inhibited this budding. Genes involved in endocytic recycling were also required for small-bud formation in actin cable-less mutants. These results suggest that budding yeast possesses a mechanism that promotes polarized growth by local recycling of endocytic vesicles. Interestingly, the type V myosin Myo2p, which was thought to use only actin cables to track, also contributed to budding in the absence of actin cables. These results suggest that some actin network may serve as the track for Myo2p-driven vesicle transport in the absence of actin cables or that Myo2p can function independent of actin filaments. Our results also show that polarity regulators including Cdc42p were still polarized in mutants defective in both actin cables and cortical actin patches, suggesting that the actin cytoskeleton does not play a major role in cortical assembly of polarity regulators in budding yeast.     

Cardiomyopathy Mutation Alters End-to-End Junction of Tropomyosin and Reduces Calcium Sensitivity

Muscle contraction is governed by tropomyosin (Tpm) shifting azimuthally between three states on F-actin (B-, C-, and M-states) in response to calcium binding to troponin and actomyosin cross-bridge formation. The Tpm coiled coil polymerizes head to tail along the long-pitch helix of F-actin to form continuous superhelical cables that wrap around the actin filaments. The end-to-end bonds formed between the N- and C-terminus of adjacent Tpm molecules define Tpm continuity and play a critical role in the ability of Tpm to cooperatively bind to actin, thus facilitating Tpm conformational switching to cooperatively propagate along F-actin. We expect that a missense mutation in this critical overlap region associated with dilated cardiomyopathy, A277V, will alter Tpm binding and thin filament activation by altering the overlap structure. Here, we used cosedimentation assays and in vitro motility assays to determine how the mutation alters Tpm binding to actin and its ability to regulate actomyosin interactions. Analytical viscometry coupled with molecular dynamics simulations showed that the A277V mutation results in enhanced Tpm end-to-end bond strength and a reduced curvature of the Tpm overlap domain. The mutant Tpm exhibited enhanced actin-Tpm binding affinity, consistent with overlap stabilization. The observed A277V-induced decrease in cooperative activation observed with regulated thin filament motility indicates that increased overlap stabilization is not correlated with Tpm-Tpm overlap binding strength or mechanical rigidity as is often assumed. Instead, A277V-induced structural changes result in local and delocalized increases in Tpm flexibility and prominent coiled-coil twisting in pseudorepeat 4. An A277V-induced decrease in Ca²⁺ sensitivity, consistent with a mutation-induced bolstering of the B-state Tpm-actin electrostatic contacts and an increased Tpm troponin T1 binding affinity, was also observed.