Cofilin from yeast and slime molds

An actin monomer-binding protein of apparent molecular weight of 15,000 has been identified and purified from Dictyostelium discoideum. The 15-kDa protein depolymerizes actin filaments in a pH-dependent manner. The protein also has an activity to decrease apparent viscosity of actin solutions in a dose-dependent manner. This activity is inhibited by phosphatidyl inositides. Molecular cloning of genes encoding this protein has revealed that the protein is 42% identical in its primary sequence to yeast cofilin. It is concluded that the 15-kDa protein of this organism is cofilin. D. discoideum cells contain two cofilin genes (DCOF1 and DCOF2) whose nucleotide sequences are entirely identical in their exons while the promoter and intron regions are different. Promoter assay experiments revealed that DCOF1 is expressed both in vegetative and differentiating cells and that DCOF2 is not expressed under any conditions examined. Gene disruption experiments have suggested that DCOF1 might be essential for the proliferation of D. discoideum cells whereas the disruption of DCOF2 did not to alter any phenotypes. Indirect immunofluorescence microscopic observations show that cofilin is distributed diffusely throughout cytoplasm in vegetative cells. In flattened cells under starvation stress, cofilin localizes at dramatically reorganizing actin-cytoskeletons in ruffling membranes of the leading edge, but not at rigid actin meshwork in focal adhesion plaques. These results suggest that cofilin may be involved in dynamic reorganization of membranous actin cytoskeletons (Aizawa, 1995).

A conventional cofilin, cofilin-1 in Dictyostelium discoideum plays significant roles in cell proliferation, phagocytosis, chemotactic movement and macropinocytosis. A new member of the cofilin family, named cofilin-2, has been described in D. discoideum. Cofilin-2 shows significant homology to a conventional Dictyostelium cofilin, cofilin-1, through its entire sequence, and contains residues conserved among the cofilin family that are responsible for actin-binding. In contrast, several residues that are conserved among the cofilin family are missing from cofilin-2. Purified cofilin-2 depolymerizes actin filaments in a dose- and pH-dependent manner and reduces the apparent viscosity of an actin solution, although cofilin-2 did not co-sediment with actin filaments at all. Cofilin-2 is not expressed in vegetative cells, but is transiently induced during the aggregation stage of development, whereas cofilin-1 is predominantly expressed in vegetative cells. Immunocytochemistry has revealed that cofilin-2 localizes at substrate adhesion sites, where cofilin-1 is almost completely excluded. Disruption of the cofilin-2 gene causes an increase in actin accumulation at the substrate adhesion sites. Cofilin-2 did not rescue Deltacof1 yeast cells, whereas cofilin-1 did. It is concluded that Cofilin-2 may play a distinct role from that of cofilin-1 in destabilization of the actin cytoskeleton during Dictyostelium development (Aizawa, 2001).

Characterization of cofilin as an actin depolymerizing factor

Actin depolymerizing factor (ADF) occurs naturally in two forms, one of which contains a phosphorylated Ser and does not bind G-actin or depolymerize F-actin. Removal of this phosphate in vitro by alkaline phosphatase restores full F-actin depolymerizing activity. To identify the phosphorylation site, [32P]pADF was purified and digested with endoproteinase Lys-C. The digest contained only one 32P-labeled peptide. Further digestion with endoproteinase Asp-N and mass spectrometric analysis showed that this peptide came from the N terminus of ADF. Alkaline phosphatase treatment of one Asp-N peptide (mass 753) converted this peptide to a mass 673, demonstrating that this peptide contains the phosphate group. Tandem mass spectrometric sequence analysis of this peptide identified the phosphorylated Ser as the encoded Ser3 (Ser2 in the processed protein). HeLa cells, transfected with either chick wild-type ADF cDNA or a cDNA mutated to code for Ala in place of Ser24 or Thr25, express and phosphorylate the exogenous ADF. Cells also express high levels of mutant ADF when Ser3 was deleted or converted to either Ala or Glu. However, none of these mutants was phosphorylated, confirming that Ser3 in the encoded ADF is the single in vivo regulatory site (Agnew, 1995).

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 ADP-bound 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 (Carlier, 1997).

Cofilin is an actin-binding protein of low molecular weight which is widely distributed in eukaryotes and is deeply involved in the dynamics of actin assembly in the cytoplasm. The actin-binding ability of cofilin is inhibited by inositol phosphates (PIP2), and the PIP2- and actin-binding site(s) has been localized in residues W104-M115 of the cofilin primary sequence. In the present study, in order to further clarify the functional domains in cofilin molecule, expression vectors were constructed containing cDNAs of different size with deletion at the 3'-region of the open reading frame. The truncated cofilin molecules produced in E. coli were purified and examined for their actin-binding and PIP2-binding ability. The truncated cofilin molecule without C-terminal residues #100-#166 including the previously-described actin-binding site, can be cross-linked with actin by EDC, a zero-length cross-linker. In addition, these truncated peptides as well as synthetic peptides corresponding to the N-terminal sequence of cofilin suppress the inhibitory action of PIP2 on actin-cofilin interaction. These results strongly suggest that additional actin- and PIP2-binding sites exist in the N-terminal region of cofilin (Kusano, 1999).

The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts has an extensively branched organization of actin filaments, which is termed the dendritic brush. Pointed ends of individual filaments are located at Y-junctions, where the Arp2/3 complex is also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments have suggested that the Arp2/3 complex also provides protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin is excluded from the distal 0.4 microM of the lamellipodial network of keratocytes and in fibroblasts it is located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Evidence supports a dendritic nucleation model for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex (Svitkina, 1999)

Cellular movements are powered by the assembly and disassembly of actin filaments. Actin dynamics are controlled by Arp2/3 complex, the Wiskott-Aldrich syndrome protein (WASp) and the related Scar protein, capping protein, profilin, and the actin-depolymerizing factor (ADF, also known as cofilin). Using an assay that both reveals the kinetics of overall reactions and allows visualization of actin filaments, it has been shown how these proteins co-operate in the assembly of branched actin filament networks. This study investigates how they work together to disassemble the networks. Actin filament branches formed by polymerization of ATP-actin in the presence of activated Arp2/3 complex were found to be metastable, dissociating from the mother filament with a half time of 500 seconds. The ADF/cofilin protein actophorin reduces the half time for both dissociation of gamma-phosphate from ADP-Pi-actin filaments and debranching to 30 seconds. Branches were stabilized by phalloidin, which inhibits phosphate dissociation from ADP-Pi-filaments, and by BeF3, which forms a stable complex with ADP and actin. Arp2/3 complex capped pointed ends of ATP-actin filaments with higher affinity (Kd approximately 40 nM) than those of ADP-actin filaments (Kd approximately 1 microM), explaining why phosphate dissociation from ADP-Pi-filaments liberates branches. Capping protein prevents annealing of short filaments after debranching and, with profilin, allows filaments to depolymerize at the pointed ends. It is concluded that the low affinity of Arp2/3 complex for the pointed ends of ADP-actin makes actin filament branches transient. By accelerating phosphate dissociation, ADF/cofilin promotes debranching. Barbed-end capping proteins and profilin allow dissociated branches to depolymerize from their free pointed ends (Blanchoin, 2000).

The actin depolymerizing factor (ADF)/cofilin family of proteins interact with actin monomers and filaments in a pH-sensitive manner. When ADF/cofilin binds F-actin it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of filaments, thereby increasing treadmilling or depolymerization. Using site-directed mutagenesis the two actin-binding sites on human cofilin were characterized. One target site was chosen because it has been shown that the villin head piece competes with ADF for binding to F-actin. Limited sequence homology between ADF/cofilin and the part of the villin headpiece essential for actin binding suggested an actin-binding site on cofilin involving a structural loop at the opposite end of the molecule to the alpha-helix already implicated in actin binding. Binding through the alpha-helix is primarily to monomeric actin, whereas the loop region is specifically involved in filament association. The actin binding properties of each site were characterized independently of one another. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Using the mutation analogous to the phosphorylated form of cofilin (S3D), it was shown that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces both the twist change and fragmentation characteristic of wild-type cofilin, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, followed by interaction with the adjacent subunit through the 'helix' site at the opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsible for the enhanced rate of actin subunit release from filaments (Pope, 2000).

LIM-kinase acts upstream of cofilin

Cell division, cell motility and the formation and maintenance of specialized structures in differentiated cells depend directly on the regulated dynamics of the actin cytoskeleton. To understand the mechanisms of these basic cellular processes, the signalling pathways that link external signals to the regulation of the actin cytoskeleton need to be characterized. A pathway has been identified for the regulation of cofilin, a ubiquitous actin-binding protein that is essential for effective depolymerization of actin filaments. LIM-kinase 1 (see Drosophila LIM-kinase1), also known as KIZ, is a protein kinase with two amino-terminal LIM motifs that induces stabilization of F-actin structures in transfected cells. Dominant-negative LIM-kinase 1 inhibits the accumulation of the F-actin. Phosphorylation experiments in vivo and in vitro provide evidence that cofilin is a physiological substrate of LIM-kinase 1. Phosphorylation by LIM-kinase 1 inactivates cofilin, leading to accumulation of actin filaments. Constitutively active Rac augments cofilin phosphorylation and LIM-kinase 1 autophosphorylation whereas phorbol ester inhibits these processes. These results define a mechanism for the regulation of cofilin and hence of actin dynamics in vivo. By modulating the stability of actin cytoskeletal structures, this pathway should play a central role in regulating cell motility and morphogenesis (Arber, 1998).

Rac is a small GTPase of the Rho family that mediates stimulus-induced actin cytoskeletal reorganization to generate lamellipodia. Little is known about the signalling pathways that link Rac activation to changes in actin filament dynamics. Cofilin is known to be a potent regulator of actin filament dynamics, and its ability to bind and depolymerize actin is abolished by phosphorylation of serine residue at position 3; however, the kinases responsible for this phosphorylation have not been identified. LIM-kinase 1 (LIMK-1), a serine/threonine kinase containing LIM and PDZ domains, phosphorylates cofilin at Ser 3, both in vitro and in vivo. When expressed in cultured cells, LIMK-1 induces actin reorganization and reverses cofilin-induced actin depolymerization. Expression of an inactive form of LIMK-1 suppresses lamellipodium formation induced by Rac or insulin. Furthermore, insulin and an active form of Rac increase the activity of LIMK-1. Taken together, these results indicate that LIMK-1 participates in Rac-mediated actin cytoskeletal reorganization, probably by phosphorylating cofilin (Yang, 1998).

Semaphorin 3A is a chemorepulsive axonal guidance molecule that depolymerizes the actin cytoskeleton and collapses growth cones of dorsal root ganglia neurons. The role of LIM-kinase 1, which phosphorylates an actin-depolymerizing protein, cofilin, in semaphorin 3A-induced growth cone collapse was investigated. Semaphorin 3A induces phosphorylation and dephosphorylation of cofilin at growth cones sequentially. A synthetic cell-permeable peptide containing a cofilin phosphorylation site inhibits LIM-kinase in vitro and in vivo, and essentially suppresses semaphorin 3A-induced growth cone collapse. A dominant-negative LIM kinase, which can not be activated by PAK or ROCK, suppressed the collapsing activity of semaphorin 3A. Phosphorylation of cofilin by LIM-kinase may be a critical signaling event in growth cone collapse by semaphorin 3A (Aizawa, 2001).

Stromal cell-derived factor 1 alpha (SDF-1alpha), the ligand for G-protein-coupled receptor CXCR4, is a chemotactic factor for T lymphocytes. LIM kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing and -severing protein, at Ser-3 and regulates actin reorganization. The role of cofilin phosphorylation by LIMK1 in SDF-1alpha-induced chemotaxis of T lymphocytes was investigated. SDF-1alpha significantly induces the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1alpha also induces cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibits SDF-1alpha-induced LIMK1 activation, thus indicating that Gi protein is involved in LIMK1 activation. Expression of dominant negative Rac (DN-Rac), but not DN-Rho or DN-Cdc42, blocks SDF-1alpha-induced activation of LIMK1, which means that SDF-1alpha-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. A cell-permeable peptide (S3 peptide) was used that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibits the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibits the SDF-1alpha-induced cofilin phosphorylation, actin reorganization, and chemotactic response of Jurkat cells. These results suggest that the phosphorylation of cofilin by LIMK1 plays a critical role in the SDF-1alpha-induced chemotactic response of T lymphocytes (Nishita, 2002)

In metastatic rat mammary adenocarcinoma cells, cell motility can be induced by epidermal growth factor. One of the early events in this process is the massive generation of actin barbed ends, which elongate to form filaments immediately adjacent to the plasma membrane at the tip of the leading edge. As a result, the membrane moves outward and forms a protrusion. To test the involvement of ADF/cofilin in the stimulus-induced barbed end generation at the leading edge, ADF/cofilin's activity was inhibited in vivo by increasing its phosphorylation level using the kinase domain of LIM-kinase 1 (GFP-K). Expression of GFP-K in rat cells results in the near total phosphorylation of ADF/cofilin, without changing either the G/F-actin ratio or signaling from the EGF receptor in vivo. Phosphorylation of ADF/cofilin is sufficient to completely inhibit the appearance of barbed ends and lamellipod protrusion, even in the continued presence of abundant G-actin. Coexpression of GFP-K, together with an active, nonphosphorylatable mutant of cofilin (S3A cofilin), rescues barbed end formation and lamellipod protrusion, indicating that the effects of kinase expression are caused by the phosphorylation of ADF/cofilin. These results indicate a direct role for ADF/cofilin in the generation of the barbed ends that are required for lamellipod extension in response to EGF stimulation (Zebda, 2000).

Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induces rapid actin polymerization into stress fibers and concomitantly activates RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants block TGF-beta1-induced actin reorganization. Since Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), it was found that TGF-beta1 induces LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitate and cofilin becomes phosphorylated in response to TGF-beta1, while RNA interference against LIMK2 blocks formation of new stress fibers by TGF-beta1. Since the kinase ROCK1 links Rho GTPases to LIMK2, it was found that inhibiting ROCK1 activity completely blocks TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. Whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization was tested. Adenoviruses expressing constitutively activated TGF-beta type I receptor leads to robust actin reorganization and Rho activation, while the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) does not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibits TGF-beta1-induced Rho activation and cytoskeletal reorganization. These data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2 (Vardouli, 2005).

Understanding the mechanisms controlling cancer cell invasion and metastasis constitutes a fundamental step in setting new strategies for diagnosis, prognosis, and therapy of metastatic cancers. LIM kinase1 (LIMK1) is a member of a novel class of serine-threonine protein kinases. Cofilin, a LIMK1 substrate, is essential for the regulation of actin polymerization and depolymerization during cell migration. Previous studies have reached opposite conclusions as to the role of LIMK1 in tumor cell motility and metastasis, claiming either an increase or decrease in cell motility and metastasis as a result of LIMK1 over expression. This paradox has been resolved by showing that the effects of LIMK1 expression on migration, intravasation, and metastasis of cancer cells can be most simply explained by its regulation of the output of the cofilin pathway. LIMK1-mediated decreases or increases in the activity of the cofilin pathway are shown to cause proportional decreases or increases in motility, intravasation, and metastasis of tumor cells (Wang, 2006).

Cofilin is reactivated by cofilin-phosphatase Slingshot

The ADF (actin-depolymerizing factor)/cofilin family is a stimulus-responsive mediator of actin dynamics. In contrast to the mechanisms of inactivation of ADF/cofilin by kinases such as LIM-kinase 1 (LIMK1), much less is known about its reactivation through dephosphorylation. Reported here is Slingshot (SSH), a family of phosphatases that have the property of F actin binding. In Drosophila, loss of ssh function dramatically increases levels of both F actin and phospho-cofilin (P cofilin) and disorganized epidermal cell morphogenesis. In mammalian cells, human SSH homologs (hSSHs) suppress LIMK1-induced actin reorganization. Furthermore, SSH and the hSSHs dephosphorylate P cofilin in cultured cells and in cell-free assays. These results strongly suggest that the SSH family plays a pivotal role in actin dynamics by reactivating ADF/cofilin in vivo (Niwa, 2002).

During cytokinesis the actomyosin-based contractile ring is formed at the equator, constricted, and then disassembled prior to cell abscission. Cofilin stimulates actin filament disassembly and is implicated in the regulation of contractile ring dynamics. However, little is known about the mechanism controlling cofilin activity during cytokinesis. Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase-1 (LIMK1) and is reactivated by a protein phosphatase Slingshot-1 (SSH1). The phosphatase activity of SSH1 decreases in the early stages of mitosis and is elevated in telophase and cytokinesis in HeLa cells, a time course correlating with that of cofilin dephosphorylation. SSH1 co-localizes with F-actin and accumulates onto the cleavage furrow and the midbody. Expression of a phosphatase-inactive SSH1 induces aberrant accumulation of F-actin and phospho-cofilin near the midbody in the final stage of cytokinesis and frequently leads to the regression of the cleavage furrow and the formation of multinucleate cells. Co-expression of cofilin rescues the inhibitory effect of phosphatase-inactive SSH1 on cytokinesis. These results suggest that SSH1 plays a critical role in cytokinesis by dephosphorylating and reactivating cofilin in later stages of mitosis (Kaji, 2003).

Cofilin mediates lamellipodium extension and polarized cell migration by stimulating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by phosphorylation at Ser-3 and reactivated by cofilin-phosphatase Slingshot-1L (SSH1L). Little is known of signaling mechanisms of cofilin activation and how this activation is spatially regulated. Cofilin-phosphatase activity of SSH1L has been shown to increase approximately 10-fold by association with actin filaments, which indicates that actin assembly at the leading edge per se triggers local activation of SSH1L and thereby stimulates cofilin-mediated actin turnover in lamellipodia. Evidence that 14-3-3 proteins inhibit SSH1L activity, dependent on the phosphorylation of Ser-937 and Ser-978 of SSH1L. Stimulation of cells with neuregulin-1beta induced Ser-978 dephosphorylation, translocation of SSH1L onto F-actin-rich lamellipodia, and cofilin dephosphorylation. These findings suggest that SSH1L is locally activated by translocation to and association with F-actin in lamellipodia in response to neuregulin-1beta and 14-3-3 proteins negatively regulate SSH1L activity by sequestering it in the cytoplasm (Nagata-Ohashi, 2004).

Cofilin plays an essential role in actin filament dynamics and membrane protrusion in motile cells. Cofilin is inactivated by phosphorylation at Ser-3 by LIM kinase and reactivated by dephosphorylation by cofilin-phosphatase Slingshot (SSH). Although cofilin is dephosphorylated in response to various extracellular stimuli, signaling pathways regulating SSH activation and cofilin dephosphorylation have remained to be elucidated. Insulin has been shown to stimulate the phosphatase activity of Slingshot-1L (SSH1L) and cofilin dephosphorylation in cultured cells, in a manner dependent on phosphoinositide 3-kinase (PI3K) activity. Consistent with this, the level of Ser-3-phosphorylated cofilin is increased in PTEN (phosphatase and tensin homolog deleted in chromosome 10)-overexpressing cells and decreased in PTEN-deficient cells. Insulin induces the accumulation of SSH1L and active Akt (a downstream effector of PI3K), together with a PI3K product phosphatidylinositol 3,4,5-trisphosphate, onto membrane protrusions. Cofilin, but not Ser-3-phosphorylated cofilin, accumulates in membrane protrusions in insulin-stimulated cells, indicating that cofilin is dephosphorylated in these areas. Finally, suppression of SSH1L expression by RNA interference abolished insulin-induced cofilin dephosphorylation and the membrane protrusion. These findings suggest that SSH1L is activated downstream of PI3K and plays a critical role in insulin-induced membrane protrusion by dephosphorylating and activating cofilin (Nishita, 2004).

Cofilin, an essential regulator of actin filament dynamics, is inactivated by phosphorylation at Ser-3 and reactivated by dephosphorylation. Although cofilin undergoes dephosphorylation in response to extracellular stimuli that elevate intracellular Ca2+ concentrations, signaling mechanisms mediating Ca2+-induced cofilin dephosphorylation have remained unknown. The role of Slingshot-1L (SSH1L), a member of a Slingshot (SSH) family of protein phosphatases, was examined in mediating Ca2+-induced cofilin dephosphorylation. The Ca2+ ionophore A23187 and Ca2+-mobilizing agonists, ATP and histamine, induce SSH1L activation and cofilin dephosphorylation in cultured cells. A23187- or histamine-induced SSH1L activation and cofilin dephosphorylation are blocked by calcineurin inhibitors or a dominant-negative form of calcineurin, indicating that calcineurin mediates Ca2+-induced SSH1L activation and cofilin dephosphorylation. Importantly, knockdown of SSH1L expression by RNA interference abolishes A23187- or calcineurin-induced cofilin dephosphorylation. Furthermore, calcineurin dephosphorylates SSH1L and increases the cofilin-phosphatase activity of SSH1L in cell-free assays. Based on these findings it is suggested that Ca2+-induced cofilin dephosphorylation is mediated by calcineurin-dependent activation of SSH1L (Wang, 2005).

Myelin-associated inhibitors (MAIs) signal through a tripartate receptor complex on neurons to limit axon regeneration in the CNS. Inhibitory influences ultimately converge on the cytoskeleton to mediate growth cone collapse and neurite outgrowth inhibition. Rho GTPase and its downstream effector Rho kinase are key signaling intermediates in response to MAIs; however, the links between Rho and the actin cytoskeleton have not been fully defined. Nogo-66, a potent inhibitory fragment of Nogo-A, signals through LIM kinase and Slingshot (SSH) phosphatase to regulate the phosphorylation profile of the actin depolymerization factor cofilin. Blockade of LIMK1 activation and subsequent cofilin phosphorylation circumvents myelin-dependent inhibition in chick dorsal root ganglion neurons, suggesting that phosphorylation and inactivation of cofilin is critical for neuronal inhibitory responses. Subsequent activation of SSH1 phosphatase mediates cofilin dephosphorylation and reactivation. Overexpression of SSH1 does not mimic the neurite outgrowth inhibitory effects of myelin, suggesting an alternative role in MAI inhibition. It is speculated that SSH-mediated persistent cofilin activation may be responsible for maintaining an inhibited neuronal phenotype in response to myelin inhibitors (Hsieh, 2006).

A multi-protein complex consisting of Slingshot, Lim kinases, actin, 14-3-3 and cofilin regulate ADF/cofilin activity

Slingshot (SSH) phosphatases and LIM kinases (LIMK) regulate actin dynamics via a reversible phosphorylation (inactivation) of serine 3 in actin-depolymerizing factor (ADF) and cofilin. A multi-protein complex consisting of SSH-1L, LIMK1, actin, and the scaffolding protein, 14-3-3zeta, is involved, along with the kinase, PAK4, in the regulation of ADF/cofilin activity. Endogenous LIMK1 and SSH-1L interact in vitro and co-localize in vivo, and this interaction results in dephosphorylation and downregulation of LIMK1 activity. The phosphatase activity of purified SSH-1L is F-actin dependent and is negatively regulated via phosphorylation by PAK4. 14-3-3zeta binds to phosphorylated slingshot, decreases the amount of slingshot that co-sediments with F-actin, but does not alter slingshot activity. A novel ADF/cofilin phosphoregulatory complex is described, and a new mechanism is suggested for the regulation of ADF/cofilin activity in mediating changes to the actin cytoskeleton (Soosairajah, 2005).

Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. Cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppresses chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produces and retaines multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. These results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. It is proposed that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement (Nishita, 2005).

Cyclase-associated protein promotes cofilin-induced actin dynamics

Cofilin-ADF (actin-depolymerizing factor) is an essential driver of actin-based motility. Two proteins, p65 and p55, were discovered that are components of the actin-cofilin complex in a human HEK293 cell extract and p55 was identified as CAP1/ASP56, a human homologue of yeast CAP/SRV2 (cyclase-associated protein). CAP is a bifunctional protein with an N-terminal domain that binds to Ras-responsive adenylyl cyclase and a C-terminal domain that inhibits actin polymerization. Surprisingly, the N-terminal domain of CAP1, but not the C-terminal domain, is responsible for the interaction with the actin-cofilin complex. The N-terminal domain of CAP1 accelerates the depolymerization of F-actin at the pointed end; depolymerization is further enhanced in the presence of cofilin and/or the C-terminal domain of CAP1. Moreover, CAP1 and its C-terminal domain facilitate filament elongation at the barbed end and stimulate ADP-ATP exchange on G-actin, a process that regenerates easily polymerizable G-actin. Although cofilin inhibits the nucleotide exchange on G-actin even in the presence of the C-terminal domain of CAP1, its N-terminal domain relieved this inhibition. Thus, CAP1 plays a key role in speeding up the turnover of actin filaments by effectively recycling cofilin and actin and through its effect on both ends of actin filament (Moriyama, 2002).

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. 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. It is concluded that two genetically and biochemically separable functions have been defined 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. The Srv2 complex is shown to be 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 (Balcer, 2003).

Cyclase-associated proteins (CAPs) are highly conserved actin monomer binding proteins present in all eukaryotes. However, gaining an understanding ofthe mechanism by which CAPs contribute to actin dynamics has been elusive. In mammals, the situation is further complicated by the presence of two CAP isoforms whose differences have not been characterized. CAP1 is widely expressed in mouse nonmuscle cells, whereas CAP2 is the predominant isoform in developing striated muscles. In cultured NIH3T3 and B16F1 cells, CAP1 is a highly abundant protein that colocalizes with cofilin-1 to dynamic regions of the cortical actin cytoskeleton. Analysis of CAP1 knockdown cells has demonstrated that this protein promotes rapid actin filament depolymerization and is important for cell morphology, migration, and endocytosis. Interestingly, depletion of CAP1 leads to an accumulation of cofilin-1 into abnormal cytoplasmic aggregates and to similar cytoskeletal defects as those seen in cofilin-1 knockdown cells, demonstrating that CAP1 is required for proper subcellular localization and function of ADF/cofilin. Together, these data provide the first direct in vivo evidence that CAP promotes rapid actin dynamics in conjunction with ADF/cofilin and is required for several central cellular processes in mammals (Bertling, 2004).

Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 microM) compared with ATP-G-actin (Kd 1.9 microM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved beta-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, evolutionarily conserved residues have been described that mediate binding to ADP-G-actin; these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, these data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly (Mattila, 2004).

Actin-interacting protein (AIP) disassembles cofilin-bound actin filaments

Actin-interacting protein 1 (AIP1) is a conserved WD-repeat protein that enhances actin filament disassembly only in the presence of actin depolymerizing factor (ADF)/cofilin. In the nematode C. elegans, an AIP1 ortholog is encoded by the unc-78 gene that is required for organized assembly of muscle actin filaments. Bacterially expressed UNC-78 protein was produced and found to enhance actin filament disassembly preferentially in the presence of a specific ADF/cofilin isoform. Extensive and rapid filament disassembly by UNC-78 was observed in the presence of UNC-60B, a muscle-specific C. elegans ADF/cofilin isoform. UNC-78 also reduces the rate of spontaneous polymerization and enhances subunit dissociation from filaments in the presence of UNC-60B. However, in the presence of UNC-60A, a non-muscle C. elegans ADF/cofilin isoform, UNC-78 only slightly enhances filament disassembly. Interestingly, UNC-78 failed to enhance disassembly by mouse muscle-type cofilin. Using mutant forms of UNC-60B, it has been demonstrated that the F-actin-specific binding site of UNC-60B at the C terminus is required for filament disassembly by UNC-78. UNC-78 was expressed in body wall muscle and co-localized with actin where UNC-60B is also present. Surprisingly, UNC-78 co-localizes with actin in unc-60B null mutants, suggesting that the AIP1-actin interaction is not dependent on ADF/cofilin in muscle. These results suggest that UNC-78 closely collaborates with UNC-60B to regulate actin dynamics in muscle cells (Mohri, 2003).

Actin-interacting protein 1 (Aip1p) is a 67-kDa WD repeat protein known to regulate the depolymerization of actin filaments by cofilin and is conserved in organisms ranging from yeast to mammals. The crystal structure of Aip1p from Saccharomyces cerevisiae was determined to a 2.3-A resolution and a final crystallographic R-factor of 0.204. The structure reveals that the overall fold is formed by two connected seven-bladed beta-propellers and has important implications for the structure of Aip1 from other organisms and WD repeat-containing proteins in general. These results were unexpected because a maximum of 10 WD repeats had been reported in the literature for this protein using sequence data. The surfaces of the beta-propellers formed by the D-A and B-C loops are positioned adjacent to one another, giving Aip1p a shape that resembles an open 'clamshell'. The mapping of conserved residues to the structure of Aip1p reveals dense patches of conserved residues on the surface of one beta-propeller and at the interface of the two beta-propellers. These two patches of conserved residues suggest a potential binding site for F-actin on Aip1p; the orientation of the beta-propellers with respect to one another plays a role in binding an actin-cofilin complex. In addition, the conserved interface between the domains is mediated by a number of interactions that appear to impart rigidity between the two domains of Aip1p and may make a large substrate-induced conformational change difficult (Voegtli, 2003).

Actin-interacting protein 1 (AIP1) is a WD40 repeat protein that enhances actin filament disassembly in the presence of actin-depolymerizing factor (ADF)/cofilin. AIP1 also caps the barbed end of ADF/cofilin-bound actin filament. However, the mechanism by which AIP1 interacts with ADF/cofilin and actin is not clearly understood. The crystal structure of Caenorhabditis elegans AIP1 (UNC-78) reveals 14 WD40 modules arranged in two seven-bladed beta-propeller domains. The structure allows for the mapping of conserved surface residues, and mutagenesis studies have identified five residues that affect the ADF/cofilin-dependent actin filament disassembly activity. Mutations of these residues, which reside in blades 3 and 4 in the N-terminal propeller domain, have significant effects on the disassembly activity but do not alter the barbed end capping activity. These data support a model in which this conserved surface of AIP1 plays a direct role in enhancing fragmentation/depolymerization of ADF/cofilin-bound actin filaments but not in barbed end capping (Mohri, 2004).

Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. The effects of these proteins on filamentous actin have been directly observed by fluorescence microscopy and novel insight has been gained into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolin-actin complex, or cytochalasin D do not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 is weak and allows filament elongation, whereas gelsolin or gelsolin-actin complex strongly caps and inhibits elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation (Ono, 2004).

Actin-filament stochastic dynamics mediated by ADF/Cofilin

The rapid dynamics of actin filaments is a fundamental process that powers a large number of cellular functions. However, the basic mechanisms that control and coordinate such dynamics remain a central question in cell biology. To reach beyond simply defining the inventory of molecules that control actin dynamics and to understand how these proteins act synergistically to modulate filament turnover, evanescent-wave microscopy was combined with a biomimetic system and the behavior of single actin filaments was followed in the presence of a physiologically relevant mixture of accessory proteins. This approach allows for the real-time visualization of actin polymerization and age-dependent filament severing. In the presence of actin-depolymerizing factor (ADF)/cofilin and profilin, actin filaments with a processive formin attached at their barbed ends were observed to oscillate between stochastic growth and shrinkage phases. Fragmentation of continuously growing actin filaments by ADF/cofilin is the key mechanism modulating the prominent and frequent shortening events. The net effect of continuous actin polymerization, driven by a processive formin that uses profilin-actin, and of ADF/cofilin-mediating severing that trims the aged ends of the growing filaments is an up to 155-fold increase in the rate of actin-filament turnover in vitro in comparison to that of actin alone. Lateral contact between actin filaments dampens the dynamics and favors actin-cable formation. A kinetic simulation accurately validates these observations. A proposed mechanism for the control of actin dynamics is dominated by ADF/cofilin-mediated filament severing that induces a stochastic behavior upon individual actin filaments. When combined with a selection process that stabilizes filaments in bundles, this mechanism could account for the emergence and extension of actin-based structures in cells (Michelot, 2007).

Cofilin function in early development

Two cDNAs, isolated from a Xenopus laevis embryonic library, encode proteins of 168 amino acids, both of which are 77% identical to chick cofilin and 66% identical to chick actin-depolymerizing factor (ADF), two structurally and functionally related proteins. These Xenopus ADF/cofilins (XADs) differ from each other in 12 residues spread throughout the sequence but do not differ in charge. Purified GST-fusion proteins have pH-dependent actin-depolymerizing and F-actin-binding activities similar to chick ADF and cofilin. Similarities in the developmental and tissue specific expression, embryonic localization, and in the cDNA sequence of the noncoding regions, suggest that the two XACs arise from allelic variants of the pseudotetraploid X. laevis. Immunofluorescence localization of XAC in oocyte sections with an XAC-specific monoclonal antibody shows it to be diffuse in the cortical cytoplasm. After fertilization, increased immunostaining is observed in two regions: along the membrane, particularly that of the vegetal hemisphere, and at the interface between the cortical and animal hemisphere cytoplasm. The cleavage furrow and the mid-body structure are stained at the end of first cleavage. Neuroectoderm derived tissues, notochord, somites, and epidermis stain heavily, either continuously or transiently from stages 18-34. A phosphorylated form of XAC (pXAC) was identified by 2D Western blotting, and it is the only species found in oocytes. Dephosphorylation of >60% of the pXAC occurs within 30 min after fertilization. Injection of one blastomere at the 2 cell stage, either with constitutively active XAC or with an XAC inhibitory antibody, blocked cleavage of only the injected blastomere in a concentration-dependent manner without inhibiting nuclear division. The cleavage furrow of eggs injected with constitutively active XAC completely regressed. These results suggest that XAC is necessary for cytokinesis and that its activity must be properly regulated for cleavage to occur (Abe, 1996).

ADF/cofilin is a key regulator for actin dynamics during cytokinesis. Its activity is suppressed by phosphorylation and reactivated by dephosphorylation. Little is known, however, about regulatory mechanisms of ADF/cofilin function during formation of contractile ring actin filaments. Using Xenopus cycling extracts, it was found that ADF/cofilin is dephosphorylated at prophase and telophase. In addition, constitutively active Rho GTPase induces dephosphorylation of ADF/cofilin in the egg extracts. This dephosphorylation is inhibited by Na3VO4 but not by other conventional phosphatase-inhibitors. A Xenopus homologue of Slingshot phosphatase (XSSH) was cloned, and antibody was raised specific for the catalytic domain of XSSH. This inhibitory antibody significantly suppresses the Rho-induced dephosphorylation of ADF/cofilin in extracts, suggesting that the dephosphorylation at telophase is dependent on XSSH. XSSH binds to actin filaments with a dissociation constant of 0.4 microM, and the ADF/cofilin phosphatase activity is increased in the presence of F-actin. When latrunculin A, a G-actin-sequestering drug, was added to extracts, both Rho-induced actin polymerization and dephosphorylation of ADF/cofilin were markedly inhibited. Jasplakinolide, an actin-stabilizing drug, alone induced actin polymerization in the extracts and led to dephosphorylation of ADF/cofilin. These results suggest that Rho-induced dephosphorylation of ADF/cofilin is dependent on the XSSH activation that is caused by increase in the amount of F-actin induced by Rho signaling. XSSH colocalized with both actin filaments and ADF/cofilin in the actin patches formed on the surface of the early cleavage furrow. Injection of inhibitory antibody blocked cleavage of blastomeres. Thus, XSSH may reorganize actin filaments through dephosphorylation and reactivation of ADF/cofilin at early stage of contractile ring formation (Tanaka, 2005).

Chronophin mediates an ATP-sensing mechanism for cofilin dephosphorylation and neuronal cofilin-actin rod formation

Actin and its key regulatory component, cofilin, are found together in large rod-shaped assemblies in neurons subjected to energy stress. Such inclusions are also enriched in Alzheimer's disease brain, and appear in transgenic models of neurodegeneration. Neuronal insults, such as energy loss and/or oxidative stress, result in rapid dephosphorylation of the cellular cofilin pool prior to its assembly into rod-shaped inclusions. Although these events implicate a role for phosphatases in cofilin rod formation, a mechanism linking energy stress, phosphocofilin turnover, and subsequent rod assembly has been elusive. This study demonstrates the ATP-sensitive interaction of the cofilin phosphatase chronophin (CIN) with the chaperone hsp90 to form a biosensor that mediates cofilin/actin rod formation. The results suggest a model whereby attenuated interactions between CIN and hsp90 during ATP depletion enhance CIN-dependent cofilin dephosphorylation and consequent rod assembly, thereby providing a mechanism for the formation of pathological actin/cofilin aggregates during neurodegenerative energy flux (Huang, 2008).

N-cofilin is essential for neural tube morphogenesis and neural crest cell migration

Cofilin/ADF proteins are a ubiquitously expressed family of F-actin depolymerizing factors found in eukaryotic cells including plants. In vitro, cofilin/ADF activity has been shown to be essential for actin driven motility, by accelerating actin filament turnover. Three actin depolymerizing factors (n-cofilin, m-cofilin, ADF) can be found in mouse and human. In the mouse the non-muscle-specific gene (n-cofilin) is essential for migration of neural crest cells as well as other cell types in the paraxial mesoderm. The main defects observed in n-cofilin mutant embryos are an impaired delamination and migration of neural crest cells, affecting the development of neural crest derived tissues. Neural crest cells lacking n-cofilin do not polarize, and F-actin bundles or fibers are not detectable. In addition, n-cofilin is required for neuronal precursor cell proliferation and scattering. These defects result in a complete lack of neural tube closure in n-cofilin mutant embryos. Although ADF is overexpressed in mutant embryos, this cannot compensate the lack of n-cofilin, suggesting that they might have a different function in embryonic development. These data suggest that in mammalian development, regulation of the actin cytoskeleton by the F-actin depolymerizing factor n-cofilin is critical for epithelial-mesenchymal type of cell shape changes as well as cell proliferation (Gurniak, 2005).

Increase in neurite outgrowth mediated by overexpression of cofilin

Growth cone motility is regulated by changes in actin dynamics. Actin depolymerizing factor (ADF) is an important regulator of actin dynamics, and extracellular signal-induced changes in ADF activity may influence growth cone motility and neurite extension. To determine this directly, ADF was overexpressed in primary neurons and neurite lengths were analyzed. Recombinant adenoviruses were constructed that express wild-type Xenopus ADF/cofilin [XAC(wt)], as well as two mutant forms of XAC, the active but nonphosphorylatable XAC(A3) and the less active, pseudophosphorylated XAC(E3). XAC expression was detectable on Western blots 24 hr after infection and peaked at 3 d in cultured rat cortical neurons. Peak expression was approximately 75% that of endogenous ADF. XAC(wt) expression caused a slight increase in growth cone area and filopodia but decreased filopodia numbers on neurite shafts. At maximal XAC levels, neurite lengths increased >50% compared with controls infected with a green fluorescent protein-expressing adenovirus. Increased neurite extension was directly related to the expression of active XAC. Expression of the XAC(E3) mutant did not increase neurite extension, whereas expression of the XAC(A3) mutant increased neurite extension but to a lesser extent than XAC(wt), which was partially phosphorylated. XAC expression had minimal, if any, impact on F-actin levels and did not result in compensatory changes in the expression of endogenous ADF or actin. However, F-actin turnover appeared to increase based on F-actin loss after treatment with drugs that block actin polymerization. These results provide direct evidence that increased ADF activity promotes process extension and neurite outgrowth (Meberg, 2000).

Control of growth cone motility and morphology by LIM kinase and Slingshot via phosphorylation and dephosphorylation of cofilin

Growth cone motility and morphology are based on actin-filament dynamics. Cofilin plays an essential role for the rapid turnover of actin filaments by severing and depolymerizing them. The activity of cofilin is repressed by phosphorylation at Ser3 by LIM kinase (LIMK, in which LIM is an acronym of the three gene products Lin-11, Isl-1, and Mec-3) and is reactivated by dephosphorylation by phosphatases, termed Slingshot (SSH). The roles of cofilin, LIMK, and SSH in the growth cone motility and morphology and neurite extension were investigated by expressing fluorescence protein-labeled cofilin, LIMK1, SSH1, or their mutants in chick dorsal root ganglion (DRG) neurons and then monitoring live images of growth cones by time-lapse video fluorescence microscopy. The expression of LIMK1 remarkably represses growth cone motility and neurite extension, whereas the expression of SSH1 or a nonphosphorylatable S3A mutant of cofilin enhances these events. The fan-like shape of growth cones was disorganized by the expression of any of these proteins. The repressive effects on growth cone behavior by LIMK1 expression were significantly rescued by the coexpression of S3A-cofilin or SSH1. These findings suggest that LIMK1 and SSH1 play critical roles in controlling growth cone motility and morphology and neurite extension by regulating the activity of cofilin and may be involved in signaling pathways that regulate stimulus-induced growth cone guidance. Using various mutants of cofilin, evidence was obtained that the actin-filament-severing activity of cofilin is critical for growth cone motility and neurite extension (Endo, 2003).

Signaling mechanisms underlying Slit2-induced collapse of Xenopus retinal growth cones

Slits mediate multiple axon guidance decisions, but the mechanisms underlying the responses of growth cones to these cues remain poorly defined. This study shows that collapse induced by Slit2-conditioned medium (Slit2-CM) in Xenopus retinal growth cones requires local protein synthesis (PS) and endocytosis. Slit2-CM elicits rapid activation of translation regulators and MAP kinases in growth cones, and inhibition of MAPKs or disruption of heparan sulfate blocks Slit2-CM-induced PS and repulsion. Interestingly, Slit2-CM causes a fast PS-dependent decrease in cytoskeletal F-actin concomitant with a PS-dependent increase in the actin-depolymerizing protein cofilin. These findings reveal an unexpected link between Slit2 and cofilin in growth cones and suggest that local translation of actin regulatory proteins contributes to repulsion (Piper, 2006).

The activation of selected MAPK proteins was monitored. To do this, Slit2-CM was applied to retinal cultures for 5 min, then active (phosphorylated) MAPK proteins were labeled with phospho-specific antibodies and digital quantitation of immunofluorescence was used to monitor relative changes in signal intensity. Five minutes was selected because a collapse assay time course showed that 40.3% of growth cones collapsed 5 min after Slit2-CM addition. Thus, it was reasoned that this time point would be optimal for assessing the signaling events leading to collapse, prior to the occurrence of overt collapse. Using a phosphospecific p38 antibody (p38-P), it was found that Slit2-CM stimulated an 80% increase in signal intensity compared to controls. The MAPK p42/p44 can be directly phosphorylated by MEK1/MEK2. After Slit2-CM application, a 2-fold rise was seen in phospho-p42/p44 fluorescent intensity. Slit2-CM did not have a significant effect on the fluorescent intensity of either p38 or p42/p44 (Piper, 2006).

The finding that cofilin immunoreactivity markedly increases in growth cones in response to both Slit2-CM and Sema3A suggests the intriguing idea that the local synthesis of actin-depolymerizing proteins contributes to repellent-induced collapse. Cofilin, a member of the ADF/cofilin family of actin-depolymerizing molecules, is a small protein of 19 kDa, and its mRNA and protein have been detected in a variety of neuronal axons and growth cones. Importantly, in vitro experiments have implicated ADF/cofilin in the control of chick retinal growth cone filopodial dynamics in response to brain-derived neurotrophic factor, and moreover, axons of cultured rat DRG axons have been shown to synthesize cofilin 1. However, the data provide a potential link between cue-induced growth cone behavior and local cofilin translation. Two Xenopus cofilin proteins have been reported, and their high level of identity at the amino acid level suggests that they are allelic variants, as Xenopus has a tetraploid genome. Locally controlling their synthesis may provide one means of influencing the balance of actin stability; in the case of Slit, perhaps resulting in an increase in growth cone cofilin and a subsequent shift toward actin depolymerization and hence growth cone collapse. This may also be consistent with respect to recent reports linking cofilin activity to shrinkage of dendritic spines in long-term synaptic depression and axon growth and neuronal morphogenesis. The idea that the mRNAs of cytoskeletal regulators are key targets for cue-induced translation is in line with recent evidence demonstrating that local synthesis of the small GTPase RhoA is necessary for Sema3A-induced collapse in mammalian sensory growth cones (Piper, 2006).

The data also suggest that regulation of cofilin in response to Slit also occurs at a posttranslational level; Slit2-CM caused a significant decrease in the growth cone signal intensity of phosphorylated cofilin. A similar phenomenon has been reported for Sema3A, which induces a rapid (1 min) increase in phospho-cofilin in embryonic murine DRG neurons, followed by a dramatic decrease after 5 min. This suggests that a cycle of inactivation/activation of cofilin, regulated by kinases such as LIM-kinase and phosphatases such as Slingshot may play a role in collapse. This may also be related to calcium (Ca2+) signaling, as high levels of focally released caged Ca2+ activate calcium-calmodulin-dependent protein kinase II (CaMKII) to induce growth cone attraction, while lower levels cause repulsion via a mechanism involving phosphatases such as calcineurin (CaN). Furthermore, in vitro cell culture studies have shown that the phosphatase responsible for dephosphorylating cofilin, Slingshot, is activated in a Ca2+/CaN-dependent fashion. Thus, Slit may increase the local actin-severing activity of cofilin in growth cones by triggering two parallel mechanisms -- local synthesis of new cofilin and dephosphorylation of existing cofilin -- that act in concert to promote changes in the cytoskeleton (Piper, 2006).

BMP gradients steer nerve growth cones by a balancing act of LIM kinase and Slingshot phosphatase on ADF/cofilin

Bone morphogenic proteins (BMPs) are involved in axon pathfinding, but how they guide growth cones remains elusive. This study reports that a BMP7 gradient elicits bidirectional turning responses from nerve growth cones by acting through LIM kinase (LIMK) and Slingshot (SSH) phosphatase to regulate actin-depolymerizing factor (ADF)/cofilin-mediated actin dynamics. Xenopus laevis growth cones from 4-8-h cultured neurons are attracted to BMP7 gradients but become repelled by BMP7 after overnight culture. The attraction and repulsion are mediated by LIMK and SSH, respectively, which oppositely regulate the phosphorylation-dependent asymmetric activity of ADF/cofilin to control the actin dynamics and growth cone steering. The attraction to repulsion switching requires the expression of a transient receptor potential (TRP) channel TRPC1 and involves Ca2+ signaling through calcineurin phosphatase for SSH activation and growth cone repulsion. Together, this study shows that spatial regulation of ADF/cofilin activity controls the directional responses of the growth cone to BMP7, and Ca2+ influx through TRPC tilts the LIMK-SSH balance toward SSH-mediated repulsion (Wen, 2007).

GABA promotes the competitive selection of dendritic spines by controlling local Ca2+ signaling

Activity-dependent competition of synapses plays a key role in neural organization and is often promoted by GABA; however, its cellular bases are poorly understood. Excitatory synapses of cortical pyramidal neurons are formed on small protrusions known as dendritic spines, which exhibit structural plasticity. This study used two-color uncaging of glutamate and GABA in rat hippocampal CA1 pyramidal neurons and found that spine shrinkage and elimination were markedly promoted by the activation of GABAA receptors shortly before action potentials. GABAergic inhibition suppressed bulk increases in cytosolic Ca2+ concentrations, whereas it preserved the Ca2+ nanodomains generated by NMDA-type receptors, both of which were necessary for spine shrinkage. Unlike spine enlargement, spine shrinkage spread to neighboring spines (<15 μm) and competed with their enlargement, and this process involved the actin-depolymerizing factor ADF/cofilin. Thus, GABAergic inhibition directly suppresses local dendritic Ca2+ transients and strongly promotes the competitive selection of dendritic spines (Hayama, 2013)..

Cofilin controls cell polarity during fibroblast migration

To migrate, normally a cell must establish morphological polarity and continuously protrude a single lamellipodium, polarized in the direction of migration. Actin filament disassembly is necessary for protrusion of the lamellipodium during fibroblast migration. Since ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, their role in polarized lamellipodium protrusion was assessed in migrating fibroblasts. The spatial distributions of AC and the inactive, phosphorylated AC (pAC), were compared in migrating cells. AC, but not pAC, localized to the lamellipodium. To investigate a role for AC in cell polarity, the proportion of pAC in migrating fibroblasts was increased by overexpressing constitutively active (CA) LIM kinase 1. In 87% of cells expressing CA LIM kinase, cell polarity was abolished. In such cells, the single polarized lamellipodium was replaced by multiple nonpolarized lamellipodia, which, in contrast to nonexpressing migrating cells, stained for pAC. Cell polarity was rescued by coexpressing an active, nonphosphorylatable Xenopus AC (CA XAC) with the CA LIMK. Furthermore, overexpressing a pseudophosphorylated (less active) XAC by itself also abolished cell polarity. It is concluded that locally maintaining ADF/cofilin in the active, nonphosphorylated state within the lamellipodium is necessary to maintain polarized protrusion during cell migration (Dawe, 2003).

Cofilin and the invasive growth of cancer cells

Invadopodia are actin-rich membrane protrusions with a matrix degradation activity formed by invasive cancer cells. The molecular mechanisms of invadopodium formation were studied in metastatic carcinoma cells. Epidermal growth factor (EGF) receptor kinase inhibitors blocked invadopodium formation in the presence of serum, and EGF stimulation of serum-starved cells induces invadopodium formation. RNA interference and dominant-negative mutant expression analyses revealed that neural WASP (N-WASP), Arp2/3 complex, and their upstream regulators, Nck1, Cdc42, and WIP, are necessary for invadopodium formation. Time-lapse analysis has revealed that invadopodia are formed de novo at the cell periphery and their lifetime varies from minutes to several hours. Invadopodia with short lifetimes are motile, whereas long-lived invadopodia tend to be stationary. Interestingly, suppression of cofilin expression by RNA interference inhibits the formation of long-lived invadopodia, resulting in formation of only short-lived invadopodia with less matrix degradation activity. These results indicate that EGF receptor signaling regulates invadopodium formation through the N-WASP-Arp2/3 pathway and cofilin is necessary for the stabilization and maturation of invadopodia (Yamaguchi, 2005).

ADF/Cofilin accelerates actin dynamics by severing filaments and promoting their depolymerization at both ends

Actin-depolymerizing factor (ADF)/cofilins (see Drosophila Twinstar) contribute to cytoskeletal dynamics by promoting rapid actin filament disassembly. In the classical view, ADF/cofilin sever filaments, and capping proteins (see Drosophila Capulet) block filament barbed ends whereas pointed ends depolymerize, at a rate that is still debated. By monitoring the activity of the three mammalian ADF/cofilin isoforms on individual skeletal muscle and cytoplasmic actin filaments, this study directly quantify the reactions underpinning filament severing and depolymerization from both ends. In the absence of monomeric actin, soluble ADF/cofilin was found to associate with bare filament barbed ends to accelerate their depolymerization. Compared to bare filaments, ADF/cofilin-saturated filaments depolymerize faster from their pointed ends and slower from their barbed ends, resulting in similar depolymerization rates at both ends. This effect is isoform specific because depolymerization is faster for ADF- than for cofilin-saturated filaments. It was also shown that, unexpectedly, ADF/cofilin-saturated filaments qualitatively differ from bare filaments: their barbed ends are very difficult to cap or elongate, and consequently undergo depolymerization even in the presence of capping protein and actin monomers. Such depolymerizing ADF/cofilin-decorated barbed ends are produced during 17% of severing events. They are also the dominant fate of filament barbed ends in the presence of capping protein, because capping allows growing ADF/cofilin domains to reach the barbed ends, thereby promoting their uncapping and subsequent depolymerization. These experiments thus reveal how ADF/cofilin, together with capping protein, control the dynamics of actin filament barbed and pointed ends. Strikingly, the results propose that significant barbed-end depolymerization may take place in cells (Wioland, 2017).

Enhanced depolymerization of actin filaments by ADF/Cofilin and monomer funneling by capping protein cooperate to accelerate barbed-end growth

A living cell's ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers, which feed polarized filament growth. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofilin (see Drosophila Twinstar) and filament cappers like capping protein (CP; see Drosophila Capulet) are essential agonists of motility, but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades. Whereas filament fragmentation by ADF/cofilin has long been demonstrated by total internal reflection fluorescence (TIRF), filament depolymerization was only inferred from bulk solution assays. Using microfluidics-assisted TIRF microscopy, this study provides the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. Using a conceptually novel assay to directly visualize ADF's effect on a population of pre-assembled filaments, it was demonstrated how ADF's enhanced pointed-end depolymerization causes an increase in polymerizable actin monomers, thus promoting faster barbed-end growth. It was further reveale that ADF-enhanced depolymerization synergizes with CP's long-predicted "monomer funneling" and leads to skyrocketing of filament growth rates, close to estimated lamellipodial rates. The "funneling model" hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few non-capped filaments transiently grow much faster, an effect proposed to be very important for motility. This study provides the first direct microscopic evidence of monomer funneling at the scale of individual filaments. These results significantly enhance understanding of the turnover of cellular actin networks (Shekhar, 2017).

twinstar: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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