Endogenous Cofilin Research Articles

Infantile spasms‐linked Nedd4‐2 mediates hippocampal plasticity and learning via cofilin signaling

Individuals affected by infantile spasms (IS), such as those carrying mutations in an IS-linked gene, neural precursor cell expressed developmentally downregulated gene 4-like (Nedd4-2), exhibit developmental delays and learning disabilities, but the underlying mechanism is unknown. Using conditional Nedd4-2 knockout mice, we uncover that Nedd4-2 functions to maintain the excitatory synapses in hippocampal neurons and allows for late-phase long-term synaptic potentiation (L-LTP) at Schaffer collateral synapses in the hippocampus. We also find that Nedd4-2 is required for multiple forms of hippocampus-dependent learning and memory. Mechanistically, we show that loss of Nedd4-2 leads to a decrease in actin polymerization caused by reduced phosphorylation of the actin depolymerizing protein cofilin. A cell-permeable peptide promoting phosphorylation of endogenous cofilin in Nedd4-2 knockout neurons restores the number of hippocampal excitatory synapses and hippocampal L-LTP and partially restores hippocampus-dependent learning in mice. Taken together, our results reveal a novel mechanism underlying IS-associated learning disabilities and may provide information for future therapeutic strategies for IS.

Exogenously Added Cofilin Increases Ex Vivo Production of Functional Platelets By Stimulating Proplatelet Formation

Platelet transfusion is essential for the treatment of thrombocytopenia. Platelet supply depends on blood donation and the ability to store platelets before use is limited by a short shelf-life. Furthermore, such supplied-platelets have the risk of bacterial or viral infection. Ex vivo platelet production could overcome these problems. Therefore, many methods of ex vivo platelet production from some stem cell sources such as umbilical cord blood (UCB) stem cells, human embryonic stem cells and induced pluripotent stem cells have been developed. However, the platelet yields achieved by these methods have not been sufficient for clinical use. The processes of platelet production are thought to involve differentiation into megakaryocytes (Mks) from hematopoietic stem cells, Mk polyploidization, proplatelet formation, and finally platelet release. Large-scale ex vivo production of functional platelets requires a system to control all these steps. Hematopoietic progenitor cell and Mk levels can be increased by the numerous hematopoietic growth factors; however, factors to stimulate proplatelet formation in Mks have not been identified.To find factors to stimulate proplatelet formation in Mks, we evaluated the effects of various conditioned media using Mks induced from UCB-CD34+ cells. The supernatants of COS cells transfected with cDNA coding for non-muscle cofilin stimulated proplatelet formation in Mks, whereas those of mock-transfected COS cells did not. Cofilin is an actin-binding protein; recently, endogenous non-muscle cofilin was reported to be associated with proplatelet formation in Mks, but that activity has not been reported for exogenous cofilin. The effects of cofilin on proplatelet formation in Mks in the presence or absence of TPO were compared with that in the presence of TPO alone. Cofilin alone increased the number of proplatelet-bearing Mks and enhanced proplatelet extension compared to TPO alone; moreover, a combination of cofilin and TPO had synergistic effects on both activities.The influences of cofilin on megakaryopoiesis from UCB-CD34+ cells were compared with those of TPO. The addition of cofilin to serum-free culture containing SCF and Flt3-L increased the size of both Mks and Mk colonies compared to SCF/Flt3-L/TPO. Next, the effects of cofilin on the platelet production in the serum-free culture system containing SCF and Flt3L were evaluated; the addition of coflin dramatically increased the number of platelets compared to that for SCF/Flt3-L/TPO. Thus, we established a suitable system for ex vivo platelet production by using cofilin. To determine whether the platelets produced using this system were functional; platelet activation in response to in vitro activation with thrombin was tested. Thrombin induces platelet granule secretion, resulting in CD62P (P-selectin) expression on the platelets surface membrane. The expression of CD62P was increased after thrombin stimulation of the platelets produced in cofilin culture. These results indicate that the platelets produced with cofilin were functional.We show here that exogenously adding cofilin to the serum-free culture system enhanced proplatelet formation in Mks, resulting in increased platelet yields. At present, many methods are under study for ex vivo platelet production for clinical use; we believe that our studies will provide the important information for the improvement of these methods. DisclosuresNo relevant conflicts of interest to declare.

Cofilin-mediated Neuronal Apoptosis via p53 Translocation and PLD1 Regulation

Amyloid β (Aβ) accumulation is an early event in the pathogenesis of Alzheimer’s disease (AD), leading to mitochondrial and synaptic dysfunction, tau accumulation, and eventual neuronal death. While the p53 apoptotic pathway has clearly been associated with Aβ deposits and neuronal apoptosis, the critical upstream factors contributing to p53 activation in AD are not well understood. We have previously shown that cofilin activation plays a pivotal role in Aβ-induced mitochondrial and synaptic dysfunction. In this study, we show that activated cofilin (S3A) preferentially forms a complex with p53 and promotes its mitochondrial and nuclear localization, resulting in transcription of p53-responsive genes and promotion of apoptosis. Conversely, reduction of endogenous cofilin by knockdown or genetic deficiency inhibits mitochondrial and nuclear translocation of p53 in cultured cells and in APP/PS1 mice. This cofilin-p53 pro-apoptotic pathway is subject to negative regulation by PLD1 thorough cofilin inactivation and inhibition of cofilin/p53 complex formation. Finally, activated cofilin is unable to induce apoptosis in cells genetically lacking p53. These findings taken together indicate that cofilin coopts and requires the nuclear and mitochondrial pro-apoptotic p53 program to induce and execute apoptosis, while PLD1 functions in a regulatory multi-brake capacity in this pathway.

State-dependent diffusion of actin-depolymerizing factor/cofilin underlies the enlargement and shrinkage of dendritic spines.

Dendritic spines are the postsynaptic sites of most excitatory synapses in the brain, and spine enlargement and shrinkage give rise to long-term potentiation and depression of synapses, respectively. Because spine structural plasticity is accompanied by remodeling of actin scaffolds, we hypothesized that the filamentous actin regulatory protein cofilin plays a crucial role in this process. Here we investigated the diffusional properties of cofilin, the actin-severing and depolymerizing actions of which are activated by dephosphorylation. Cofilin diffusion was measured using fluorescently labeled cofilin fusion proteins and two-photon imaging. We show that cofilins are highly diffusible along dendrites in the resting state. However, during spine enlargement, wild-type cofilin and a phosphomimetic cofilin mutant remain confined to the stimulated spine, whereas a nonphosphorylatable mutant does not. Moreover, inhibition of cofilin phosphorylation with a competitive peptide disables spine enlargement, suggesting that phosphorylated-cofilin accumulation is a key regulator of enlargement, which is localized to individual spines. Conversely, spine shrinkage spreads to neighboring spines, even though triggered by weaker stimuli than enlargement. Diffusion of exogenous cofilin injected into a pyramidal neuron soma causes spine shrinkage and reduced PSD95 in spines, suggesting that diffusion of dephosphorylated endogenous cofilin underlies the spreading of spine shrinkage and long-term depression.

Cofilin contributes to phagocytosis of IgG-opsonized particles but not non-opsonized particles in RAW264 macrophages

Cofilin is an actin-binding protein that severs actin filaments. It plays a key role in regulating actin cytoskeletal remodeling, thereby contributing to diverse cellular functions. However, the involvement of cofilin in phagocytosis remains to be elucidated. We examined the spatiotemporal localization of cofilin during phagocytosis of IgG-opsonized erythrocytes, IgG-opsonized latex beads and non-opsonized latex beads. Live-cell imaging showed that GFP-cofilin accumulates in the sites of IgG-opsonized particle binding and in phagocytic cups. Moreover, immunofluorescence microscopy revealed that endogenous cofilin localizes to phagocytic cups engulfing IgG-opsonized particles, but not non-opsonized latex beads. Scanning electron microscopy demonstrated a notable difference in morphology between phagocytic structures in IgG-dependent and IgG-independent phagocytosis. In phagocytosis of IgG-opsonized particles, sheet-like pseudopodia extended along the surface of IgG-opsonized particles to form phagocytic cups. In contrast, in opsonin-independent phagocytosis, long finger-like filopodia captured non-opsonized latex beads. Importantly, non-opsonized beads sank into the cells without extending phagocytic cups. Our analysis of cofilin mutant expression demonstrates that phagocytosis of IgG-opsonized particles is enhanced in cells expressing wild-type cofilin or active mutant cofilin-S3A, whereas the uptake of non-opsonized latex beads is not. These data suggest that cofilin promotes actin cytoskeletal remodeling to form phagocytic cups by accelerating actin turnover and thereby facilitating phagosome formation. In contrast, cofilin is not involved in opsonin-independent phagocytosis of latex beads.

A genetically encoded reporter for real-time imaging of cofilin-actin rods in living neurons.

Filament bundles (rods) of cofilin and actin (1:1) form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP) and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30–60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.

Actin Dynamics Regulated by the Balance of Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) and Cofilin Activities Determines the Biphasic Response of Glucose-induced Insulin Secretion

Actin dynamics in pancreatic β-cells is involved in insulin secretion. However, the molecular mechanisms of the regulation of actin dynamics by intracellular signals in pancreatic β-cells and its role in phasic insulin secretion are largely unknown. In this study, we elucidate the regulation of actin dynamics by neuronal Wiskott-Aldrich syndrome protein (N-WASP) and cofilin in pancreatic β-cells and demonstrate its role in glucose-induced insulin secretion (GIIS). N-WASP, which promotes actin polymerization through activation of the actin nucleation factor Arp2/3 complex, was found to be activated by glucose stimulation in insulin-secreting clonal pancreatic β-cells (MIN6-K8 β-cells). Introduction of a dominant-negative mutant of N-WASP, which lacks G-actin and Arp2/3 complex-binding region VCA, into MIN6-K8 β-cells or knockdown of N-WASP suppressed GIIS, especially the second phase. We also found that cofilin, which severs F-actin in its dephosphorylated (active) form, is converted to the phosphorylated (inactive) form by glucose stimulation in MIN6-K8 β-cells, thereby promoting F-actin remodeling. In addition, the dominant-negative mutant of cofilin, which inhibits activation of endogenous cofilin, or knockdown of cofilin reduced the second phase of GIIS. However, the first phase of GIIS occurs in the G-actin predominant state, in which cofilin activity predominates over N-WASP activity. Thus, actin dynamics regulated by the balance of N-WASP and cofilin activities determines the biphasic response of GIIS.

Cofilin oligomer formation occurs in vivo and is regulated by cofilin phosphorylation.

BackgroundADF/cofilin proteins are key regulators of actin dynamics. Their function is inhibited by LIMK-mediated phosphorylation at Ser-3. Previous in vitro studies have shown that dependent on its concentration, cofilin either depolymerizes F-actin (at low cofilin concentrations) or promotes actin polymerization (at high cofilin concentrations).Methodology/Principal FindingsWe found that after in vivo cross-linking with different probes, a cofilin oligomer (65 kDa) could be detected in platelets and endothelial cells. The cofilin oligomer did not contain actin. Notably, ADF that only depolymerizes F-actin was present mainly in monomeric form. Furthermore, we found that formation of the cofilin oligomer is regulated by Ser-3 cofilin phosphorylation. Cofilin but not phosphorylated cofilin was present in the endogenous cofilin oligomer. In vitro, formation of cofilin oligomers was drastically reduced after phosphorylation by LIMK2. In endothelial cells, LIMK-mediated cofilin phosphorylation after thrombin-stimulation of EGFP- or DsRed2-tagged cofilin transfected cells reduced cofilin aggregate formation, whereas inhibition of cofilin phosphorylation after Rho-kinase inhibitor (Y27632) treatment of endothelial cells promoted formation of cofilin aggregates. In platelets, cofilin dephosphorylation after thrombin-stimulation and Y27632 treatment led to an increased formation of the cofilin oligomer.Conclusion/SignificanceBased on our results, we propose that an equilibrium exists between the monomeric and oligomeric forms of cofilin in intact cells that is regulated by cofilin phosphorylation. Cofilin phosphorylation at Ser-3 may induce conformational changes on the protein-protein interacting surface of the cofilin oligomer, thereby preventing and/or disrupting cofilin oligomer formation. Cofilin oligomerization might explain the dual action of cofilin on actin dynamics in vivo.

Both NMDA and non-NMDA receptors mediate glutamate stimulation induced cofilin rod formation in cultured hippocampal neurons

Cofilin is the major actin-depolymerizing factor in the CNS for the regulation of actin dynamics. Neurodegenerative stimuli can induce the formation of cofilin rod, a pathological structure composed of cofilin and actin. The formation of cofilin rod was found to disrupt synapse function and cause neurite loss. The aim of the present study is to study the whole process of cofilin rod formation pattern in cultured hippocampal neurons under excitotoxic stimulation and to explore its underlying pharmacological mechanism. By using live cell imaging of neurons overexpressing EGFP-tagged wild type cofilin, we found a two-phase pattern of rod formation induced by glutamate stimulation. The early phase of rod formation occurred shortly after stimulation (∼0.5h) but quickly dissolved within 2h. The second phase happened within a much longer time window, 8h after stimulation. Immunostaining of endogenous cofilin in neurons also confirmed this glutamate stimulation induced two-phase rod formation pattern. The first phase was co-related with intracellular calcium concentration and pH increase while the second phase was not. These two phases of cofilin rod formation induced by glutamate stimulation was antagonized by both non-NMDA and NMDA receptor antagonist DNQX and AP5, respectively. Our results for the first time demonstrate the dynamic cofilin rod formation pattern under stress stimulation in detail by time lapse imaging. These findings reveal a novel time course of excitotoxicity induced neuronal damage and indicate a potential target of neuropathy treatment of neurodegenerative diseases.

Cofilin cooperates with fascin to disassemble filopodial actin filaments

Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.

Abstract 1087: The expression and activity of actin-regulating protein cofilin are associated with the cell cycle progression

Abstract Cofilin is a member of actin depolymerizing factor (ADF)/cofilin family that can regulates actin dynamics for cancer growth and invasion/metastasis. Several lines of evidence have demonstrated that cofilin is associated with the cell cycle regulation, while the underlying mechanisms remain to be addressed. To better understand the involvement of cofilin in cell cycle progression, we dissected the cofilin expression and activity in different phases of the cell cycle. We synchronized human non-small lung A549 cancer cells in G1, S and G2/M phase by serum starvation, double thymidine blockage and nocodazole treatment, respectively. It was found that the expression of endogenous cofilin was weakest in G1 phase, while it was strongest in S phase and was modest in G2/M phase. This observation was further confirmed by detecting the down-regulation of highly expressed cofilin in S phase-synchronized cells entering mid-late S, G2/M and G1 phase of the cell cycle. Interestingly, it also showed that inactive form of cofilin (ser-3 phosphorylated) weakly expressed in S phase and gradually increased when cells progress through G2/M and G1 phases. These results suggest that cofilin expression and activity are coordinately regulated during the cell cycle progression. It is also consistent with our previous findings that enforced expression of cofilin results in G1 phase arrest but not S and G2/M phases. Moreover, the level of cofilin transcripts was not significantly altered in different phases of cell cycle. On the other hand, pretreatment of the proteasome inhibitor MG132 resulted in increase of cofilin in G1 phase, suggesting that the fluctuation of cofilin during cell cycle progression is associated with the protein stability. In addition, transduction of undegradable Y68F form of mutant cofilin potentially led to increase of the G1 percentage. Taken together, precisely regulation of cofilin expression and activity is important for cell cycle progression. Targeting on cofilin may provide alternative therapeutic strategy for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1087.

Unbalancing the Phosphatidylinositol-4,5-bisphosphate–Cofilin Interaction Impairs Cell Steering

Cofilin is a key player in actin dynamics during cell migration. Its activity is regulated by (de)phosphorylation, pH, and binding to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P(2)]. Here, we here use a human cofilin-1 (D122K) mutant with increased binding affinity for PI(4,5)P(2) and slower release from the plasma membrane to study the role of the PI(4,5)P(2)-cofilin interaction in migrating cells. In fibroblasts in a background of endogenous cofilin, D122K cofilin expression negatively affects cell turning frequency. In carcinoma cells with down-regulated endogenous cofilin, D122K cofilin neither rescues the drastic morphological defects nor restores the effects in cell turning capacity, unlike what has been reported for wild-type cofilin. In cofilin knockdown cells, D122K cofilin expression promotes outgrowth of an existing lamellipod in response to epidermal growth factor (EGF) but does not result in initiation of new lamellipodia. This indicates that, next to phospho- and pH regulation, the normal release kinetics of cofilin from PI(4,5)P(2) is crucial as a local activation switch for lamellipodia initiation and as a signal for migrating cells to change direction in response to external stimuli. Our results demonstrate that the PI(4,5)P(2) regulatory mechanism, that is governed by EGF-dependent phospholipase C activation, is a determinant for the spatial and temporal control of cofilin activation required for lamellipodia initiation.

Oxidant-induced apoptosis is mediated by oxidation of the actin-regulatory protein cofilin

Physiological oxidants that are generated by activated phagocytes comprise the main source of oxidative stress during inflammation. Oxidants such as taurine chloramine (TnCl) and hydrogen peroxide (H(2)O(2)) can damage proteins and induce apoptosis, but the role of specific protein oxidation in this process has not been defined. We found that the actin-binding protein cofilin is a key target of oxidation. When oxidation of this single regulatory protein is prevented, oxidant-induced apoptosis is inhibited. Oxidation of cofilin causes it to lose its affinity for actin and to translocate to the mitochondria, where it induces swelling and cytochrome c release by mediating opening of the permeability transition pore (PTP). This occurs independently of Bax activation and requires both oxidation of cofilin Cys residues and dephosphorylation at Ser 3. Knockdown of endogenous cofilin using targeted siRNA inhibits oxidant-induced apoptosis, which is restored by re-expression of wild-type cofilin but not by cofilin containing Cys to Ala mutations. Exposure of cofilin to TnCl results in intramolecular disulphide bonding and oxidation of Met residues to Met sulphoxide, but only Cys oxidation causes cofilin to induce mitochondrial damage.

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. We demonstrate the ATP-sensitive interaction of the cofilin phosphatase chronophin (CIN) with the chaperone hsp90 to form a biosensor that mediates cofilin/actin rod formation. Our 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.

Phosphorylated Cofilin Not Inactive After All

Cofilin is an actin-depolymerizing factor, and this activity is inhibited when the protein is phosphorylated by LIM-kinase or TES-kinase. In an attempt to identify the mechanism by which the muscarinic acetylcholine receptor activated phospholipase D (PLD) downstream of the Rho effector Rho-kinase, Han et al . found that in the fibroblast cell line HEK293 and the neuroblastoma cell line N1E-115, PLD1 was not directly phosphorylated by Rho-kinase, but instead carbachol-stimulated PLD1 activation was inhibited by expression of a kinase-deficient LIM-kinase 1, which is a target for Rho-kinase. However, PLD1 was not a substrate for LIM-kinase 1 either, suggesting another step in the path from the receptor to PLD1. As cofilin is a known substrate for LIM-kinase 1, the authors overexpressed cofilin or a nonphosphorylatable mutant S3A cofilin and showed that carbachol-mediated stimulation of PLD1 was enhanced in the presence of excess wild-type cofilin and inhibited by S3A cofilin. RNAi-mediated depletion of cofilin also reduced carbachol-mediated activation of PLD1, consistent with a role for endogenous cofilin. Expression of a phosphorylation-mimic mutant of cofilin S3D also enhanced carbachol-stimulated PLD1 activation, whereas overexpression of the cofilin phosphatase slingshot inhibited PLD activation. Pull-down assays, as well as colocalization assays and coimmunoprecipitation assays with transfected cells, showed a direct interaction between PLD1 and phosphorylated, but not unphosphorylated, cofilin. If the interaction between cofilin and PLD1 was disrupted by expressing a fragment of PLD1 (F-3 fragment) responsible for mediating the interaction, then carbachol-mediated PLD1 activation was inhibited. Carbachol stimulation of stress fiber formation was also inhibited by the F-3 fragment. Carbachol stimulated the transient phosphorylation of cofilin and increased the interaction between the cofilin and PLD1 in coimmunoprecipitation assays. Phosphorylated cofilin stimulated PLD1 activity in vitro. Thus, phosphorylated cofilin is not an inactive entity but rather interacts with partners other than actin, such as PLD1. L. Han, M. B. Stope, M. López de Jesús, P. A. Oude Weernink, M. Urban, T. Wieland, D. Rosskopf, K. Mizuno, K. H. Jakobs, M. Schmidt, Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin. EMBO J. 26 , 4189-4202 (2007). [PubMed]

LIM Kinase and Slingshot Are Critical for Neurite Extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.

Spatial and Temporal Control of Cofilin Activity Is Required for Directional Sensing during Chemotaxis

Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.

Suppression of cofilin phosphorylation in insulin-stimulated ruffling membrane formation in KB cells.

Various cellular events such as cell motility and division are directed by the actin cytoskeleton under the control of its regulatory system. Cofilin is a low molecular weight actin-modulating protein that severs and depolymerizes F-actin and is shown to enhance actin filament dynamics. The activity of cofilin is negatively regulated by phosphorylation at Ser-3. In human epidermoid carcinoma KB cells, insulin treatment induces characteristic ruffling membranes, and it was reported that LIMK1, a cofilin kinase, was activated in these cells treated with insulin. Since cofilin is a key protein responsible for establishing the rapid turnover of actin filaments, it appears to be contradictory that cofilin is phosphorylated (inactivated) by a stimulus that is known to induce the highly dynamic actin structure, ruffling membranes. Therefore, we examined the phosphorylation state of endogenous cofilin in KB cells treated with insulin. The dephosphorylated form of cofilin increased with insulin treatment, as analyzed by nonequilibrium pH gradient gel electrophoresis (NEpHGE)-immunoblotting. Cell labeling with (32)P orthophosphate indicated that cofilin was being continuously phosphorylated and dephosphorylated, and that the apparent insulin-induced dephosphorylation was due to suppression of continuous phosphorylation and not to enhanced dephosphorylation. Further, we examined the localization of the phosphorylated form of cofilin using phospho-specific antibody raised against phosphorylated cofilin. Surprisingly, phosphorylated cofilin was concentrated in the ruffling membranes induced by insulin. These results suggest that the examination of the kinetics and spatial regulation of phosphorylation is critical for the elucidation of the role of cofilin and upstream kinases in actin reorganization.

Site-directed mutagenesis of the phosphorylation site of cofilin: Its role in cofilin-actin interaction and cytoplasmic localization

It has been demonstrated that the activity of ADF and cofilin, which constitute a functionally related protein family, is markedly altered by phosphorylation, and that the phosphorylation site is Ser 3 in their amino acid sequences [Agnew et al., 1995: J. Biol. Chem. 270:17582-17587; Moriyama et al., 1996: Genes Cells 1:73-86]. In order to clarify the function of the phosphorylated and unphosphorylated forms of cofilin in living cells especially in the process of cytokinesis, we generated analogs of the unphosphorylated form (A3-cofilin) and phosphorylated form (D3-cofilin) by converting the phosphorylation site (Ser 3) of cofilin to Ala and Asp, respectively. The mutated proteins were produced in an Escherichia coli expression system, and conjugated with fluorescent dyes. In in vitro functional assay, labeled A3-cofilin retained the authentic ability to bind to and sever F-actin, while labeled D3-cofilin failed to interact with actin. They were then injected into living cells to examine their cellular distribution. They exhibited distinct localization patterns in the cytoplasm; A3-cofilin was highly concentrated at the membrane ruffles and cleavage furrow, where endogenous cofilin is also known to be enriched. In contrast, D3-cofilin showed only diffuse distribution both in the cytoplasm and nucleus. These results suggest that the subcellular distribution of cofilin as well as its interacting with actin in vivo is regulated by its phosphorylation and dephosphorylation.

Effects of cofilin on actin filamentous structures in cultured muscle cells: intracellular regulation of cofilin action

The previous investigation (Abe et al. (1989) J. Biochem. 106, 696-702) suggested that cofilin is deeply involved in the regulation of actin assembly in developing skeletal muscle. In this study, to examine further the function of cofilin in living myogenic cells in culture, recombinant cofilin having extra Cys residues at the N terminus was produced in Escherichia coli and was labeled with tetramethylrhodamine-iodoacetamide (IATMR). When the cofilin labeled with IATMR (IATMR-cofilin) was introduced into myogenic cells, actin filaments in the cytoplasm or nascent myofibrils were promptly disrupted, and many cytoplasmic rods which contained both IATMR-cofilin and actin were generated. Sarcomeric myofibrillar structures were not disrupted but tropomyosin was dissociated from the structures by the exogenous cofilin, and the IATMR-cofilin became localized in I-band regions. 24 hours after the injection, however, the actin-cofilin rods disappeared completely and the IATMR-cofilin became diffused in the cytoplasm as endogenous cofilin. Concomitantly, actin filaments were recovered and tropomyosin was re-associated with sarcomeric I-bands. At this point, the IATMR-cofilin in the cells still retained the functional activity to form intranuclear actin-cofilin rods in response to stimulation by DMSO just as endogenous cofilin. FITC-labeled actin introduced into myogenic cells at first failed to assemble into filamentous structures in the presence of the exogenous cofilin, but was gradually incorporated into myofibrils with time. The drastic effects of the exogenous cofilin on actin assembly were suppressed by phosphatidylinositol 4,5-bisphosphate (PIP2). These results indicate that the exogenous cofilin is active and alters actin dynamics remarkably in muscle cells, but its activity in the cytoplasm gradually becomes regulated by the action of some factors including PIP2-binding.