It is known that retinoid receptor function is attenuated during T cell activation, a phenomenon that involves actin remodeling, suggesting that actin modification may play a role in such inhibition. Here we have investigated the role of actin dynamics and the effect of actin cytoskeleton modifying agents on retinoid receptor-mediated transactivation.
Agents that disturb the F-actin assembly or disassembly attenuated receptor-mediated transcription indicating that actin cytoskeletal homeostasis is important for retinoid receptor function. Overexpression or siRNA-induced knockdown of cofilin-1 (CFL1), a key regulator of F-actin assembly, induced the loss of receptor function. In addition, expression of either constitutively active or inactive/dominant-negative mutants of CFL1or CFL1 kinase LIMK1 induced loss of receptor function suggesting a critical role of the LIMK1-mediated CFL1 pathway in receptor-dependent transcription. Further evidence of the role of LMK1/CFL1-mediated actin dynamics, was provided by studying the effect of Nef, an actin modifying HIV-1 protein, on receptor function. Expression of Nef induced phosphorylation of CFL1 at serine 3 and LIMK1 at threonine 508, inhibited retinoid-receptor mediated reporter activity, and the expression of a number of genes that contain retinoid receptor binding sites in their promoters. The results suggest that the Nef-mediated inhibition of receptor function encompasses deregulation of actin filament dynamics by LIMK1 activation and phosphorylation of CFL1.
We have identified a critical role of LIMK1-mediated CFL1 pathway and actin dynamics in modulating retinoid receptor mediated function and shown that LIMK1-mediated phosphocycling of CFL1 plays a crucial role in maintaining actin homeostasis and receptor activity. We suggest that T cell activation-induced repression of nuclear receptor-dependent transactivation is in part through the modification of actin dynamics.
ADF/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).
We 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.
Based 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.
Chemokine binding to cognate receptors induces actin dynamics that are a major driving force for T cell migration and chemotactic motility. HIV-1 binding to the chemokine coreceptor CXCR4 initiates chemotactic signaling, mimicking chemokine-induced actin dynamics to facilitate infection processes such as entry, early DNA synthesis, and nuclear migration. Recently, we identified that HIV-triggered early actin polymerization is mediated through the Rac1-PAK1/2-LIMK1-cofilin pathway. Inhibition of LIMK1 (LIM domain kinase 1), a kinase phosphorylating cofilin, through shRNA knockdown decreases actin polymerization and T cell chemotaxis toward SDF-1. The LIMK1 knockdown T cells also supported lower viral entry, DNA synthesis and nuclear migration, suggesting a critical role of LIMK1-mediated actin dynamics in the initiation of HIV-1 infection. Surprisingly, LIMK1 knockdown in CEM-SS T cells did not lead to an overall change in the ratio of phospho-cofilin to total cofilin although there was a measurable decrease in the amount of actin filaments in cells. The decrease in filamentous actin in LIMK1 knockdown cells was found to mainly occur in polarized cap region rich in F-actin. These results suggest that LIMK1 may be involved in spontaneous actin polarization in transformed T cells. The inhibition of T cell chemotaxis by LIMK1 knockdown likely result from inhibition of localized LIMK1 activation and cofilin phosphorylation that are required for polarized actin polymerization for directional cell migration. The inhibition of HIV-1 infection by LIMK1 knockdown may also result from the decrease of actin-rich membrane protrusions that may be preferred viral entry sites in T cells.
LIMK1; cofilin; chemotaxis; SDF-1; CXCR4; HIV-1; CD4 T cells; Rac1; Pak1; Pak2
Stromal cell-derived factor 1 α (SDF-1α), 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. We investigated the role of cofilin phosphorylation by LIMK1 in SDF-1α-induced chemotaxis of T lymphocytes. SDF-1α significantly induced the activation of LIMK1 in Jurkat human leukemic T cells and peripheral blood lymphocytes. SDF-1α also induced cofilin phosphorylation, actin reorganization, and activation of small GTPases, Rho, Rac, and Cdc42, in Jurkat cells. Pretreatment with pertussis toxin inhibited SDF-1α-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, blocked SDF-1α-induced activation of LIMK1, which means that SDF-1α-induced LIMK1 activation is mediated by Rac but not by Rho or Cdc42. We used a cell-permeable peptide (S3 peptide) that contains the phosphorylation site (Ser-3) of cofilin to inhibit the cellular function of LIMK1. S3 peptide inhibited the kinase activity of LIMK1 in vitro. Treatment of Jurkat cells with S3 peptide inhibited the SDF-1α-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-1α-induced chemotactic response of T lymphocytes.
We previously provided evidence that cadherin-6B induces de-epithelialization of the neural crest prior to delamination and is required for the overall epithelial mesenchymal transition (EMT). Furthermore, de-epithelialization induced by cadherin-6B was found to be mediated by BMP receptor signaling independent of BMP. We now find that de-epithelialization is mediated by non-canonical BMP signaling through the BMP type II receptor (BMPRII) and not by canonical Smad dependent signaling through BMP Type I receptor. The LIM kinase/cofilin pathway mediates non-canonical BMPRII induced de-epithelialization, in response to either cadherin-6B or BMP. LIMK1 induces de-epithelialization in the neural tube and dominant negative LIMK1 decreases de-epithelialization induced by either cadherin-6B or BMP. Cofilin is the major known LIMK1 target and a S3A phosphorylation deficient mutated cofilin inhibits de-epithelialization induced by cadherin-6B as well as LIMK1. Importantly, LIMK1 as well as cadherin-6B can trigger ectopic delamination when co-expressed with the competence factor SOX9, showing that this cadherin-6B stimulated signaling pathway can mediate the full EMT in the appropriate context. These findings suggest that the de-epithelialization step of the neural crest EMT by cadherin-6B/BMPRII involves regulation of actin dynamics via LIMK/cofilin.
EMT; Cadherin-6B; BMP; Non-canonical; LIMK; Cofilin; Neural Crest
Cofilin is a major regulator of actin dynamics involved in the regulation of cell spreading and migration through its actin depolymerizing and severing activities. V-Src is an activated Src tyrosine kinase and a potent oncogene known to phosphorylate a variety of cellular proteins in cell transformation process including altered cell adhesion, spreading and migration. Recently, it has been suggested that cofilin is a potential substrate of v-Src (Rush et al., 2005). Here, we show direct tyrosine phosphorylation of cofilin by v-Src and identify Y68 as the major phosphorylation site. Cofilin phosphorylation at Y68 did not change its activity per se, but induced increased ubiquitination of cofilin and its degradation through the proteosome pathway. Furthermore, the negative effect of cofilin on cellular F-actin contents was inhibited by co-expression of v-Src, whereas that of cofilin mutant Y68F (Y68 mutated to F) was not affected, suggesting that v-Src-mediated cofilin phosphorylation at Y68 is required for degradation of cofilin in vivo. Lastly, inhibition of cell spreading by v-Src was rescued partially by co-expression of cofilin, and to a greater extent by the Y68F mutant which is not subjected to v-Src induced degradation through phosphorylation, suggesting that v-Src mediated changes in cell spreading is, at least in part, through inhibiting the function of cofilin via phosphorylating it at Y68. Together, these results suggest a novel mechanism by which cofilin is regulated by v-Src through tyrosine phosphorylation at Y68 that triggers degradation of cofilin via ubiquitination-proteosome pathway and consequently inhibits cofilin activity in reducing cellular F-actin contents and cell spreading.
cofilin; protein phosphorylation; protein degradation; cell spreading
Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content.
Dendritic spines are the postsynaptic sites of most excitatory synapses in the brain and are highly enriched in polymerized F-actin, which drives the formation and maintenance of mature dendritic spines and synapses. We propose that suppressing the activity of the actin-severing protein cofilin plays an important role in the stabilization of mature dendritic spines, and is accomplished through an EphB receptor–focal adhesion kinase (FAK) pathway. Our studies revealed that Cre-mediated knock-out of loxP-flanked fak prompted the reversion of mature dendritic spines to an immature filopodial-like phenotype in primary hippocampal cultures. The effects of FAK depletion on dendritic spine number, length, and morphology were rescued by the overexpression of the constitutively active FAKY397E, but not FAKY397F, indicating the significance of FAK activation by phosphorylation on tyrosine 397. Our studies demonstrate that FAK acts downstream of EphB receptors in hippocampal neurons and EphB2–FAK signaling controls the stability of mature dendritic spines by promoting cofilin phosphorylation, thereby inhibiting cofilin activity. While constitutively active nonphosphorylatable cofilinS3A induced an immature spine profile, phosphomimetic cofilinS3D restored mature spine morphology in neurons with disrupted EphB activity or lacking FAK. Further, we found that EphB-mediated regulation of cofilin activity at least partially depends on the activation of Rho-associated kinase (ROCK) and LIMK-1. These findings indicate that EphB2-mediated dendritic spine stabilization relies, in part, on the ability of FAK to activate the RhoA–ROCK–LIMK-1 pathway, which functions to suppress cofilin activity and inhibit cofilin-mediated dendritic spine remodeling.
Testicular protein kinase 1 (TESK1) is a serine/threonine kinase
with a structure composed of a kinase domain related to those of
LIM-kinases and a unique C-terminal proline-rich domain. Like
LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both
in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated
the formation of actin stress fibers and focal adhesions. In contrast
to LIM-kinases, the kinase activity of TESK1 was not enhanced by
Rho-associated kinase (ROCK) or p21-activated kinase, indicating that
TESK1 is not their downstream effector. Both the kinase activity of
TESK1 and the level of cofilin phosphorylation increased by plating
cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited
LIM-kinase-induced cofilin phosphorylation but did not affect
fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa
cells. Expression of a kinase-negative TESK1 suppressed cofilin
phosphorylation and formation of stress fibers and focal adhesions
induced in cells plated on fibronectin. These results suggest that
TESK1 functions downstream of integrins and plays a key role in
integrin-mediated actin reorganization, presumably through
phosphorylating and inactivating cofilin. We propose that TESK1 and
LIM-kinases commonly phosphorylate cofilin but are regulated in
different ways and play distinct roles in actin reorganization in
The Drosophila neuromuscular junction (NMJ) is capable of rapidly budding new presynaptic varicosities over the course of minutes in response to elevated neuronal activity. Using live imaging of synaptic growth, we characterized this dynamic process and demonstrated that rapid bouton budding requires retrograde bone morphogenic protein (BMP) signaling and local alteration in the presynaptic actin cytoskeleton. BMP acts during development to provide competence for rapid synaptic growth by regulating the levels of the Rho-type guanine nucleotide exchange factor Trio, a transcriptional output of BMP–Smad signaling. In a parallel pathway, we find that the BMP type II receptor Wit signals through the effector protein LIM domain kinase 1 (Limk) to regulate bouton budding. Limk interfaces with structural plasticity by controlling the activity of the actin depolymerizing protein Cofilin. Expression of constitutively active or inactive Cofilin in motor neurons demonstrates that increased Cofilin activity promotes rapid bouton formation in response to elevated synaptic activity. Correspondingly, the overexpression of Limk, which inhibits Cofilin, inhibits bouton budding. Live imaging of the presynaptic F-actin cytoskeleton reveals that activity-dependent bouton addition is accompanied by the formation of new F-actin puncta at sites of synaptic growth. Pharmacological disruption of actin turnover inhibits bouton budding, indicating that local changes in the actin cytoskeleton at pre-existing boutons precede new budding events. We propose that developmental BMP signaling potentiates NMJs for rapid activity-dependent structural plasticity that is achieved by muscle release of retrograde signals that regulate local presynaptic actin cytoskeletal dynamics.
actin; BMP; Drosophila; neuromuscular junction; synapse formation; synaptic plasticity
The ovarian hormone estrogen increases the axospinous synapse density in the hippocampal CA1 region of young female rats but fails to do so in aged rats. This estrogen-mediated alteration of spine synapse structures suggests the coincident requirement for the structural reorganization of the underlying actin cytoskeleton network. Actin reorganization is known to require the deactivation of Cofilin, an actin depolymerization factor. Cofilin is deactivated by LIM Kinase (LIMK), and LIMK activity is modulated by the phosphorylation of specific residues. We have previously demonstrated that estrogen is able to increase phosphorylated LIMK (pLIMK) immunoreactivity (IR) in the hippocampus in vivo and that this estrogen-stimulated pLIMK-IR is decreased in the aged brain. Because Cofilin phosphorylation allows for actin filament elongation and spine synapse growth, we sought to determine if estrogen acts through Cofilin and if such estrogen action requires the observed LIMK activity. Using both hippocampal neurons and the NG108-15 neuroblastoma cell line, we demonstrate here that estrogen stimulates the phosphorylation of Cofilin in vitro. Furthermore, this estrogen action on Cofilin requires LIMK. Lastly, while initiating the phosphorylation of LIMK and Cofilin, estrogen can also stimulate the formation of filopodial extensions, an early step in the formation of nascent spines, demonstrating that estrogen can alter the actin-dependent neuronal morphology. This linkage of estrogen communication to Cofilin via LIMK provides the functionality to the age-sensitive pLIMK-IR that we have observed in vivo.
Herpes simplex virus type 1 (HSV-1) establishes latency in neurons and can cause severe disseminated infection with neurological impairment and high mortality. This neurodegeneration is thought to be tightly associated with virus-induced cytoskeleton disruption. Currently, the regulation pattern of the actin cytoskeleton and the involved molecular mechanisms during HSV-1 entry into neurons remain unclear. Here, we demonstrate that the entry of HSV-1 into neuronal cells induces biphasic remodeling of the actin cytoskeleton and an initial inactivation followed by the subsequent activation of cofilin, a member of the actin depolymerizing factor family that is critical for actin reorganization. The disruption of F-actin dynamics or the modulation of cofilin activity by mutation, knockdown, or overexpression affects HSV-1 entry efficacy and virus-mediated cell ruffle formation. Binding of the HSV-1 envelope initiates the epidermal growth factor receptor (EGFR)-phosphatidylinositide 3-kinase (PI3K) signaling pathway, which leads to virus-induced early cofilin phosphorylation and F-actin polymerization. Moreover, the extracellular signal-regulated kinase (ERK) kinase and Rho-associated, coiled-coil-containing protein kinase 1 (ROCK) are recruited as downstream mediators of the HSV-1-induced cofilin inactivation pathway. Inhibitors specific for those kinases significantly reduce the virus infectivity without affecting virus binding to the target cells. Additionally, lipid rafts are clustered to promote EGFR-associated signaling cascade transduction. We propose that HSV-1 hijacks cofilin to initiate infection. These results could promote a better understanding of the pathogenesis of HSV-1-induced neurological diseases.
The actin cytoskeleton is involved in many crucial cellular processes and acts as an obstacle to pathogen entry into host cells. Because HSV-1 establishes lifelong latency in neurons and because neuronal cytoskeletal disruption is thought to be the main cause of HSV-1-induced neurodegeneration, understanding the F-actin remodeling pattern by HSV-1 infection and the molecular interactions that facilitate HSV-1 entry into neurons is important. In this study, we showed that HSV-1 infection induces the rearrangement of the cytoskeleton as well as the initial inactivation and subsequent activation of cofilin. Then, we determined that activation of the EGFR-PI3K-Erk1/2 signaling pathway inactivates cofilin and promotes F-actin polymerization. We postulate that by regulating actin cytoskeleton dynamics, cofilin biphasic activation could represent the specific cellular machinery usurped by pathogen infection, and these results will greatly contribute to the understanding of HSV-1-induced early and complex changes in host cells that are closely linked to HSV-1 pathogenesis.
Cofilin is an actin-binding protein that depolymerizes and/or severs actin filaments. This dual function of cofilin makes it one of the major regulators of actin dynamics important for T-cell activation and migration. The activity of cofilin is spatio-temporally regulated. Its main control mechanisms comprise a molecular toolbox of phospho-, phospholipid, and redox regulation. Phosphorylated cofilin is inactive and represents the dominant cofilin fraction in the cytoplasm of resting human T cells. A fraction of dephosphorylated cofilin is kept inactive at the plasma membrane by binding to phosphatidylinositol 4,5-bisphosphate. Costimulation via the T-cell receptor/CD3 complex (signal 1) together with accessory receptors (signal 2) or triggering through the chemokine SDF1α (stromal cell-derived factor 1α) induce Ras-dependent dephosphorylation of cofilin, which is important for immune synapse formation, T-cell activation, and T-cell migration. Recently, it became evident that cofilin is also highly sensitive for microenvironmental changes, particularly for alterations in the redox milieu. Cofilin is inactivated by oxidation, provoking T-cell hyporesponsiveness or necrotic-like programmed cell death. In contrast, in a reducing environment, even phosphatidylinositol 4,5-bisphosphate -bound cofilin becomes active, leading to actin dynamics in the vicinity of the plasma membrane. In addition to the well-established three signals for T-cell activation, this microenvironmental control of cofilin delivers a modulating signal for T-cell-dependent immune reactions. This fourth modulating signal highly impacts both initial T-cell activation and the effector phase of T-cell-mediated immune responses.
costimulation; T-cell activation; immune synapse; microenvironment; redox; actin cytoskeleton
Developmental cognitive deficits including X-linked mental retardation (XLMR) can be caused by mutations in P21-activated kinase 3 (PAK3) that disrupt actin dynamics in dendritic spines. Neurodegenerative diseases such as Alzheimer disease (AD), where both PAK1 and PAK3 are dysregulated, may share final common pathways with XLMR. Independent of familial mutation, cognitive deficits emerging with aging, notably AD, begin after decades of normal function. This prolonged prodromal period involves the buildup of amyloid-β (Aβ) extracellular plaques and intraneuronal neurofibrillary tangles (NFT). Subsequently region dependent deficits in synapses, dendritic spines and cognition coincide with dysregulation in PAK1 and PAK. Specifically proximal to decline, cytoplasmic levels of actin-regulating Rho GTPase and PAK1 kinase are decreased in moderate to severe AD, while aberrant activation and translocation of PAK1 appears around the onset of cognitive deficits. Downstream to PAK1, LIM kinase inactivates cofilin, contributing to cofilin pathology, while the activation of Rho-dependent kinase ROCK increases Aβ production. Aβ activation of fyn disrupts neuronal PAK1 and ROCK-mediated signaling, resulting in synaptic deficits. Reductions in PAK1 by the anti-amyloid compound curcumin suppress synaptotoxicity. Similarly other neurological disorders, including Huntington disease (HD) show dysregulation of PAKs. PAK1 modulates mutant huntingtin toxicity by enhancing huntingtin aggregation, and inhibition of PAK activity protects HD as well as fragile X syndrome (FXS) symptoms. Since PAK plays critical roles in learning and memory and is disrupted in many cognitive disorders, targeting PAK signaling in AD, HD and XLMR may be a novel common therapeutic target for AD, HD and XLMR.
Alzheimer disease; curcumin; PAK; ROCK; signaling pathways; synapses
The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.
Bone morphogenic proteins (BMPs) are involved in axon pathfinding, but how they guide growth cones remains elusive. In this study, we report 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, we show 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.
A current model posits that cofilin-dependent actin severing negatively impacts dendritic spine volume. Studies suggested that increased cofilin activity underlies activity-dependent spine shrinkage, and that reduced cofilin activity induces activity-dependent spine growth. We suggest instead that both types of structural plasticity correlate with decreased cofilin activity. However, the mechanism of inhibition determines the outcome for spine morphology. RNAi in rat hippocampal cultures demonstrates that cofilin is essential for normal spine maintenance. Cofilin-F-actin binding and filament barbed-end production decrease during the early phase of activity-dependent spine shrinkage; cofilin concentration also decreases. Inhibition of the cathepsin B/L family of proteases prevents both cofilin loss and spine shrinkage. Conversely, during activity-dependent spine growth, LIM kinase stimulates cofilin phosphorylation, which activates phospholipase D-1 to promote actin polymerization. These results implicate novel molecular mechanisms and prompt a revision of the current model for how cofilin functions in activity-dependent structural plasticity.
The functions of the actin cytoskeleton in post-Golgi trafficking are still poorly understood. Here, we report the role of LIM Kinase 1 (LIMK1) and its substrate cofilin in the trafficking of apical and basolateral proteins in Madin-Darby canine kidney cells. Our data indicate that LIMK1 and cofilin organize a specialized population of actin filaments at the Golgi complex that is selectively required for the emergence of an apical cargo route to the plasma membrane (PM). Quantitative pulse-chase live imaging experiments showed that overexpression of kinase-dead LIMK1 (LIMK1-KD), or of LIMK1 small interfering RNA, or of an activated cofilin mutant (cofilin S3A), selectively slowed down the exit from the trans-Golgi network (TGN) of the apical PM marker p75-green fluorescent protein (GFP) but did not interfere with the apical PM marker glycosyl phosphatidylinositol-YFP or the basolateral PM marker neural cell adhesion molecule-GFP. High-resolution live imaging experiments of carrier formation and release by the TGN and analysis of peri-Golgi actin dynamics using photoactivatable GFP suggest a scenario in which TGN-localized LIMK1-cofilin regulate a population of actin filaments required for dynamin-syndapin-cortactin–dependent generation and/or fission of precursors to p75 transporters.
Dynamic remodeling of the actin cytoskeleton is required for cell spreading, motility, and migration and can be regulated by tyrosine kinase activity. Phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein annexin A2 (AnxA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering. Expression of the phospho-mimicking mutant Y23E-AnxA2 itself was sufficient to induce actin reorganization and cell scattering in MDCK cells. While Y23E-AnxA2, but not Y23A-AnxA2, enhanced Src- or hepatocyte growth factor (HGF)-induced cell scattering, short hairpin RNA-mediated knockdown of AnxA2 inhibited both v-Src- and HGF-induced cell scattering. Three-dimensional branching morphogenesis was induced in wild-type-AnxA2-expressing cells only in the presence of HGF, while Y23E-AnxA2 induced HGF-independent branching morphogenesis. Knockdown of AnxA2 prevented lumen formation during cystogenesis. The Y23E-AnxA2-induced scattering was associated with dephosphorylation/activation of the actin-severing protein cofilin. Likewise, inactive S3E-cofilin and constitutively active LIM kinase, a direct upstream kinase of cofilin, inhibited Y23E-AnxA2-induced scattering. Together, our studies indicate an essential role for AnxA2 phosphorylation in regulating cofilin-dependent actin cytoskeletal dynamics in the context of cell scattering and branching morphogenesis.
Myelination is a complex process requiring coordination of directional motility and an increase in glial cell size to generate a multilamellar myelin sheath. Regulation of actin dynamics during myelination is poorly understood. However, it is known that myelin thickness is related to the abundance of neuregulin-1 (NRG1) expressed on the axon surface. Here we identify cofilin1, an actin depolymerizing and severing protein, as a downstream target of NRG1 signaling in rat Schwann cells (SCs). In isolated SCs, NRG1 promotes dephosphorylation of cofilin1 and its upstream regulators, LIM kinase (LIMK) and Slingshot-1 phosphatase (SSH1), leading to cofilin1 activation and recruitment to the leading edge of the plasma membrane. These changes are associated with rapid membrane expansion yielding a 35–50% increase in SC size within 30 min. Cofilin1-deficient SCs increase phosphorylation of ErbB2, ERK, focal adhesion kinase, and paxillin in response to NRG1, but fail to increase in size possibly due to stabilization of unusually long focal adhesions. Cofilin1-deficient SCs cocultured with sensory neurons do not myelinate. Ultrastructural analysis reveals that they unsuccessfully segregate or engage axons and form only patchy basal lamina. After 48 h of coculturing with neurons, cofilin1-deficient SCs do not align or elongate on axons and often form adhesions with the underlying substrate. This study identifies cofilin1 and its upstream regulators, LIMK and SSH1, as end targets of a NRG1 signaling pathway and demonstrates that cofilin1 is necessary for dynamic changes in the cytoskeleton needed for axon engagement and myelination by SCs.
Vascular endothelial cells and their actin microfilaments align in the direction of fluid shear stress (FSS) in vitro and in vivo. To determine whether cofilin, an actin severing protein, is required in this process, the levels of phospho-cofilin (serine-3) were evaluated in cells exposed to FSS. Phospho-cofilin levels decreased in the cytoplasm and increased in the nucleus during FSS exposure. This was accompanied by increased nuclear staining for activated LIMK, a cofilin kinase. Blocking stress kinases JNK and p38, known to play roles in actin realignment during FSS, decreased cofilin phosphorylation under static conditions, and JNK inhibition also resulted in decreased phospho-cofilin during FSS exposure. Inhibition of dynamic changes in cofilin phosphorylation through cofilin mutants decreased correct actin realignment. The mutants also decreased barrier integrity as did inhibition of the stress kinases. These results identify the importance of cofilin in the process of actin alignment and the requirement for actin realignment in endothelial barrier integrity during FSS.
Vascular endothelial cells; Fluid shear stress; Actin realignment; Cofilin; Barrier integrity
Neurofibromatosis Type 2 (NF2) is caused by mutations in the neurofibromatosis 2 (NF2) gene that encodes a tumor suppressor protein called merlin. NF2 is characterized by formation of multiple schwannomas, meningiomas and ependymomas. Merlin loss of function is associated with increased activity of Rac and p21-activated kinases (PAK) and deregulation of cytoskeletal organization. LIM domain kinases (LIMK1 and 2) are substrate for Cdc42/Rac-PAK, and modulate actin dynamics by phosphorylating cofilin at serine-3. This modification inactivates cofilin’s actin severing and depolymerizing activity. LIMKs also translocate into the nucleus and regulate cell cycle progression. Significantly, LIMKs are overexpressed in several tumor types, including skin, breast, lung, liver and prostate. Here we report that mouse Schwann cells (MSCs) in which merlin function is lost as a result of Nf2 exon2 deletion (Nf2ΔEx2) exhibited increased levels of LIMK1, LIMK2, and active phospho-Thr508/505-LIMK1/2, as well as phospho-Ser3-cofilin, compared to wild-type normal MSCs. Similarly, levels of LIMK1 and 2 total protein and active phosphorylated forms were elevated in human vestibular schwannomas compared to normal human Schwann cells (SCs). Reintroduction of wild-type NF2 into Nf2ΔEx2 MSC reduced LIMK1 and LIMK2 levels. We show that pharmacological inhibition of LIMK with BMS-5, decreased the viability of Nf2ΔEx2 MSCs in a dose-dependent manner, but did not affect viability of control MSCs. Similarly, LIMK knockdown decreased viability of Nf2ΔEx2 MSCs. The decreased viability of Nf2ΔEx2 MSCs was not due to caspase-dependent or -independent apoptosis, but rather, to inhibition of cell cycle progression as evidenced by accumulation of cells in G2/M phase. Inhibition of LIMKs arrest cells in early mitosis by decreasing Aurora A activation. Our results suggest that LIMKs are potential drug targets for NF2 and tumors associated with merlin deficiency.
LIMK; cell proliferation; Neurofibromatosis; schwannomas; cytoskeleton dynamics; cell cycle
Cofilin is an essential actin filament severing protein that functions in the dynamic remodeling of the actin cytoskeleton. Filament severing activity is most efficient at sub-stoichiometric cofilin binding densities (i.e. <1 cofilin per actin filament subunit), and peaks when the number density of boundaries (i.e. junctions) between bare and cofilin-decorated segments is maximal. A model in which local topological and mechanical discontinuities lead to preferential fragmentation at boundaries accounts for available experimental data, including direct visualization of cofilin and actin during real-time severing events. The boundary-severing model predicts that ligands (e.g. other actin-binding proteins) that compete with cofilin for actin filament binding and modulate cofilin occupancy on filaments will alter the bare-decorated segment boundary density, and thus, the filament severing activity of cofilin. Here, we directly test this model prediction by evaluating the effects of phalloidin and myosin, two ligands that compete with cofilin for filament binding, on the actin filament binding and severing activities of cofilin. Our experiments demonstrate that competitive displacement of cofilin lowers cofilin occupancy and promotes severing when initial cofilin occupancy is high (i.e. >50%). Even in the presence of competitive ligands, maximum severing activity occurs when cofilin-decorated boundary density is highest, consistent with preferential fragmentation at boundaries. We propose a general “severodyne” framework for the modulation of cofilin-mediated actin filament severing by small molecule or actin-binding protein ligands that compete with cofilin for actin filament binding.
actin binding protein; competition; cytoskeleton; myosin; phalloidin
Postsynaptic receptor localization is crucial for synapse development and function, but the underlying cytoskeletal mechanisms remain elusive. Using Xenopus neuromuscular junctions as a model, we here report that actin depolymerizing factor (ADF)/cofilin regulates actin-dependent vesicular trafficking of acetylcholine receptors (AChRs) to the postsynaptic membrane. We found that active ADF/cofilin was concentrated in small puncta adjacent to AChR clusters and spatiotemporally correlated with the formation and maintenance of surface AChR clusters. Importantly, increased actin dynamics, vesicular markers, and intracellular AChRs were all enriched at the sites of ADF/cofilin localization. Furthermore, a substantial amount of new AChRs was detected at these ADF/cofilin-enriched sites. Manipulation of either ADF/cofilin activity through its serine-3 phosphorylation or ADF/cofilin localization via 14-3-3 proteins markedly attenuated AChR insertion and clustering. These results suggest that spatiotemporally restricted ADF/cofilin-mediated actin dynamics regulate AChR trafficking during the development of neuromuscular synapses.
ADF/cofilin; receptor trafficking; actin dynamics; neuromuscular junction; synapse
Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin towards strong preponderance of filamentous actin, and formation of actin stress fibers. A549 cells expressing FDH demonstrated a much slower recovery of GFP-actin fluorescence in a FRAP assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the JNK inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phospho-cofilin, indicating that the observed effects are independent from FDH function in apoptosis. Interestingly, cofilin siRNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can be also prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate-specific and not a general response to nutrient starvation. Overall, this study demonstrates the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.
ALDH1L1; cofilin; phosphatases; actin; folate