Role of cofilin­1 in arsenic trioxide­induced apoptosis of NB4­R1 cells.

All­trans retinoic acid (ATRA) and arsenic trioxide (As2O3) are currently first­line treatments for acute promyelocytic leukemia (APL). However, a number of patients with APL are resistant to ATRA but still sensitive to As2O3, and the underlying mechanisms of this remain unclear. In the present study, two­dimensional gel electrophoresis, mass spectrometry and other proteomic methods were applied to screen and identify the differentially expressed proteins between the retinoic acid­sensitive cell lines and drug­resistant cell lines. The results demonstrated that in retinoic acid­resistant NB4­R1 cells, the protein expression of cofilin­1 was markedly increased compared with that in the drug­sensitive NB4 cells. Subsequently, the effects of cofilin­1 on As2O3­induced apoptosis in NB4­R1 cells were further investigated. The results revealed that cell viability was markedly suppressed and apoptosis was increased in the As2O3­treated NB4­R1 cells, with increased expression levels of cleaved­poly (ADP­ribose) polymerase and cleaved­caspase 12. Cofilin­1 expression was significantly decreased at both the mRNA and protein levels in the As2O3­treated group compared with the control. Western blotting further revealed that As2O3 treatment decreased the cytoplasmic cofilin­1 level but increased its expression in the mitochondrion. However, the opposite effects of As2O3 on the cytochrome C distribution were found in NB4­R1 cells. This suggested that As2O3 can induce the transfer of cofilin­1 from the cytoplasm to mitochondria and trigger the release of mitochondrial cytochrome C in NB4­R1 cells. Moreover, cofilin­1 knockdown by its specific short hairpin RNA significantly suppressed As2O3­induced NB4­R1 cell apoptosis and inhibited the release of mitochondrial cytochrome C. Whereas, overexpression of cofilin­1 using a plasmid vector carrying cofilin­1 increased the release of cytochrome C into the cytoplasm from the mitochondria in As2O3­treated NB4­R1 cells. In conclusion, cofilin­1 played a role in As2O3­induced NB4­R1 cell apoptosis and it might be a novel target for APL treatment.

Cofilin dysregulation alters actin turnover in frataxin-deficient neurons.

Abnormalities in actin cytoskeleton have been linked to Friedreich's ataxia (FRDA), an inherited peripheral neuropathy characterised by an early loss of neurons in dorsal root ganglia (DRG) among other clinical symptoms. Despite all efforts to date, we still do not fully understand the molecular events that contribute to the lack of sensory neurons in FRDA. We studied the adult neuronal growth cone (GC) at the cellular and molecular level to decipher the connection between frataxin and actin cytoskeleton in DRG neurons of the well-characterised YG8R Friedreich's ataxia mouse model. Immunofluorescence studies in primary cultures of DRG from YG8R mice showed neurons with fewer and smaller GCs than controls, associated with an inhibition of neurite growth. In frataxin-deficient neurons, we also observed an increase in the filamentous (F)-actin/monomeric (G)-actin ratio (F/G-actin ratio) in axons and GCs linked to dysregulation of two crucial modulators of filamentous actin turnover, cofilin-1 and the actin-related protein (ARP) 2/3 complex. We show how the activation of cofilin is due to the increase in chronophin (CIN), a cofilin-activating phosphatase. Thus cofilin emerges, for the first time, as a link between frataxin deficiency and actin cytoskeleton alterations.

Cofilin-1-induced actin reorganization in stored platelets.

BACKGROUND: During platelet storage, there are extensive changes in cytoskeleton and phosphatidylserine exposure. The intrinsic mitochondrial pathway of apoptosis, activated in stored platelets, is a major mediator these changes. Cofilin-1 is an effector of actin reorganization. We examined the effect of cofilin-1 deficiency on cytoskeleton and phosphatidylserine exposure during storage and following activation of apoptosis. METHODS AND RESULTS: We assessed actin filaments by Alexa-647-phalloidin and phosphatidylserine exposure by fluorescein isothiocyanate-lactadherin by fluorescence microscopy. In fresh platelets, actin filaments are distributed in the subcortical region, and they do not express phosphatidylserine in the outer surface. In stored platelets, there is retraction of actin filaments from the subcortical region with increased phosphatidylserine expression. These changes are seen in 20% of platelets of 6 days old and increases further with storage. Treatment with ABT-737, which activates the mitochondrial apoptosis, induces similar cytoskeletal changes in actin filaments with increased phosphatidylserine. Cofilin-1 is activated in stored platelets as well as in ABT-737 treated platelets by dephosphorylation. In cofilin-1 deficient murine platelets actin filaments are abnormal and ABT-737 induces less phosphatidylserine. Despite these changes in vitro, platelet survival of cofilin-1 deficient platelets in mice was not significantly different from their wild-type controls. CONCLUSION: These results show that cofilin-1 plays a role in apoptosis-induced actin rearrangement and phosphatidylserine exposure during storage. Despite the defects in platelet cytoskeleton and phosphatidylserine exposure in cofilin-1-deficient platelets, the in vivo life span of platelets is similar to littermate controls, indicating multiple redundant pathways for the clearance of platelets in vivo.

Actin(g) on mitochondria - a role for cofilin1 in neuronal cell death pathways.

Actin dynamics, the coordinated assembly and disassembly of actin filaments (F-actin), are essential for fundamental cellular processes, including cell shaping and motility, cell division or organelle transport. Recent studies highlighted a novel role for actin dynamics in the regulation of mitochondrial morphology and function, for example, through mitochondrial recruitment of dynamin-related protein 1 (Drp1), a key factor in the mitochondrial fission machinery. Mitochondria are dynamic organelles, and permanent fission and fusion is essential to maintain their function in energy metabolism, calcium homeostasis and regulation of reactive oxygen species (ROS). Here, we summarize recent insights into the emerging role of cofilin1, a key regulator of actin dynamics, for mitochondrial shape and function under physiological conditions and during cellular stress, respectively. This is of peculiar importance in neurons, which are particularly prone to changes in actin regulation and mitochondrial integrity and function. In neurons, cofilin1 may contribute to degenerative processes through formation of cofilin-actin rods, and through enhanced mitochondrial fission, mitochondrial membrane permeabilization, and the release of cytochrome c. Overall, mitochondrial impairment induced by dysfunction of actin-regulating proteins such as cofilin1 emerge as important mechanisms of neuronal death with relevance to acute brain injury and neurodegenerative diseases, such as Parkinson's or Alzheimer's disease.

Torsional stress generated by ADF/cofilin on cross-linked actin filaments boosts their severing.

Proteins of the actin depolymerizing factor (ADF)/cofilin family are the central regulators of actin filament disassembly. A key function of ADF/cofilin is to sever actin filaments. However, how it does so in a physiological context, where filaments are interconnected and under mechanical stress, remains unclear. Here, we monitor and quantify the action of ADF/cofilin in different mechanical situations by using single-molecule, single-filament, and filament network techniques, coupled to microfluidics. We find that local curvature favors severing, while tension surprisingly has no effect on cofilin binding and weakly enhances severing. Remarkably, we observe that filament segments that are held between two anchoring points, thereby constraining their twist, experience a mechanical torque upon cofilin binding. We find that this ADF/cofilin-induced torque does not hinder ADF/cofilin binding, but dramatically enhances severing. A simple model, which faithfully recapitulates our experimental observations, indicates that the ADF/cofilin-induced torque increases the severing rate constant 100-fold. A consequence of this mechanism, which we verify experimentally, is that cross-linked filament networks are severed by cofilin far more efficiently than nonconnected filaments. We propose that this mechanochemical mechanism is critical to boost ADF/cofilin's ability to sever highly connected filament networks in cells.

The role of cofilin-l in vulvar squamous cell carcinoma: A marker of carcinogenesis, progression and targeted therapy.

Numerous studies have revealed that cofilin-l (CFL1) is associated with cancer cell migration and invasion in various types of tumor tissues. We investigated the roles of CFL1 in vulvar squamous cell carcinoma (VSCC). CFL1 expression was detected in VSCC and normal vulvar tissues using immunohistochemistry and western blotting. The vulvar carcinoma SW962 cell line was transfected with CFL1 small interfering RNA (siRNA) and exposed to periplocoside. We then assessed changes in cell proliferation, apoptosis, invasion and metastasis. We detected changes in CFL1 mRNA and protein expression by RT-PCR and western blotting, and alterations in protein expression of various relevant molecules by western blotting. CFL1 expression was found to be significantly upregulated in the VSCC tissues compared with the normal vulvar tissues by immunohistochemistry and western blotting (P<0.05) and was positively correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, differentiation and lymphatic metastasis (P<0.05). After CFL1 knockdown by siRNA transfection, SW962 cells exhibited a decrease in growth, G1 phase cell cycle arrest, induction of apoptotic, low invasion and metastasis, and disrupted lamellipodium formation. We found that the protein expression of Bcl-xL, cyclin A1, MMP2, MMP9 and STAT3 was decreased, while expression of Bax was increased. Periplocoside inhibited SW962 cell growth, promoted apoptosis, suppressed invasion and migration, and lamellipodium formation. Periplocoside exposure resulted in lower CFL1, Bcl-xL, cyclin A1, MMP2, MMP9 and STAT3 levels, but a higher Bax level compared with the control group. We demonstrated that abnormal CFL1 expression may affect vulvar carcinogenesis and subsequent progression. CFL1 silencing by siRNA significantly inhibited VSCC cell progression, which suggests that CFL1 is a potential therapeutic target for vulvar cancer. Periplocoside, which was utilized in the present study for the clinical treatment of vulvar cancer, showed strong antitumor effects by suppression of CFL1 expression.

Chronophin coordinates cell leading edge dynamics by controlling active cofilin levels.

Cofilin, a critical player of actin dynamics, is spatially and temporally regulated to control the direction and force of membrane extension required for cell locomotion. In carcinoma cells, although the signaling pathways regulating cofilin activity to control cell direction have been established, the molecular machinery required to generate the force of the protrusion remains unclear. We show that the cofilin phosphatase chronophin (CIN) spatiotemporally regulates cofilin activity at the cell edge to generate persistent membrane extension. We show that CIN translocates to the leading edge in a PI3-kinase-, Rac1-, and cofilin-dependent manner after EGF stimulation to activate cofilin, promotes actin free barbed end formation, accelerates actin turnover, and enhances membrane protrusion. In addition, we establish that CIN is crucial for the balance of protrusion/retraction events during cell migration. Thus, CIN coordinates the leading edge dynamics by controlling active cofilin levels to promote MTLn3 cell protrusion.

Aß Influences Cytoskeletal Signaling Cascades with Consequences to Alzheimer's Disease.

Abnormal signal transduction events can impact upon the cytoskeleton, affecting the actin and microtubule networks with direct relevance to Alzheimer's disease (AD). Cytoskeletal anomalies, in turn, promote atypical neuronal responses, with consequences for cellular organization and function. Neuronal cytoskeletal modifications in AD include neurofibrillary tangles, which result from aggregates of hyperphosphorylated tau protein. The latter is a microtubule (MT)-binding protein, whose abnormal phosphorylation leads to MT instability and consequently provokes irregularities in the neuronal trafficking pathways. Early stages of AD are also characterized by synaptic dysfunction and loss of dendritic spines, which correlate with cognitive deficit and impaired brain function. Actin dynamics has a prominent role in maintaining spine plasticity and integrity, thus providing the basis for memory and learning processes. Hence, factors that disrupt both actin and MT network dynamics will compromise neuronal function and survival. The peptide Aß is the major component of senile plaques and has been described as a pivotal mediator of neuronal dystrophy and synaptic loss in AD. Here, we review Aß-mediated effects on both MT and actin networks and focus on the relevance of the elicited cytoskeletal signaling events targeted in AD pathology.

Attention-Deficit/Hyperactivity Disorder-like Phenotype in a Mouse Model with Impaired Actin Dynamics.

BACKGROUND: Actin depolymerizing proteins of the actin depolymerizing factor (ADF)/cofilin family are essential for actin dynamics, which is critical for synaptic function. Two ADF/cofilin family members, ADF and n-cofilin, are highly abundant in the brain, where they are present in excitatory synapses. Previous studies demonstrated the relevance of n-cofilin for postsynaptic plasticity, associative learning, and anxiety. These studies also suggested overlapping functions for ADF and n-cofilin. METHODS: We performed pharmacobehavioral, electrophysiologic, and electron microscopic studies on ADF and n-cofilin single mutants and double mutants (named ACC mice) to characterize the importance of ADF/cofilin activity for synapse physiology and mouse behavior. RESULTS: The ACC mice, but not single mutants, exhibited hyperlocomotion, impulsivity, and impaired working memory. Hyperlocomotion and impulsive behavior were reversed by methylphenidate, a psychostimulant commonly used for the treatment of attention-deficit/hyperactivity disorder (ADHD). Also, ACC mice displayed a disturbed morphology of striatal excitatory synapses, accompanied by strongly increased glutamate release. Blockade of dopamine or glutamate transmission resulted in normal locomotion. CONCLUSIONS: Our study reveals that ADHD can result from a disturbed balance between excitation and inhibition in striatal circuits, providing novel insights into the mechanisms underlying this neurobehavioral disorder. Our results link actin dynamics to ADHD, suggesting that mutations in actin regulatory proteins may contribute to the etiology of ADHD in humans.

Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

The actin filament severing protein cofilin-1 (CFL-1) is required for actin and P-type ATPase secretory pathway calcium ATPase (SPCA)-dependent sorting of secretory proteins at the trans-Golgi network (TGN). How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known. We used purified proteins to assess interaction of the cytoplasmic domains of SPCA1 with actin and CFL-1. A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner. This domain, coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads, specifically recruited F-actin in the presence of CFL-1 and, when expressed in HeLa cells, inhibited Ca(2+) entry into the TGN and secretory cargo sorting. Mutagenesis of four amino acids in SPCA1 that represent the CFL-1 binding site also affected Ca(2+) import into the TGN and secretory cargo sorting. Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.

Genome-wide pathway analysis in neuroblastoma.

The aim of this study was to identify candidate single-nucleotide polymorphisms (SNPs) that might play a role in susceptibility to neuroblastoma, elucidate their potential mechanisms, and generate SNP-to-gene-to-pathway hypotheses. A genome-wide association study (GWAS) dataset of neuroblastoma that included 442,976 SNPs from 1,627 neuroblastoma patients and 3,254 control subjects of European descent was used in this study. The identify candidate causal SNPs and pathways (ICSNPathway) analysis was applied to the GWAS dataset. ICSNPathway analysis identified 15 candidate SNPs, 10 genes, and 31 pathways, which revealed 10 hypothetical biological mechanisms. The strongest hypothetical biological mechanism was one wherein SNPrs40401 modulates the role of IL3 in several pathways and conditions, including the stem pathway, asthma (hsa05310), the dendritic cell pathway, and development (0.001 < p < 0.004; 0.001 < FDR < 0.033). The second strongest mechanism identified was that in which rs1048108 and rs16852600 alter the function of BARD1, which negatively regulates developmental process and modulates processes including cell development and programmed cell death (0.001 < p < 0.004; 0.001 < FDR < 0.033). The third mechanism identified was one wherein rs1939212 modulated CFL1, resulting in negative regulation of development, cell death, neural crest cell migration, and apoptosis (0.001 < p < 0.004; 0.001 < FDR < 0.033). By using the ICSNPathway to analyze neuroblastoma GWAS data, 15 candidate SNPs, 10 genes including IL3, BARD1, and CFL, and 31 pathways were identified that might contribute to the susceptibility of patients to neuroblastoma.

Cofilin and Vangl2 cooperate in the initiation of planar cell polarity in the mouse embryo.

The planar cell polarity (PCP; non-canonical Wnt) pathway is required to orient the cells within the plane of an epithelium. Here, we show that cofilin 1 (Cfl1), an actin-severing protein, and Vangl2, a core PCP protein, cooperate to control PCP in the early mouse embryo. Two aspects of planar polarity can be analyzed quantitatively at cellular resolution in the mouse embryo: convergent extension of the axial midline; and posterior positioning of cilia on cells of the node. Analysis of the spatial distribution of brachyury(+) midline cells shows that the Cfl1 mutant midline is normal, whereas Vangl2 mutants have a slightly wider midline. By contrast, midline convergent extension fails completely in Vangl2 Cfl1 double mutants. Planar polarity is required for the posterior positioning of cilia on cells in the mouse node, which is essential for the initiation of left-right asymmetry. Node cilia are correctly positioned in Cfl1 and Vangl2 single mutants, but cilia remain in the center of the cell in Vangl2 Cfl1 double mutants, leading to randomization of left-right asymmetry. In both the midline and node, the defect in planar polarity in the double mutants arises because PCP protein complexes fail to traffic to the apical cell membrane, although other aspects of apical-basal polarity are unaffected. Genetic and pharmacological experiments demonstrate that F-actin remodeling is essential for the initiation, but not maintenance, of PCP. We propose that Vangl2 and cofilin cooperate to target Rab11(+) vesicles containing PCP proteins to the apical membrane during the initiation of planar cell polarity.

AIP1 acts with cofilin to control actin dynamics during epithelial morphogenesis.

During epithelial morphogenesis, cells not only maintain tight adhesion for epithelial integrity but also allow dynamic intercellular movement to take place within cell sheets. How these seemingly opposing processes are coordinated is not well understood. Here, we report that the actin disassembly factors AIP1 and cofilin are required for remodeling of adherens junctions (AJs) during ommatidial precluster formation in Drosophila eye epithelium, a highly stereotyped cell rearrangement process which we describe in detail in our live imaging study. AIP1 is enriched together with F-actin in the apical region of preclusters, whereas cofilin displays a diffuse and uniform localization pattern. Cofilin overexpression completely rescues AJ remodeling defects caused by AIP1 loss of function, and cofilin physically interacts with AIP1. Pharmacological reduction of actin turnover results in similar AJ remodeling defects and decreased turnover of E-cadherin, which also results from AIP1 deficiency, whereas an F-actin-destabilizing drug affects AJ maintenance and epithelial integrity. Together with other data on actin polymerization, our results suggest that AIP1 enhances cofilin-mediated actin disassembly in the apical region of precluster cells to promote remodeling of AJs and thus intercellular movement, but also that robust actin polymerization promotes AJ general adhesion and integrity during the remodeling process.

Complement receptor-3 negatively regulates the phagocytosis of degenerated myelin through tyrosine kinase Syk and cofilin.

BACKGROUND: Intact myelin, which normally surrounds axons, breaks down in Wallerian degeneration following axonal injury and during neurodegenerative diseases such as multiple sclerosis. Clearance of degenerated myelin by phagocytosis is essential since myelin impedes repair and exacerbates damage. CR3 (complement receptor-3) is a principal phagocytic receptor in myelin phagocytosis. We studied how tyrosine kinase Syk (spleen tyrosine kinase) and cofilin control phagocytosis of degenerated myelin by CR3 in microglia and macrophages. Syk is a non-receptor tyrosine kinase that CR3 recruits to convey cellular functions. Cofilin is an actin-depolymerizing protein that controls F-actin (filamentous actin) remodeling (i.e., disassembly and reassembly) by shifting between active unphosphorylated and inactive phosphorylated states. RESULTS: Syk was continuously activated during prolonged phagocytosis. Phagocytosis increased when Syk activity and expression were reduced, suggesting that normally Syk down regulates CR3-mediated myelin phagocytosis. Levels of inactive p-cofilin (phosphorylated cofilin) decreased transiently during prolonged phagocytosis. In contrast, p-cofilin levels decreased continuously when Syk activity and expression were continuously reduced, suggesting that normally Syk advances the inactive state of cofilin. Observations also revealed inverse relationships between levels of phagocytosis and levels of inactive p-cofilin, suggesting that active unphosphorylated cofilin advances phagocytosis. Active cofilin could advance phagocytosis by promoting F-actin remodeling, which supports the production of membrane protrusions (e.g., filopodia), which, as we also revealed, are instrumental in myelin phagocytosis. CONCLUSIONS: CR3 both activates and downregulates myelin phagocytosis at the same time. Activation was previously documented. We presently demonstrate that downregulation is mediated through Syk, which advances the inactive phosphorylated state of cofilin. Self-negative control of phagocytosis by the phagocytic receptor can be useful in protecting phagocytes from excessive phagocytosis (i.e., "overeating") during extended exposure to particles that are destined for ingestion.

The actin-severing protein cofilin is downstream of neuregulin signaling and is essential for Schwann cell myelination.

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.

α-Crystallin promotes rat axonal regeneration through regulation of RhoA/rock/cofilin/MLC signaling pathways.

Intravitreal injection of α-crystallin can promote axons from optic nerve regeneration after crushing in rats. We have previously demonstrated that α-crystallin can counteract the effect of myelin inhibitory factors and stimulate neurite growth. And a common crucial signaling event for myelin inhibitory factors is the activation of RhoA. To investigate whether α-crystallin counteracts the inhibitory effect of myelin inhibitory factors through regulation of RhoA/Rock signaling pathway, α-crystallin (10(-4) g/L) was injected into rat vitreous at the time the optic nerve crushed. The RhoA protein activity and the expression of RhoA and Rock were evaluated after 3 days of optic nerve axotomy. Rock downstream effectors, phosphorylated cofilin, and phosphorylated myosin light chain were detected when retinal neurons were cultured for 3 days. Axonal regeneration and neurites growth of cultured cells were observed also. Our results showed that α-crystallin decreased the RhoA protein activity and the phosphorylation of both cofilin and myosin light chain, and promoted the axonal growth. However, the expression of RhoA and Rock was not affected by α-crystallin. These findings indicated that α-crystallin could counteract the effect of myelin inhibitory factors through the regulation of RhoA/Rock signaling pathway.

Cofilin activation mediates Bax translocation to mitochondria during excitotoxic neuronal death.

During excitotoxic neuronal death, Bax translocates to the mitochondria where it plays an important role by contributing to the release of proapoptotic factors. However, how Bax translocates to the mitochondria during excitotoxicity remains poorly understood. Herein, our data suggest the presence of a novel signalling mechanism by which NMDA receptor stimulation promotes Bax translocation. This signalling pathway is triggered by dephosphorylation of cofilin. Once dephosphorylated, cofilin might interact physically with Bax acting as a carrier for it, translocating it to the mitochondria, where it contributes to mitochondrial membrane despolarization, permeabilization and to the release of apoptotic factors, thus leading to neuronal death. Lack-of-function studies indicate that only the Slingshot family of phosphatases, more specifically the enzyme Slingshot 1L phosphatase, but not cronophin participates in the cofilin activation process during excitotoxicity. Indeed, cofilin-mediated Bax translocation seems to be a key event in excitotoxic neuronal death as knock down of either cofilin or Slingshot 1L phosphatase has a marked neuroprotective effect on NMDA-mediated neuronal death. This novel biochemical pathway may therefore be a good target to develop future therapeutic molecules for neurodegenerative diseases.

N-cofilin can compensate for the loss of ADF in excitatory synapses.

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.

Validation of cofilin-1 as a biomarker in non-small cell lung cancer: application of quantitative method in a retrospective cohort.

PURPOSE: Cofilin is a cytoskeletal protein whose overexpression has been associated with aggressiveness in several types of malignancies. Here, we established and optimized a simple semi-quantitative immunohistochemistry (SQ-IHC) method for cofilin quantification in tumor biopsies, and applied it in a retrospective cohort of NSCLC patients aiming at validating the use of cofilin-1 as a prognostic biomarker. METHODS: The SQ-IHC method for cofilin-1 quantification was established and applied in a NSCLC cohort. An archival collection of biopsies from 50 patients with clinicopathological information and 5 years follow-up was accessed. Association between cofilin-1 immunocontent and clinical outcome was assessed using standard Kaplan-Meier mortality curves and the log-rank test. To evaluate the robustness of our findings, three different partitional clustering strategies were used to stratify patients into two groups according to the biomarker expression level (hierarchical clustering, Kmeans and median cutoff). RESULTS: In all the three different partitional clustering we used, survival analysis showed that patient with high cofilin-1 immunocontent had a lower overall survival rate (P < 0.05), and could be used to discriminate between good and bad prognosis. No other correlation was found when the variables age, sex or histological type were tested in association with patients outcome or with cofilin immunocontent. CONCLUSIONS: Our method showed good sensitivity/specificity to indicate the outcome of patients according to their cofilin immunocontent in biological samples. Its application in a retrospective cohort and the results presented here are an important step toward the validation process of cofilin-1 as a prognostic biomarker.

Regulation of AMPA receptor channels and synaptic plasticity by cofilin phosphatase Slingshot in cortical neurons.

Cofilin, the major actin depolymerizing factor, modulates actin dynamics that contribute to spine morphology, synaptic transmission and plasticity. Much evidence implicates the cofilin inactivation kinase LIMK in synaptic function, but little is known about the cofilin activation phosphatase Slingshot in this regard. In this study, we found that suppressing endogenous Slingshot with small RNA interference or function-blocking antibody led to a dramatic reduction of AMPA receptor-mediated excitatory postsynaptic currents (EPSCs) in cortical neurons. Perturbation of Slingshot function also diminished the ability to express synaptic plasticity. Inactivating cofilin or disturbing actin dynamics reduced AMPAR-EPSCs in a Slingshot-dependent manner. Moreover, surface GluR 1 and synaptic GluR2/3 clusters were reduced by Slingshot knockdown. Our data suggest that Slingshot plays a pivotal role in AMPAR trafficking and synaptic transmission by controlling actin cytoskeleton via cofilin activation.