Cofilin-Membrane Interactions: Electrostatic Effects in Phosphoinositide Lipid Binding.

The actin cytoskeleton interacts with the cell membrane primarily through the indirect interactions of actin-binding proteins such as cofilin-1. The molecular mechanisms underlying the specific interactions of cofilin-1 with membrane lipids are still unclear. Here, we performed coarse-grain molecular dynamics simulations of cofilin-1 with complex lipid bilayers to analyze the specificity of protein-lipid interactions. We observed the maximal interactions with phosphoinositide (PIP) lipids, especially PIP2 and PIP3 lipids. A good match was observed between the residues predicted to interact and previous experimental studies. The clustering of PIP lipids around the membrane bound protein leads to an overall lipid demixing and gives rise to persistent membrane curvature. Further, through a series of control simulations, we observe that both electrostatics and geometry are critical for specificity of lipid binding. Our current study is a step towards understanding the physico-chemical basis of cofilin-PIP lipid interactions.

Wnt5a modulates dendritic spine dynamics through the regulation of Cofilin via small Rho GTPase activity in hippocampal neurons.

Dendritic spines are small, actin-rich protrusions that act as the receiving sites of most excitatory inputs in the central nervous system. The remodeling of the synapse architecture is mediated by actin cytoskeleton dynamics, a process precisely regulated by the small Rho GTPase family. Wnt ligands exert their presynaptic and postsynaptic effects during formation and consolidation of the synaptic structure. Specifically, Wnt5a has been identified as an indispensable synaptogenic factor for the regulation and organization of the postsynaptic side; however, the molecular mechanisms through which Wnt5a induces morphological changes resulting from actin cytoskeleton dynamics within dendritic spines remain unclear. In this work, we employ primary rat hippocampal cultures and HT22 murine hippocampal neuronal cell models, molecular and pharmacological tools, and fluorescence microscopy (laser confocal and epifluorescence) to define the Wnt5a-induced molecular signaling involved in postsynaptic remodeling mediated via the regulation of the small Rho GTPase family. We report that Wnt5a differentially regulates the phosphorylation of Cofilin in neurons through both Ras-related C3 botulinum toxin substrate 1 and cell division cycle 42 depending on the subcellular compartment and the extracellular calcium levels. Additionally, we demonstrate that Wnt5a increases the density of dendritic spines and promotes their maturation via Ras-related C3 botulinum toxin substrate 1. Accordingly, we find that Wnt5a requires the combined activation of small Rho GTPases to increase the levels of filamentous actin, thus promoting the stability of actin filaments. Altogether, these results provide evidence for a new mechanism by which Wnt5a may target actin dynamics, thereby regulating the subsequent morphological changes in dendritic spine architecture.

Inhibition of human prostate smooth muscle contraction by the LIM kinase inhibitors, SR7826 and LIMKi3.

BACKGROUND AND PURPOSE: In men with benign prostatic hyperplasia, increased smooth muscle tone in the prostate may lead to bladder outlet obstruction and subsequent lower urinary tract symptoms. Consequently, medical treatment aims to inhibit prostate smooth muscle contraction. However, the efficacy of the treatment options available is limited, and improved understanding of mechanisms of prostate smooth muscle contraction and identification of new targets for medical intervention are mandatory. Several studies suggest that LIM kinases (LIMKs) promote smooth muscle contraction; however, this has not yet been examined. Here, we studied effects of the LIMK inhibitors on prostate smooth muscle contraction. EXPERIMENTAL APPROACH: Human prostate tissues were obtained from radical prostatectomy. Phosphorylation of cofilin, a LIMK substrate, was examined using a phospho-specific antibody. Smooth muscle contractions were studied in organ bath experiments. KEY RESULTS: Real-time PCR, Western blot and immunofluorescence suggested LIMKs are expressed in smooth muscle cells of prostate tissues. Two different LIMK inhibitors, SR7826 (1 µM) and LIMKi3 (1 µM), inhibited contractions of prostate strips, which were induced by electrical field stimulation, α1 -adrenoceptor agonists phenylephrine and methoxamine and the TXA2 analogue, U46619. LIMK inhibition in prostate tissues and cultured stromal cells (WPMY-1) was confirmed by cofilin phosphorylation, which was reduced by SR7826 and LIMKi3. In WPMY-1 cells, SR7826 and LIMKi3 caused breakdown of actin filaments and reduced viability. CONCLUSIONS AND IMPLICATIONS: Smooth muscle tone in the hyperplastic human prostate may underlie the effects of LIMKs, which promote contraction. Contraction of prostate strips can be inhibited by small molecule LIMK inhibitors.

LIMK/cofilin pathway and Slingshot are implicated in human colorectal cancer progression and chemoresistance.

Cofilin phospho-regulation is important for actin filament turnover and is implicated in cancer. Phosphorylation of cofilin is mediated by LIM kinases (LIMKs) and dephosphorylation by Slingshot phosphatases (SSH). LIMKs and SSH promote cancer cell invasion and metastasis and represent novel anti-cancer targets. However, little is known regarding LIMK/cofilin and SSH in human colorectal cancer (CRC). In this study, we aimed to address their expression and significance in human CRC. We evaluated expression of non-phosphorylated (active) and phosphorylated cofilin, LIMK1, LIMK2, and SSH1 by immunohistochemistry in 143 human CRC samples in relation to clinicopathologic parameters, response of metastatic disease to chemotherapy, and epithelial-mesenchymal transition (EMT) markers ß-catenin, E-cadherin, and ZEB. We show that active cofilin, LIMK1, LIMK2, and SSH1 are overexpressed in human CRC and are associated with tumor progression parameters. SSH1 is an independent predictor of lymph node metastasis by multivariate analysis. LIMK1 and SSH1 expression is also higher in non-responders to chemotherapy, and SSH1 is shown by multivariate analysis to independently predict response of metastatic disease to chemotherapy. Active cofilin, LIMK1, LIMK2, and SSH1 also correlated with the EMT markers examined. In addition, immunofluorescence analysis showed increased expression of active cofilin, LIMK1, LIMK2, and SSH1 in HT29 colon cancer cells resistant to 5-fluorouracil compared to parental HT29 cells. Our results suggest that F-actin regulators LIMK/cofilin pathway and SSH1 are associated with CRC progression and chemoresistance representing promising tumor biomarkers and therapeutic targets in CRC.

Aerobic exercise regulates Rho/cofilin pathways to rescue synaptic loss in aged rats.

PURPOSE: The role of exercise to prevent or reverse aging-induced cognitive decline has been widely reported. This neuroprotection is associated with changes in the synaptic structure plasticity. However, the mechanisms of exercise-induced synaptic plasticity in the aging brain are still unclear. Thus, the aim of the present study is to investigate the aging-related alterations of Rho-GTPase and the modulatory influences of exercise training. METHODS: Young and old rats were used in this study. Old rats were subjected to different schedules of aerobic exercise (12 m/min, 60 min/d, 3d/w or 5d/w) or kept sedentary for 12 w. After 12 w of aerobic exercise, the synapse density in the cortex and hippocampus was detected with immunofluorescent staining using synaptophysin as a marker. The total protein levels of RhoA, Rac1, Cdc42 and cofilin in the cortex and hippocampus were detected with Western Blot. The activities of RhoA, Rac1 and Cdc42 were determined using a pull down assay. RESULTS: We found that synapse loss occurred in aging rats. However, the change of expression and activity of RhoA, Rac1 and Cdc42 was different in the cortex and hippocampus. In the cortex, the expression and activity of Rac1 and Cdc42 was greatly increased with aging, whereas there were no changes in the expression and activity of RhoA. In the hippocampus, the expression and activity of Rac1 and Cdc42 was greatly decreased and there were no changes in the expression and activity of RhoA. As a major downstream substrate of the Rho GTPase family, the increased expression of cofilin was only observed in the cortex. High frequency exercise ameliorated all aging-related changes in the cortex and hippocampus. CONCLUSIONS: These data suggest that aerobic exercise reverses synapse loss in the cortex and hippocampus in aging rats, which might be related to the regulation of Rho GTPases.

The role of cytoplasmic p57 in invasion of hepatocellular carcinoma.

BACKGROUND: Our previous research suggested that p57 downregulation could accelerate the growth and invasion of hepatocellular carcinoma in vitro and in vivo. AIM: To evaluate the role of cytoplasmic p57 and its regulatory mechanism during hepatocellular carcinoma invasion. METHODS: We examined the subcellular localization of p57 by immunohistochemistry in 45 pairs of cancerous tissues and adjacent non-cancerous tissues. Moreover, we generated stable p57 knockdown hepatoma cell lines to investigate the mechanism of cytoplasmic p57-mediated regulation of invasion by immunoprecipitation, confocal immunofluorescence microscopy and western blot of nuclear and cytoplasmic extracts. RESULTS: Our results showed that cytoplasmic expression of p57 was reduced in specimens from patients with capsular invasion and metastasis (P < 0.05). Moreover, the level of p-cofilin was decreased in the group lacking cytoplasmic p57 expression (P < 0.05). Co-expression of p57 and p-cofilin was reduced in specimens from patients with tumors at later stages (III + IV), tumors showing capsular invasion and metastatic tumors. We further observed that p57 downregulation decreased the assembly of p57 and LIM domain kinase 1 and its kinase activity, subsequently reducing the level of p-cofilin in the cytoplasm. CONCLUSIONS: Cytoplasmic p57 might be a key regulator in hepatocellular carcinoma invasion via the LIM domain kinase 1/p-cofilin pathway.

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

BACKGROUND: 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). METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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.

MPC polymer regulates fibrous tissue formation by modulating cell adhesion to the biomaterial surface.

The aim of this study was to analyze the effects of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer on fibrous tissue formation and cell adhesion plaque (CAP)-forming reactions. Silastic elastomer (SE) plates coated (experimental group) and uncoated (control group) with MPC polymer were prepared for in vivo and in vitro experiments. For the in vivo animal experiments, SE plates were implanted subcutaneously in the rat dorsal region. At 4, 8, and 12 weeks, thicknesses of the fibrous tissue capsules in the experimental group were lower than in the control group. Likewise, the amount of collagen in the experimental group was lower than that of the control group. For the in vitro cell culture experiments, KMST-6 fibroblast cells in the experimental group demonstrated enhanced cell migration, accompanied with a weaker expression of vinculin and a larger amount of filopodia. Furthermore, weaker expressions of paxillin, talin, and ROCK1, but stronger expression of cofilin, were observed in the experimental group. Taken together, these results suggested that MPC polymer regulated fibrous tissue formation by modulating cell adhesion through changes in local CAPs and downstream signaling.

Proteomic analysis of proteins associated with tt-DDE induced toxicity in BEAS-2B cells.

Trans, trans-2,4-decadienal (tt-DDE), a specific type of dienaldehyde, is abundant in heated oils or cooking oil fumes. Ingestion of heated oils and exposure to cooking oil fumes has been suggested to have a great health impact in a variety of organs, including the lungs. Previous studies have demonstrated that acute exposures to high doses of tt-DDE have induced oxidative stress, genotoxicity, and cytotoxicity in human lung cells. The objective in utilizing proteomic techniques of this study was to identify protein biomarkers associated with tt-DDE-induced oxidative stress and cytotoxicity in human bronchial epithelial cells BEAS-2B. Experimental results suggested that DJ-1 and cofilin proteins were protein biomarkers for tt-DDE-induced cytotoxicity and oxidative stress in lung cells. DJ-1 was especially an early biomarker for tt-DDE exposure.

Ins and outs of ADF/cofilin activity and regulation.

The actin-binding proteins of the actin-depolymerisation factor (ADF)/cofilin family were first described more than three decades ago, but research on these proteins still occupies a front role in the actin and cell migration field. Moreover, cofilin activity is implicated in the malignant, invasive properties of cancer cells. The effects of ADF/cofilins on actin dynamics are diverse and their regulation is complex. In stimulated cells, multiple signalling pathways can be initiated resulting in different activation/deactivation switches that control ADF/cofilin activity. The output of this entire regulatory system, in combination with spatial and temporal segregation of the activation mechanisms, underlies the contribution of ADF/cofilins to various cell migration/invasion phenotypes. In this framework, we describe current views on how ADF/cofilins function in migrating and invading cells.

EGF-induced PIP2 hydrolysis releases and activates cofilin locally in carcinoma cells.

Lamellipodial protrusion and directional migration of carcinoma cells towards chemoattractants, such as epidermal growth factor (EGF), depend upon the spatial and temporal regulation of actin cytoskeleton by actin-binding proteins (ABPs). It is generally hypothesized that the activity of many ABPs are temporally and spatially regulated by PIP(2); however, this is mainly based on in vitro-binding and structural studies, and generally in vivo evidence is lacking. Here, we provide the first in vivo data that directly visualize the spatial and temporal regulation of cofilin by PIP(2) in living cells. We show that EGF induces a rapid loss of PIP(2) through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, we find that cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation. Moreover, our data provide evidence for how PLC is involved in the formation of protrusions in breast carcinoma cells during chemotaxis and metastasis towards EGF.

Oscillatory increases in alkalinity anticipate growth and may regulate actin dynamics in pollen tubes of lily.

Lily (Lilium formosanum or Lilium longiflorum) pollen tubes, microinjected with a low concentration of the pH-sensitive dye bis-carboxyethyl carboxyfluorescein dextran, show oscillating pH changes in their apical domain relative to growth. An increase in pH in the apex precedes the fastest growth velocities, whereas a decline follows growth, suggesting a possible relationship between alkalinity and cell extension. A target for pH may be the actin cytoskeleton, because the apical cortical actin fringe resides in the same region as the alkaline band in lily pollen tubes and elongation requires actin polymerization. A pH-sensitive actin binding protein, actin-depolymerizing factor (ADF), together with actin-interacting protein (AIP) localize to the cortical actin fringe region. Modifying intracellular pH leads to reorganization of the actin cytoskeleton, especially in the apical domain. Acidification causes actin filament destabilization and inhibits growth by 80%. Upon complete growth inhibition, the actin fringe is the first actin cytoskeleton component to disappear. We propose that during normal growth, the pH increase in the alkaline band stimulates the fragmenting activity of ADF/AIP, which in turn generates more sites for actin polymerization. Increased actin polymerization supports faster growth rates and a proton influx, which inactivates ADF/AIP, decreases actin polymerization, and retards growth. As pH stabilizes and increases, the activity of ADF/AIP again increases, repeating the cycle of events.

Spatial organization of cofilin in dendritic spines.

Synaptic plasticity is associated with morphological changes in dendritic spines. The actin-based cytoskeleton plays a key role in regulating spine structure, and actin reorganization in spines is critical for the maintenance of long term potentiation. To test the hypothesis that a stable pool of F-actin rests in the spine "core," while a dynamic pool lies peripherally in its "shell," we performed immunoelectron microscopy in the stratum radiatum of rat hippocampus to elucidate the subcellular distribution of cofilin, an actin-depolymerizing protein that mediates reorganization of the actin cytoskeleton. We provide direct evidence that cofilin in spines avoids the core, and instead concentrates in the shell and within the postsynaptic density. These data suggest that cofilin may link synaptic plasticity to the actin remodeling that underlies changes in spine morphology.