Actin Stabilization Research Articles

A Two-step Mechanism for the Folding of Actin by the Yeast Cytosolic Chaperonin

Actin requires the chaperonin containing TCP1 (CCT), a hexadecameric ATPase essential for cell viability in eukaryotes, to fold to its native state. Following binding of unfolded actin to CCT, the cavity of the chaperone closes and actin is folded and released in an ATP-dependent folding cycle. In yeast, CCT forms a ternary complex with the phosducin-like protein PLP2p to fold actin, and together they can return nascent or chemically denatured actin to its native state in a pure in vitro folding assay. The complexity of the CCT-actin system makes the study of the actin folding mechanism technically challenging. We have established a novel spectroscopic assay through selectively labeling the C terminus of yeast actin with acrylodan and observe significant changes in the acrylodan fluorescence emission spectrum as actin is chemically unfolded and then refolded by the chaperonin. The variation in the polarity of the environment surrounding the fluorescent probe during the unfolding/folding processes has allowed us to monitor actin as it folds on CCT. The rate of actin folding at a range of temperatures and ATP concentrations has been determined for both wild type CCT and a mutant CCT, CCT4anc2, defective in folding actin in vivo. Binding of the non-hydrolysable ATP analog adenosine 5'-(β,γ-imino)triphosphate to the ternary complex leads to 3-fold faster release of actin from CCT following addition of ATP, suggesting a two-step folding process with a conformational change occurring upon closure of the cavity and a subsequent final folding step involving packing of the C terminus to the native-like state.

Forgetting to remember in epilepsy

Amyloid-β (Aβ) drives the synaptic impairment and dendritic spine loss characteristic of Alzheimer9s disease (AD), but how Aβ affects the actin cytoskeleton remains unknown and contentious. The actin-binding protein, cofilin-1 (cof1), is a major regulator of actin dynamics in dendritic spines, and is subject to phospho-regulation by multiple pathways, including the Rho-associated protein kinase (ROCK) pathway. While cof1 is implicated as a driver of the synaptotoxicity characteristic of the early phases of AD pathophysiology, questions remain about the molecular mechanisms involved. Cofilin-actin rods are observed in neurons exposed to Aβ oligomers (Aβo) and in tissue from AD patients, and others have described an increased cofilin phosphorylation (p-cof1) in AD patients. Here, we report elevated p-cof1 of the postsynaptic enriched fraction of synaptosomes from cortical samples of male APP/PS1 mice and human AD cases of either sex. In primary cortical neurons, Aβo induced rapid actin stabilization and increased p-cof1 in the postsynaptic compartment of excitatory synapses within 30 min. Fluorescence recovery after photobleaching of actin-GFP and calcium imaging in live neurons expressing active or inactive cof1 mutants suggest that cof1 phosphorylation is necessary and sufficient for Aβo-induced synaptic impairment via actin stabilization before the reported formation of cofilin-actin rods. Moreover, the clinically available and well-tolerated ROCK inhibitor, fasudil, prevented Aβo-induced actin stabilization, synaptic impairment, and synaptic loss by blocking cofilin phosphorylation. Aβo also blocked the LTP-induced insertion of the AMPAR subunit, GluA1, at the postsynaptic density, in a fasudil-sensitive manner. These data support an important role for ROCKs and cofilin in mediating Aβ-induced synaptic impairment. <b>SIGNIFICANCE STATEMENT</b> We report that amyloid-β oligomers rapidly induce aberrant stabilization of F-actin within dendritic spines, which impairs synaptic strength and plasticity. Activation of the Rho-associated protein kinase (ROCK) pathway results in phosphorylation of cof1 and is sufficient to mediate Aβo-induced actin stabilization synaptic impairment and synaptic loss. Further, the ROCK inhibitor, fasudil, prevents cofilin phosphorylation, acute synaptic disruption, and synaptotoxicity in primary cortical neurons. Together, the herein presented data provide strong support for further study of the ROCK pathway as a therapeutic target for the cognitive decline and synaptotoxicity in Alzheimer9s disease.

Establishment of HIV-1 latency in resting CD4 + T cells depends on chemokine-induced changes in the actin cytoskeleton

Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4(+) T cells. We now show that HIV-1 latency can be established in resting CD4(+) T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4(+) T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4(+) T cells during normal chemokine-directed recirculation of CD4(+) T cells between blood and tissue.

Specific cleavage of the DNase-I binding loop dramatically decreases the thermal stability of actin

Differential scanning calorimetry was used to investigate the thermal unfolding of actin specifically cleaved within the DNaseI-binding loop between residues Met47-Gly48 or Gly42-Val43 by two bacterial proteases, subtilisin or ECP32/grimelysin (ECP), respectively. The results obtained show that both cleavages strongly decreased the thermal stability of monomeric actin with either ATP or ADP as a bound nucleotide. An even more pronounced difference in the thermal stability between the cleaved and intact actin was observed when both actins were polymerized into filaments. Similar to intact F-actin, both cleaved F-actins were significantly stabilized by phalloidin and aluminum fluoride; however, in all cases, the thermal stability of the cleaved F-actins was much lower than that of intact F-actin, and the stability of ECP-cleaved F-actin was lower than that of subtilisin-cleaved F-actin. These results confirm that the DNaseI-binding loop is involved in the stabilization of the actin structure, both in monomers and in the filament subunits, and suggest that the thermal stability of actin depends, at least partially, on the conformation of the nucleotide-binding cleft. Moreover, an additional destabilization of the unstable cleaved actin upon ATP/ADP replacement provides experimental evidence for the highly dynamic actin structure that cannot be simply open or closed, but rather should be considered as being able to adopt multiple conformations.

Trio Is a Key Guanine Nucleotide Exchange Factor Coordinating Regulation of the Migration and Morphogenesis of Granule Cells in the Developing Cerebellum

Orchestrated regulation of neuronal migration and morphogenesis is critical for neuronal development and establishment of functional circuits, but its regulatory mechanism is incompletely defined. We established and analyzed mice with neural-specific knock-out of Trio, a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Knock-out mice showed defective cerebella and severe signs of ataxia. Mutant cerebella had no granule cells in the internal granule cell layer due to aberrant granule cell migration as well as abnormal neurite growth. Trio-deficient granule cells showed reduced extension of neurites and highly branched and misguided processes with perturbed stabilization of actin and microtubules. Trio deletion caused down-regulation of the activation of Rac1, RhoA, and Cdc42, and mutant granule cells appeared to be unresponsive to neurite growth-promoting molecules such as Netrin-1 and Semaphorin 6A. These results suggest that Trio may be a key signal module for the orchestrated regulation of neuronal migration and morphogenesis during cerebellar development. Trio may serve as a signal integrator decoding extrinsic signals to Rho GTPases for cytoskeleton organization.

Protective effect of phosphorylated Hsp27 in coronary arteries through actin stabilization

There is evidence for an inverse association between cellular expression of Hsp27 and vascular disease with carotid plaques, endarterectomy specimens, and cardiac biopsies investigated to date. Here we compare non-diseased coronary arteries from human heart transplant donors and patients with dilated cardiomyopathy (DCM) with no evidence of coronary artery disease, to coronary arteries from patients with ischemic heart disease (IHD) in order to determine abundance of phosphorylated Hsp27 (phospho-Hsp27) in plaque-free diseased vessels and elucidate how this protective effect is brought about through protein regulation. Western blotting identified phospho-Hsp27, phosphorylated on Ser82, Ser78, and Ser15, to be specifically decreased in IHD, but not DCM, compared to non-diseased vessels. Immunohistochemistry confirmed these results and revealed phospho-Hsp27 was located within both smooth muscle and endothelial cells. Disease-free coronary arteries and from patients with IHD were then subjected to 2-Dimensional Difference Gel Electrophoresis (2D-DIGE) analysis to detect proteins with altered abundance, which were subsequently identified by mass spectrometry. Hsp27 showed decreased abundance in ischemic vessels as expected. The expression of cytoskeletal proteins, namely vimentin was significantly reduced, while transgelin and tropomyosin showed significantly increased abundance in vessels with IHD. Immunohistochemistry studies suggested an increase in G-actin abundance to be present within IHD vessels. The results are consistent with the hypothesis that phospho-Hsp27 protects against vascular disease possibly by stabilizing the actin cytoskeleton within endothelial and/or smooth muscle cells.

Phosphatase of Regenerating Liver-1 Promotes Cell Migration and Invasion and Regulates Filamentous Actin Dynamics

The phosphatases of regenerating liver (PRLs) are a unique family of plasma membrane-associated protein tyrosine phosphatases that have been hypothesized to be involved in metastatic cancer. How PRLs control cancer cell migration, invasion, and proliferation remains largely unknown. In the current study, we demonstrate a role for PRL-1 in the regulation of filamentous actin dynamics, which could promote cell metastatic processes. Human A549 non-small-cell lung cancer cells stably expressing wild-type PRL-1 exhibited a 60% increase in migration and a 3-fold increase in invasion. Cells expressing catalytic mutants of PRL-1 (C104S and D72A) lacked increased cell migration and invasion, indicating that these phenotypic changes required PRL-1 phosphatase activity. In contrast, PRL-1 small interfering RNA decreased in vitro lung cancer cell migration and invasion. The cadherin-catenin complex and dynamic filamentous actin are believed to control cellular invasiveness. Expression of wild-type PRL-1, but not phosphatase-inactive PRL-1 (C104S or D72A), decreased E-cadherin, vinculin, and paxillin expression. Ectopic expression of wild-type PRL-1 increased RhoA levels, which have an important role in actin filament assembly and stabilization of focal adhesion, and decreased activated Cdc42 and Rac. The Rho-associated protein kinase inhibitor, (R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride (Y-27632), decreased RhoA activity, actin filament levels, and cellular migration and invasion in PRL-1-expressing cells. These results suggest that PRL-1 could be a productive cancer therapeutic target and support further efforts to identify its substrates.

Abstract 5210: PRL-1 promotes cell metastatic processes and regulates filamentous actin dynamics

Abstract The phosphatases of regenerating liver (PRL) are a family of the protein tyrosine phosphatases that have been hypothesized to be involved in metastatic cancer. While the PRLs appear to regulate cancer cell migration, invasion and proliferation, we do not fully understand mechanistically how they control these processes. In the current study, we present new findings demonstrating that PRL-1 regulates filamentous actin dynamics, which could promote cell metastatic processes. Human A549 non-small cell lung cancer cells stably expressing wildtype PRL-1 exhibited a 60% increase in cell migration at 72 hours and a three-fold increase in cell invasion at 24 hours. Cells expressing catalytic mutants of PRL-1 (C104S and D72A) did not demonstrate increased cell migration and invasion, indicating that these phenotypic changes required the PRL-1 phosphatase activity. PRL-1 siRNA decreased PRL-1 protein levels by 50% and decreased cell migration by 25%. Cellular invasiveness is controlled by the cadherin-catenin complex and dynamic filamentous actin. We found wildtype PRL-1 interrupted E-cadherin and regulated Rho family kinase function, which have important roles in actin filament assembly and stabilization of focal adhesion. We also observed that expression of wildtype PRL-1 caused diffusely disassembled F-actin with reduced F-actin levels as compared to that seen in cells expressing the vector control or the catalytic mutant. We conclude the PRL-1 enhanced lung cancer cell migration and invasion. These results suggest PRL-1 could be a productive therapeutic target for cancer metastasis and support further efforts to identify its substrates. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5210.

Abstract 2539: Reactivating tumor suppression with small-molecule deregulators of the Rho-regulated diaphanous-related formin autoinhibitory mechanism

Abstract Loss of all or part of the long-arm of chromosome 5 is a frequent karyotypic defect in myeloproliferitive neoplasms, including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). To date, there is no clear mechanism that accounts for how any of the 5q tumor suppressor candidates contribute to hematopoietic stem cell growth control or how defects in their expression/function contribute to malignant progression. An attractive 5q- MDS tumor suppressor gene is DIAPH1 (5q31.3)]. DIAPH1 encodes the canonical mammalian Diaphanous-related (mDia) formin mDia1. Consistent with a role in tumor suppression, mDia1 knockout (KO) mice develop an age-dependent MDS-like phenotype. mDia1 is an effector for the Rho-family small GTP-binding protein RhoB, which is known to harbor tumor suppressor activity. mDia1 has a central role in the actin and microtubule dynamics that underlie cell division and directed migration. In this study we sought to determine if we could manipulate mDia activity to ‘reactivate’ tumor suppression by exploiting our working knowledge of the Rho-controlled mDia autoregulatory mechanism. GTP-bound Rho proteins activate mDia formins by binding to and disrupting an intramolecular autoinhibitory mechanism mediated by regulatory Dia-inhibitory (DID) and Dia-autoregulatory (DAD) domains that flank the conserved formin homology-2 (FH2) domain found in all formins that is responsible for assembling linear actin filaments and modulating microtubule stability. To identify small molecule activators of mDia formins, a screen for inhibitors of DID-DAD binding was conducted in a high-throughput format. We identified two structurally-related semi-thiocarbazones that bind within the DAD binding pocket of DID. These compounds, called Intramimics (IMM)-1 and −2, possess IC50 values for in vitro inhibition of DAD binding of 99 and 140 nM, respectively; the values were comparable to a DAD-derived peptide (280 nM). Consistent with mDia activation, exposure of fibroblasts and AML-derived cells leads to stabilization of actin and microtubule dynamics. The consequence is the induction of apoptosis, which was assayed by a variety of approaches. Using IMMs as tool compounds to explore tumor suppression mechanisms, IMM treatment activated serum response factor (SRF)-regulated gene expression. SRF is a growth factor-regulated transcription factor that also senses changes in actin assembly and is a potent activator of Egr1 expression. Egr1 is another 5q tumor suppressor candidate and is also a transcription factor that appears to be a master regulator of the well-known tumor suppressors PTEN and p53. Our next goal is to test IMMs in preclinical models for their ability to modulate myeloproliferation and to inhibit tumor growth, alone and in combination with other anti-tumor agents. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2539.

Interdependence of Endomembrane Trafficking and Actin Dynamics during Polarized Growth of Arabidopsis Pollen Tubes

During polarized growth of pollen tubes, endomembrane trafficking and actin polymerization are two critical processes that establish membrane/wall homeostasis and maintain growth polarity. Fine-tuned interactions between these two processes are therefore necessary but poorly understood. To better understand such cross talk in the model plant Arabidopsis (Arabidopsis thaliana), we first established optimized concentrations of drugs that interfere with either endomembrane trafficking or the actin cytoskeleton, then examined pollen tube growth using fluorescent protein markers that label transport vesicles, endosomes, or the actin cytoskeleton. Both brefeldin A (BFA) and wortmannin disturbed the motility and structural integrity of ARA7- but not ARA6-labeled endosomes, suggesting heterogeneity of the endosomal populations. Disrupting endomembrane trafficking by BFA or wortmannin perturbed actin polymerization at the apical region but not in the longitudinal actin cables in the shank. The interference of BFA/wortmannin with actin polymerization was progressive rather than rapid, suggesting an indirect effect, possibly due to perturbed endomembrane trafficking of certain membrane-localized signaling proteins. Both the actin depolymerization drug latrunculin B and the actin stabilization drug jasplakinolide rapidly disrupted transport of secretory vesicles, but each drug caused distinct responses on different endosomal populations labeled by ARA6 or ARA7, indicating that a dynamic actin cytoskeleton was critical for some steps in endomembrane trafficking. Our results provide evidence of cross talk between endomembrane trafficking and the actin cytoskeleton in pollen tubes.

The Actin-Bundling Protein Fascin Stabilizes Actin in Invadopodia and Potentiates Protrusive Invasion

Fascin is an actin-bundling protein involved in filopodia assembly and cancer invasion and metastasis of multiple epithelial cancer types. Fascin forms stable actin bundles with slow dissociation kinetics in vitro and is regulated by phosphorylation of serine 39 by protein kinase C (PKC). Cancer cells use invasive finger-like protrusions termed invadopodia to invade into and degrade extracellular matrix. Invadopodia have highly dynamic actin that is assembled by both Arp2/3 complex and formins; they also contain components of membrane trafficking machinery such as dynamin and cortactin and have been compared with focal adhesions and podosomes. We show that fascin is an integral component of invadopodia and that it is important for the stability of actin in invadopodia. The phosphorylation state of fascin at S39, a PKC site, contributes to its regulation at invadopodia. We further implicate fascin in invasive migration into collagen I-Matrigel gels and particularly in cell types that use an elongated mesenchymal type of motility in 3D. We provide a potential molecular mechanism for how fascin increases the invasiveness of cancer cells, and we compare invadopodia with invasive filopod-like structures in 3D.

An Evolutionary Structural Bioinformatics Analysis of the Actin Binding Protein, Tropomyosin

Tropomyosin is a two-chained, α-helical coiled-coil protein that associates end-to-end to form a continuous strand along actin filaments and regulates the functions and stability of actin. Mutations in tropomyosin cause skeletal and cardiomyopathies. We have carried out a phylogenetic analysis of tropomyosin to identify its conserved residues and to elucidate their importance for binding actin. Actin is a highly conserved protein and our hypothesis is that the actin binding sites of tropomyosin have been conserved through evolution to retain its actin-binding function. Phylogenetic trees of 60 coding sequences were constructed from tropomyosin genes from 19 species within the phyla Chordata, Hemichordata and Echinoderm. The rates of substitution (ω) at amino acid sites (codons) in the protein sequence were calculated to identify the most conserved sites (lowest ω) using CODEML in PAML 4.1. A total of 103 out of 284 residues were identified as highly conserved (ω ≤ 0.015), of which 24 are in a and d positions of the heptad repeat involved in the hydrophobic coiled coil interface, 38 are in potential interhelical e-g position salt bridges important for folding of the coiled coil, and 41 are in b, c, or f surface positions available for binding to other proteins, such as actin. The conserved residues at the b, c, and f surface positions were selected for initial mutagenesis studies with mutations to alanine. Preliminary actin binding co-sedimentation assays carried out for three tropomyosin mutants showed a 2- to 3-fold decrease in actin-binding affinity compared to the wild-type protein. Further analysis of mutations at other conserved surface positions and their effect on actin-binding affinity and stability of the mutant proteins are in progress. Supported by Muscle Dystrophy Association.

Identification of Molecular Targets of AML by Phosphoproteomic Screening of Valproic Acid Treated BNML and C. Elegans RNAi Validation.

Abstract 4151Disease stabilisation, instead of cure, is proposed as the therapeutic strategy of choice in elderly or chemoresistant acute myeloid leukemia (AML). This approach may also be of particular benefit to patients for whom allogeneic bone marrow (re)transplantation is not an option. Previously, we have clinically investigated the addition of valproic acid (VPA) to various combination chemotherapies with initial results in AML indicating prolonged survival, with follow-up on a predominantly out-patient basis. Consequently, we aimed to identify further molecular targets of VPA, which may enhance its therapeutic efficacy through screening for VPA-modulated phosphoproteins in relevant preclinical models of AML, and validation of these targets in RNAi screen of Caenorhabditis elegans (C. elegans).Brown Norwegian Myeloid Leukemia (BNML) has previously been described as a particularly relevant preclinical rat model of AML. Indeed, leukemic rats treated with 170 mg/kg VPA twice-daily achieved therapeutic serum levels of VPA and demonstrated significant increases in survival in comparison to controls (p = 0.004). To screen for molecular targets of VPA effect in this responsive model, we investigated the differences in control and VPA treated BNML phosphoproteomes by difference gel electrophoresis (DIGE) separation and subsequent differential gel software analysis. This was achieved through harvest of phosphoproteins from leukemic blasts, isolated from the spleens of treated and control BNML rats by immobilized metal ion affinity chromatography (IMAC) and subsequent protein identification via Orbitrap mass-spectrometry. Significant differential expression of 9 phosphoproteins was found in VPA treated BNML rats compared to controls, including Tubulin α-1B chain (TBA1B) and Actin β (ACTB), indicating these genes as possible targets of VPA therapy.To validate the functionality of 7 of these genes, RNAi was performed in wild type Bristol N2 strain of C. elegans at larval stage L1, 24 hours prior to exposure to 15 mM VPA for 72 hours. Knockdown of 4 of 7 genes resulted in larval developmental arrest, defined as synthetic lethality. In order to ascertain if synthetic lethality induced by these 4 genes was resultant of apoptosis, we employed the CED-1::GFP transgenic reporter assay to quantify germline cell death following RNAi depletion and subsequent exposure to VPA (15 mM, 24 hours). Increased numbers of apoptotic corpses in the germline was determined for all genes examined. To further examine the role of p53 in the observed apoptotic induction we used the transgenic strain cep-1::CED-1::GFP, which expresses the C. elegans ortholog of p53, CEP-1. Successive RNAi knockdown of our 4 candidate genes, again effected increased basal number of apoptotic corpses independently of CEP-1. These results suggest that similar combinational treatment of AML may be beneficial, irrespective of p53 status.To further investigate this thesis in a human AML cell line, MOLM-13 cells were co-treated with VPA and small molecule inhibitors of prospective targets TBA1B and ACTB, namely paclitaxel, and cytochalasin B. Inhibition of actin polymerization or stabilisation of tubulin polymerization resulted in increased apoptosis when supplemented with VPA, as determined by DNA specific staining with Hoechst 33342. These results suggest that use of these combinations may be beneficial in the treatment of AML. In conclusion, this study indicates that phosphoproteomic screening of BNML and subsequent target verification in C. elegans worms has the potential to identify future drugable targets for effective combinatorial therapy with valproic acid in acute myeloid leukemia. Disclosures:No relevant conflicts of interest to declare.

Modification of actin fibers changes the electrical phenotype of cardiac myofibroblasts

Background: Slow conduction and ectopic activity are major determinants of cardiac arrhythmogenesis. Both of these conditions can be elicited by myofibroblasts (MFBs) following establishment of heterocellular gap junctional coupling with cardiomyocytes. MFBs appear during structural remodeling of the heart and are characterized by the expression of α-smooth muscle actin (α-SMA) containing stress fibers. In this study, we investigated whether pharmacological interference with the actin cytoskeleton affects myofibroblast arrhythmogeneicity. Methods: Experiments were performed with patterned growth strands of neonatal rat ventricular cardiomyocytes coated with cardiac MFBs. Impulse conduction velocity (θ) and maximal upstroke velocities of propagated action potentials (dV/dtmax), expressed as % action potential amplitude change (%APA) per ms, were measured optically using voltage sensitive dyes. Actin was destabilized by latrunculin B (LtB) and cytochalasin D and stabilized with jasplakinolide. Data are given as mean ± S.D. (n = 5-22). Single cell electrophysiology was assessed using standard patch-clamp techniques. Results: As revealed by immunocytochemistry, exposure of MFBs to LtB (0.01-10 μmol/L) profoundly disrupted stress fibers which led to drastic changes in cell morphology with MFBs assuming an astrocyte-like shape. In control cardiomyocyte strands (no MFB coat), LtB had negligible effects on θ and dV/dtmax. In contrast, LtB applied to MFB-coated strands increased θ dose-dependently from 197 ± 35 mm/s to 344 ± 26 mm/s and dV/dtmax from 38 ± 5 to 78 ± 3% APA/ms, i.e., to values virtually identical to those of cardiomyocyte control strands (339 ± 24 mm/s; 77 ± 3% APA/ms). Highly similar results were obtained when exposing the preparations to cytochalasin D. In contrast, stabilization of actin with increasing concentrations of jasplakinolide exerted no significant effects on impulse conduction characteristics in MFB-coated strands. Whole-cell patch-clamp experiments showed that LtB hyperpolarized MFBs from -25 mV to -50 mV, thus limiting their depolarizing effect on cardiomyocytes which was shown before to cause arrhythmogenic slow conduction and ectopic activity. Conclusion: Pharmacological interference with the actin cytoskeleton of cardiac MFBs affects their electrophysiological phenotype to such an extent that they loose their detrimental effects on cardiomyocyte electrophysiology. This result might form a basis for the development of therapeutic strategies aimed at limiting the arrhythmogenic potential of MFBs.

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.

Nephrin Is Expressed on the Surface of Insulin Vesicles and Facilitates Glucose-Stimulated Insulin Release

OBJECTIVENephrin, an immunoglobulin-like protein essential for the function of the glomerular podocyte and regulated in diabetic nephropathy, is also expressed in pancreatic β-cells, where its function remains unknown. The aim of this study was to investigate whether diabetes modulates nephrin expression in human pancreatic islets and to explore the role of nephrin in β-cell function.RESEARCH DESIGN AND METHODSNephrin expression in human pancreas and in MIN6 insulinoma cells was studied by Western blot, PCR, confocal microscopy, subcellular fractionation, and immunogold labeling. Islets from diabetic (n = 5) and nondiabetic (n = 7) patients were compared. Stable transfection and siRNA knockdown in MIN-6 cells/human islets were used to study nephrin function in vitro and in vivo after transplantation in diabetic immunodeficient mice. Live imaging of green fluorescent protein (GFP)-nephrin–transfected cells was used to study nephrin endocytosis.RESULTSNephrin was found at the plasma membrane and on insulin vesicles. Nephrin expression was decreased in islets from diabetic patients when compared with nondiabetic control subjects. Nephrin transfection in MIN-6 cells/pseudoislets resulted in higher glucose-stimulated insulin release in vitro and in vivo after transplantation into immunodeficient diabetic mice. Nephrin gene silencing abolished stimulated insulin release. Confocal imaging of GFP-nephrin–transfected cells revealed nephrin endocytosis upon glucose stimulation. Actin stabilization prevented nephrin trafficking as well as nephrin-positive effect on insulin release.CONCLUSIONSOur data suggest that nephrin is an active component of insulin vesicle machinery that may affect vesicle-actin interaction and mobilization to the plasma membrane. Development of drugs targeting nephrin may represent a novel approach to treat diabetes.

Ca2+/Calmodulin-dependent Protein Kinase IV-mediated LIM Kinase Activation Is Critical for Calcium Signal-induced Neurite Outgrowth

Actin cytoskeletal remodeling is essential for neurite outgrowth. LIM kinase 1 (LIMK1) regulates actin cytoskeletal remodeling by phosphorylating and inactivating cofilin, an actin filament-disassembling factor. In this study, we investigated the role of LIMK1 in calcium signal-induced neurite outgrowth. The calcium ionophore ionomycin induced LIMK1 activation and cofilin phosphorylation in Neuro-2a neuroblastoma cells. Knockdown of LIMK1 or expression of a kinase-dead mutant of LIMK1 suppressed ionomycin-induced cofilin phosphorylation and neurite outgrowth in Neuro-2a cells. Ionomycin-induced cofilin phosphorylation and neurite outgrowth were also blocked by KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaMKs), and STO-609, an inhibitor of CaMK kinase. An active form of CaMKIV but not CaMKI enhanced Thr-508 phosphorylation of LIMK1 and increased the kinase activity of LIMK1. Moreover, the active form of CaMKIV induced cofilin phosphorylation and neurite outgrowth, and a dominant negative form of CaMKIV suppressed ionomycin-induced neurite outgrowth. Taken together, our results suggest that LIMK1-mediated cofilin phosphorylation is critical for ionomycin-induced neurite outgrowth and that CaMKIV mediates ionomycin-induced LIMK1 activation.

Phosphatidylinositol (4,5)-Bisphosphate Regulation ofN-Methyl-d-aspartate Receptor Channels in Cortical Neurons

The membrane phospholipid phosphatidylinositol (4,5)-bisphosphate (PIP(2)) has been implicated in the regulation of several ion channels and transporters. In this study, we examined the impact of PIP(2) on N-methyl-D-aspartate receptors (NMDARs) in cortical neurons. Blocking PIP(2) synthesis by inhibiting phosphoinositide-4 kinase, or stimulating PIP(2) hydrolysis via activation of phospholipase C (PLC), or blocking PIP(2) function with an antibody caused a significant reduction of NMDAR-mediated currents. On the other hand, inhibition of PLC or application of PIP(2) caused an enhancement of NMDAR currents. These electrophysiological effects were accompanied by changes in NMDAR surface clusters induced by agents that manipulate PIP(2) levels. The PIP(2) regulation of NMDAR currents was abolished by the dynamin inhibitory peptide, which blocks receptor internalization. Agents perturbing actin stability prevented PIP(2) regulation of NMDAR currents, suggesting the actin-dependence of this effect of PIP(2). Cofilin, a major actin depolymerizing factor, which has a common binding sequence for actin and PIP(2), was required for PIP(2) regulation of NMDAR currents. It is noteworthy that the PIP(2) regulation of NMDAR channels was impaired in a transgenic mouse model of Alzheimer's disease, probably because of the amyloid-beta disruption of PIP(2) metabolism. Taken together, our data suggest that continuous synthesis of PIP(2) at the membrane might be important for the maintenance of NMDARs at the cell surface. When PIP(2) is lost, cofilin is released from the PIP(2) complex and is rendered free to depolymerize actin. With the actin cytoskeleton no longer intact, NMDARs are internalized via a dynamin/clathrin-dependent mechanism, leading to reduced NMDAR currents.

Moesin orchestrates cortical polarity of melanoma tumour cells to initiate 3D invasion

Tumour cell dissemination through corporal fluids (blood, lymph and body cavity fluids) is a distinctive feature of the metastatic process. Tumour cell transition from fluid to adhesive conditions involves an early polarization event and major rearrangements of the submembrane cytoskeleton that remain poorly understood. As regulation of cortical actin-membrane binding might be important in this process, we investigated the role of ezrin and moesin, which are key crosslinking proteins of the ERM (ezrin, radixin, moesin) family. We used short interfering RNA (siRNA) to show that moesin is crucial for invasion by melanoma cells in 3D matrices and in early lung colonization. Using live imaging, we show that following initial adhesion to the endothelium or 3D matrices, moesin is redistributed away from the region of adhesion, thereby generating a polarized cortex: a stable cortical actin dome enriched in moesin and an invasive membrane domain full of blebs. Using Lifeact-GFP, a 17-amino-acid peptide that binds F-actin, we show the initial symmetry breaking of cortical actin cytoskeleton during early attachment of round cells. We also demonstrated that ezrin and moesin are differentially distributed during initial invasion of 3D matrices, and, specifically, that moesin controls adhesion-dependent activation of Rho and subsequent myosin II contractility. Our results reveal that polarized moesin plays a role in orienting Rho activation, myosin II contractility, and cortical actin stability, which is crucial for driving directional vertical migration instead of superficial spreading on the fluid-to-solid tissue interface. We propose that this mechanism of cortical polarization could sustain extravasation of fluid-borne tumour cells during the process of metastasis.

Influence of cytoskeletal structure and mechanics on epithelial cell injury during cyclic airway reopening

Although patients with acute respiratory distress syndrome require mechanical ventilation, these ventilators often exacerbate the existing lung injury. For example, the cyclic closure and reopening of fluid-filled airways during ventilation can cause epithelial cell (EpC) necrosis and barrier disruption. Although much work has focused on minimizing the injurious mechanical forces generated during ventilation, an alternative approach is to make the EpC less susceptible to injury by altering the cell's intrinsic biomechanical/biostructural properties. In this study, we hypothesized that alterations in cytoskeletal structure and mechanics can be used to reduce the cell's susceptibility to injury during airway reopening. EpC were treated with jasplakinolide to stabilize actin filaments or latrunculin A to depolymerize actin and then exposed to cyclic airway reopening conditions at room temperature using a previously developed in vitro cell culture model. Actin stabilization did not affect cell viability but significantly improved cell adhesion primarily due to the development of more numerous focal adhesions. Surprisingly, actin depolymerization significantly improved both cell viability and cell adhesion but weakened focal adhesions. Optical tweezer based measurements of the EpC's micromechanical properties indicate that although latrunculin-treated cells are softer, they also have increased viscous damping properties. To further investigate the effect of "fluidization" on cell injury, experiments were also conducted at 37 degrees C. Although cells held at 37 degrees C exhibited no changes in cytoskeletal structure, they did exhibit increased viscous damping properties and improved cell viability. We conclude that fluidization of the actin cytoskeleton makes the EpC less susceptible to the injurious mechanical forces generated during cyclic airway reopening.