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PubMed2002

14-3-3 regulates actin dynamics by stabilizing phosphorylated cofilin.

The functionality of the actin cytoskeleton depends on a dynamic equilibrium between filamentous and monomeric actin. Proteins of the ADF/cofilin family are essential for the high rates of actin filament turnover observed in motile cells through regulation of actin polymerization/depolymerization cycles. Rho GTPases act through p21-activated kinase-1 (Pak-1) and Rho kinase to inhibit cofilin activity via the LIM kinase (LIMK)-mediated phosphorylation of cofilin on Ser3. We report the identification of 14-3-3zeta as a novel phosphocofilin binding protein involved in the maintenance of the cellular phosphocofilin pool. A Ser3 phosphocofilin binding protein was purified from bovine brain and was identified as 14-3-3zeta by mass spectrometry. The phosphorylation-dependent interaction between cofilin and 14-3-3zeta was confirmed in pulldown and coimmunoprecipitation experiments. Both Ser3 phosphorylation and a 14-3-3 recognition motif in cofilin are necessary for 14-3-3 binding. The expression of 14-3-3zeta increases phosphocofilin levels, and the coexpression of 14-3-3zeta with LIMK further elevates phosphocofilin levels and potentiates LIMK-dependent effects on the actin cytoskeleton. This potentiation of cofilin action appears to be a result of the protection of phosphocofilin from phosphatase-mediated dephosphorylation at Ser3 by bound 14-3-3zeta. Taken together, these results suggest that 14-3-3zeta proteins may play a dynamic role in the regulation of cellular actin structures through the maintenance of phosphocofilin levels.

14-3-3 Proteins↗
PubMed2003

Cryptococcus neoformans induces alterations in the cytoskeleton of human brain microvascular endothelial cells.

The fungal pathogen Cryptococcus neoformans has a predilection for the central nervous system (CNS), resulting in devastating meningoencephalitis. At present, it is unclear how C. neoformans traverses the blood-brain barrier (BBB) and causes CNS infection. The present study has examined and characterized the interaction of C. neoformans with human brain microvascular endothelial cells (HBMEC), which constitute the BBB. Adhesion of and transcytosis of HBMEC by C. neoformans was inoculum- and time-dependent and occurred with both encapsulated and acapsulated strains. C. neoformans induced marked morphological changes in HBMEC, for example membrane ruffling, irregular nuclear morphology and swelling of the mitochondria and the ER. These findings suggest that C. neoformans induced actin cytoskeletal reorganization of the host cells. In addition, it was observed that the dephosphorylated form of cofilin was increased during cryptococcal adherence to HBMEC, concomitant with the actin rearrangement. Cryptococcal binding to HBMEC was increased in the presence of Y27632, a Rho kinase (ROCK)-specific inhibitor. Since ROCK activates LIM kinase (LIMK), which phosphorylates cofilin (inactive form), this suggests the involvement of the ROCK-->LIMK-->cofilin pathway. In contrast, the phosphatase inhibitor sodium orthovanadate decreased adherence of Cryptococcus to HBMEC, concomitant with the increase of phosphorylation of cofilin. Furthermore, the tight junction marker protein occludin became Triton-extractable, indicating alteration of tight junctions in brain endothelial cells. This is the first demonstration that C. neoformans is able to adhere to and transcytose across the HBMEC monolayer and alter the cytoskeleton morphology in HBMEC. Further characterization of the interactions between C. neoformans and HBMEC should help the development of novel strategies to prevent cryptococcal meningitis and its associated morbidity.

Actin Depolymerizing Factors↗
PubMed2006

The Cdi/TESK1 kinase is required for Sevenless signaling and epithelial organization in the Drosophila eye.

How cellular behaviors such as cell-to-cell communication, epithelial organization and cell shape reorganization are coordinated during development is poorly understood. The developing Drosophila eye offers an ideal model system to study these processes. Localized actin polymerization is required to constrict the apical surface of epithelial cells of the eye imaginal disc to maintain the refined arrangement of the developing ommatidia. The identity of each photoreceptor cell within the epithelium is determined by cell-to-cell contacts involving signal transduction events. The R7 photoreceptor cell requires the activity of the Sevenless RTK to adopt a proper cell fate. We performed an EP screen for negative regulators of this inductive process, and we identified the serine/threonine kinase Center divider (cdi) as a suppressor of the phenotype caused by an activated Sevenless receptor. Cdi is homologous to the human testis-specific kinase 1 (TESK1), a member of the LIM kinases involved in cytoskeleton control through ADF/cofilin phosphorylation. We have analyzed the effects of gain- and loss-of-function of cdi and found alterations in actin organization and in the adherens junctions proteins DE-cadherin and beta-catenin, as well as in Sevenless apical localization. Interference with the function of the ADF/cofilin phosphatase Slingshot (ssh), which antagonizes Cdi, also results in a suppression of signaling triggered by the Sevenless RTK. Our results reveal a critical interplay between the localization of molecules involved in epithelial organization and signal transduction.

Actins↗
PubMed2001

A role for a RhoA/ROCK/LIM-kinase pathway in the regulation of cytotoxic lymphocytes.

Polarization of lipid rafts and granules to the site of target contact is required for the development of cell-mediated killing by cytotoxic lymphocytes. We have previously shown that these events require the activation of proximal protein tyrosine kinases. However, the downstream intracellular signaling molecules involved in the development of cell-mediated cytotoxicity remain poorly defined. We report here that a RhoA/ROCK/LIM-kinase axis couples the receptor-initiated protein tyrosine kinase activation to the reorganization of the actin cytoskeleton required for the polarization of lipid rafts and the subsequent generation of cell-mediated cytotoxicity. Pharmacologic and genetic interruption of any element of this RhoA/ROCK/LIM-kinase pathway inhibits both the accumulation of F-actin and lipid raft polarization to the site of target contact and the subsequent delivery of the lethal hit. These data define a specialized role for a RhoA-->ROCK-->LIM-kinase pathway in cytotoxic lymphocyte activation.

Actins↗
PubMed2004

p120 catenin is required for morphogenetic movements involved in the formation of the eyes and the craniofacial skeleton in Xenopus.

During Xenopus development, p120 transcripts are enriched in highly morphogenetic tissues. We addressed the developmental function of p120 by knockdown experiments and by expressing E-cadherin mutants unable to bind p120. This resulted in defective eye formation and provoked malformations in the craniofacial cartilage structures, derivatives of the cranial neural crest cells. Closer inspection showed that p120 depletion impaired evagination of the optic vesicles and migration of cranial neural crest cells from the neural tube into the branchial arches. These morphogenetic processes were also affected by p120-uncoupled cadherins or E-cadherin containing a deletion of the juxtamembrane domain. Irrespective of the manipulation that caused the malformations, coexpression of dominant-negative forms of either Rac1 or LIM kinase rescued the phenotypes. Wild-type RhoA and constitutively active Rho kinase caused partial rescue. Our results indicate that, in contrast to invertebrates, p120 is an essential factor for vertebrate development and an adequate balance between cadherin activity and cytoskeletal condition is critical for correct morphogenetic movements.

Animals↗
PubMed2004

B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts.

Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.

Actin Depolymerizing Factors↗
PubMed2006

Photodynamic treatment (ALA-PDT) suppresses the expression of the oncogenic Bcr-Abl kinase and affects the cytoskeleton organization in K562 cells.

K562 is the chronic myelogenous leukemia (CML)-derived cell line that expresses high levels of chimeric oncoprotein Bcr-Abl. The deregulated (permanent) kinase activity of Bcr-Abl leads to continuous proliferation of K562 cells and their resistance to the apoptosis promotion by conventional drugs. The photodynamic treatment (PDT) based on the application of 5-aminolevulinic acid (ALA) and irradiation with blue light (ALA-PDT) resulted in the suppression of K562 cells proliferation. It was followed by a necrosis-like cell death [K. Kuzelová, D. Grebenová, M. Pluskalová, I. Marinov, Z. Hrkal, J. Photochem. Photobiol. B 73 (2004) 67-78]. ALA-PDT led to the perturbation of the Hsp90/p23 multichaperone complex of which the Bcr-Abl is the client protein. Bcr-Abl protein was suppressed whereas the bcr-abl mRNA level was not affected. Further on, we observed several changes in the cytoskeleton organization. We detected ALA-PDT-mediated disruption of filamental actin structure using FITC-Phalloidin staining. In connection with this we uncovered certain cytoskeleton organizing proteins involved in the cell response to the treatment. Among these proteins, Septin2, which plays a role in maintaining actin bundles, was suppressed. Another one, PDZ-LIM domain protein 1 (CLP36) was altered. This protein acts as an adaptor molecule for LIM-kinase which phosphorylates and thus inactivates cofilin. Cofilin was indeed dephosphorylated and could thus be activated and operate as an actin-depolymerizing factor. We propose the scheme of molecular response of K562 cells to ALA-PDT.

Aminolevulinic Acid↗
PubMed2006

Actin turnover-dependent fast dissociation of capping protein in the dendritic nucleation actin network: evidence of frequent filament severing.

Actin forms the dendritic nucleation network and undergoes rapid polymerization-depolymerization cycles in lamellipodia. To elucidate the mechanism of actin disassembly, we characterized molecular kinetics of the major filament end-binding proteins Arp2/3 complex and capping protein (CP) using single-molecule speckle microscopy. We have determined the dissociation rates of Arp2/3 and CP as 0.048 and 0.58 s(-1), respectively, in lamellipodia of live XTC fibroblasts. This CP dissociation rate is three orders of magnitude faster than in vitro. CP dissociates slower from actin stress fibers than from the lamellipodial actin network, suggesting that CP dissociation correlates with actin filament dynamics. We found that jasplakinolide, an actin depolymerization inhibitor, rapidly blocked the fast CP dissociation in cells. Consistently, the coexpression of LIM kinase prolonged CP speckle lifetime in lamellipodia. These results suggest that cofilin-mediated actin disassembly triggers CP dissociation from actin filaments. We predict that filament severing and end-to-end annealing might take place fairly frequently in the dendritic nucleation actin arrays.

Actin Capping Proteins↗
PubMed2006

Cofilin mediates ATP depletion-induced endothelial cell actin alterations.

Ischemia and sepsis lead to endothelial cell damage, resulting in compromised microvascular flow in many organs. Much remains to be determined regarding the intracellular structural events that lead to endothelial cell dysfunction. To investigate potential actin cytoskeletal-related mechanisms, ATP depletion was induced in mouse pancreatic microvascular endothelial cells (MS1). Fluorescent imaging and biochemical studies demonstrated a rapid and progressive increase in F-actin along with a decrease in G-actin at 60 min. Confocal microscopic analysis showed ATP depletion resulted in destruction of actin stress fibers and accumulation of F-actin aggregates. We hypothesized these actin alterations were secondary to dephosphorylation/activation of actin-depolymerizing factor (ADF)/cofilin proteins. Cofilin, the predominant isoform expressed in MS1 cells, was rapidly dephosphorylated/activated during ATP depletion. To directly investigate the role of cofilin activation on the actin cytoskeleton during ischemia, MS1 cells were infected with adenoviruses containing the cDNAs for wild-type Xenopus laevis ADF/cofilin green fluorescent protein [XAC(wt)-GFP], GFP, and the constitutively active and inactive isoforms XAC(S3A)-GFP and XAC(S3E)-GFP. The rate and extent of cortical actin destruction and actin aggregate formation were increased in ATP-depleted XAC(wt)-GFP- and XAC(S3A)-GFP-expressing cells, whereas increased actin stress fibers were observed in XAC(S3E)-GFP-expressing cells. To investigate the upstream signaling pathway of ADF/cofilin, LIM kinase 1-GFP (LIMK1-GFP) was expressed in MS1 cells. Cells expressing LIMK1-GFP protein had higher levels of phosphorylated ADF/cofilin, increased stress fibers, and delayed F-actin cytoskeleton destruction during ATP depletion. These results strongly support the importance of cofilin regulation in ischemia-induced endothelial cell actin cytoskeleton alterations leading to cell damage and microvascular dysfunction.

Actin Depolymerizing Factors↗
PubMed2004

LIMK1 and CLIP-115: linking cytoskeletal defects to Williams syndrome.

Williams Syndrome is a developmental disorder that is characterized by cardiovascular problems, particular facial features and several typical behavioral and neurological abnormalities. In Williams Syndrome patients, a heterozygous deletion is present of a region on chromosome 7q11.23 (the Williams Syndrome critical region), which spans approximately 20 genes. Two of these genes encode proteins that regulate dynamic aspects of the cytoskeleton of the cell, either via the actin filament system (LIM kinase 1, or LIMK1), or through the microtubule network (cytoplasmic linker protein of 115 kDa, or CLIP-115). The recent findings that knockout mice lacking LIMK1 or CLIP-115 have distinct neurological and behavioural phenotypes, indicates that cytoskeletal defects might play a role in the development of neurological symptoms in Williams Syndrome patients. In this review, we discuss the properties of LIMK and CLIP family proteins, their function in the regulation of the actin and microtubule cytoskeletal systems, respectively, and the relationship with neurodevelopmental aspects of Williams Syndrome.

Actins↗
PubMed2004

[Approach to depressogenic genes from genetic analyses of animal models].

Human depression or mood disorder is defined as a complex disease, making positional cloning of susceptibility genes a formidable task. We have undertaken genetic analyses of three different animal models for depression, comparing our results with advanced database resources. We first performed quantitative trait loci (QTL) analysis on two mouse models of "despair", namely, the forced swim test (FST) and tail suspension test (TST), and detected multiple chromosomal loci that control immobility time in these tests. Since one QTL detected on mouse chromosome 11 harbors the GABA A receptor subunit genes, we tested these genes for association in human mood disorder patients. We obtained significant associations of the alpha 1 and alpha 6 subunit genes with the disease, particularly in females. This result was striking, because we had previously detected an epistatic interaction between mouse chromosomes 11 and X that regulates immobility time in these animals. Next, we performed genome-wide expression analyses using a rat model of depression, learned helplessness (LH). We found that in the frontal cortex of LH rats, a disease implicated region, the LIM kinase 1 gene (Limk 1) showed greatest alteration, in this case down-regulation. By combining data from the QTL analysis of FST/TST and DNA microarray analysis of mouse frontal cortex, we identified adenylyl cyclase-associated CAP protein 1 (Cap 1) as another candidate gene for depression susceptibility. Both Limk 1 and Cap 1 are key players in the modulation of actin G-F conversion. In summary, our current study using animal models suggests disturbances of GABAergic neurotransmission and actin turnover as potential pathophysiologies for mood disorder.

Actins↗
PubMed2003

Cofilin interacts with ClC-5 and regulates albumin uptake in proximal tubule cell lines.

Receptor-mediated endocytosis is a constitutive high capacity pathway for the reabsorption of proteins from the glomerular filtrate by the renal proximal tubule. ClC-5 is a voltage-gated chloride channel found in the proximal tubule where it has been shown to be essential for protein uptake, based on evidence from patients with Dent's disease and studies in ClC-5 knockout mice. To further delineate the role of ClC-5 in albumin uptake, we performed a yeast two-hybrid screen with the C-terminal tail of ClC-5 to identify any interactions of the channel with proteins involved in endocytosis. We found that the C-terminal tail of ClC-5 bound the actin depolymerizing protein, cofilin, a result that was confirmed by GST-fusion pulldown assays. In cultured proximal tubule cells, cofilin was distributed in nuclear, cytoplasmic, and microsomal fractions and co-localized with ClC-5. Phosphorylation of cofilin by overexpressing LIM kinase 1 resulted in a stabilization of the actin cytoskeleton. Phosphorylation of cofilin in two proximal tubule cell models (porcine renal proximal tubule and opossum kidney) was also accompanied by a pronounced inhibition of albumin uptake. This study identifies a novel interaction between the C-terminal tail of ClC-5 and cofilin, an actin-associated protein that is crucial in the regulation of albumin uptake by the proximal tubule.

Actin Depolymerizing Factors↗
PubMed2002

Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice.

In vitro studies indicate a role for the LIM kinase family in the regulation of cofilin phosphorylation and actin dynamics. In addition, abnormal expression of LIMK-1 is associated with Williams syndrome, a mental disorder with profound deficits in visuospatial cognition. However, the in vivo function of this family of kinases remains elusive. Using LIMK-1 knockout mice, we demonstrate a significant role for LIMK-1 in vivo in regulating cofilin and the actin cytoskeleton. Furthermore, we show that the knockout mice exhibited significant abnormalities in spine morphology and in synaptic function, including enhanced hippocampal long-term potentiation. The knockout mice also showed altered fear responses and spatial learning. These results indicate that LIMK-1 plays a critical role in dendritic spine morphogenesis and brain function.

Actin Cytoskeleton↗
PubMed2003

Rho-ROCK-LIMK-cofilin pathway regulates shear stress activation of sterol regulatory element binding proteins.

Previous studies have shown that integrin activation and fluid shear stress can modulate the activity of sterol regulatory element binding proteins (SREBPs) in vascular endothelial cells. We investigated the role of small GTPase Rho-mediated signal transduction pathway in this mode of SREBP activation. Fluid shear stress activates the Rho downstream effectors ROCK, LIM kinase (LIMK), and cofilin. The various negative mutants of RhoA, ROCK, LIMK, and cofilin can block the shear stress activation of SREBPs. The shear stress-activated SREBP depends on S2P proteases but not caspase-3. Mechanistically, the endoplasmic reticulum-to-Golgi transport of SREBP cleavage-activating protein requires the actin-based cytoskeleton and is enhanced by the Rho-ROCK-LIMK-cofilin pathway. By enhancing the SREBP-mediated cholesterol metabolism, this unique mechanism may contribute to endothelial cell functions under flow.

Actin Depolymerizing Factors↗
PubMed2006

Initiation of cofilin activity in response to EGF is uncoupled from cofilin phosphorylation and dephosphorylation in carcinoma cells.

It has been demonstrated that the actin-severing activity of cofilin can be downregulated by LIM kinase (LIMK)-dependent phosphorylation at residue Ser3. Chemotactic stimulation in various cell types induces cofilin dephosphorylation, suggesting that cofilin activation in these cells occurs by a dephosphorylation mechanism. However, resting metastatic carcinoma cells have the majority of their cofilin in a dephosphorylated but largely inactive state. Stimulation with epidermal growth factor (EGF) induces an increase in cofilin activity after 60 seconds together with an increase in phosphorylated cofilin (p-cofilin), indicating that cofilin dephosphorylation is not coupled to cofilin activation in these cells. Suppression of LIMK function by inhibiting Rho-associated protein kinase (ROCK) or LIMK siRNA inhibited the EGF-induced cofilin phosphorylation but had no effect on cofilin activity or cofilin-dependent lamellipod protrusion induced by EGF. Correlation analysis revealed that cofilin, p-cofilin and LIMK are not colocalized, and changes in the location of these proteins upon stimulation with EGF indicate that they are not functionally coupled. Phospholipase C, which has been implicated in cofilin activation following stimulation with EGF, does not regulate p-cofilin levels following stimulation with EGF. Therefore, our results do not support a model for the initial activation of cofilin by dephosphorylation in response to chemoattractant stimulation in metastatic carcinoma cells.

Actin Depolymerizing Factors↗
PubMed2004

Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis.

The growth and morphological differentiation of dendrites are critical events in the establishment of proper neuronal connectivity and neural function. One extrinsic factor, BMP7, has been shown to specifically affect dendritic morphogenesis; however, the underlying mechanism by which this occurs is unknown. Here we show that LIM kinase 1 (LIMK1), a key downstream effector of Rho GTPases, colocalizes with the BMP receptor, BMPRII, in the tips of neurites and binds to BMPRII. This interaction is required for BMP-dependent induction of the dendritic arbor in cortical neurons. Furthermore, we demonstrate that the physical interaction of LIMK1 with BMPRII synergizes with the Rho GTPase, Cdc42, to activate LIMK1 catalytic activity. These studies thus define a Smad-independent pathway that directly links the BMP receptor to regulation of actin dynamics and provides insights into how extracellular signals modulate LIMK1 activity to permit fine spatial control over cytoskeletal remodelling during dendritogenesis.

Actin Depolymerizing Factors↗
PubMed1999

Molecular and clinical correlation study of Williams-Beuren syndrome: No evidence of molecular factors in the deletion region or imprinting affecting clinical outcome.

Williams-Beuren syndrome (WBS) results from a deletion of 7q11.23 in 90-95% of all clinically typical cases. Clinical manifestation can be variable and therefore, deletion size, inherited elastin (ELN) and LIM kinase 1 (LIMK1) alleles, gender, and parental origin of deletion have been investigated for associations with clinical outcome. In an analysis of 85 confirmed deletion cases, no statistically significant associations were found after Bonferroni's correction for multiple pairwise comparisons. Furthermore, the present data do not support presence of imprinted genes in the WBS common deletion despite a nonsignificant excess of maternal over paternal deletions. Maternal deletion cases were more likely to have a large head circumference in the present data. Also, pairwise comparisons between individual WBS clinical features have been conducted and revealed significant associations between (1) low birth weight and poor postnatal weight gain (<10th percentile at the time of examination) and (2) transient infantile hypercalcemia and a stellate iris pattern. The latter association could indicate a common underlying etiology.

Alleles↗
PubMed2003

Cell cycle-associated changes in Slingshot phosphatase activity and roles in cytokinesis in animal cells.

During cytokinesis the actomyosin-based contractile ring is formed at the equator, constricted, and then disassembled prior to cell abscission. Cofilin stimulates actin filament disassembly and is implicated in the regulation of contractile ring dynamics. However, little is known about the mechanism controlling cofilin activity during cytokinesis. Cofilin is inactivated by phosphorylation on Ser-3 by LIM-kinase-1 (LIMK1) and is reactivated by a protein phosphatase Slingshot-1 (SSH1). Here we show that the phosphatase activity of SSH1 decreases in the early stages of mitosis and is elevated in telophase and cytokinesis in HeLa cells, a time course correlating with that of cofilin dephosphorylation. SSH1 co-localizes with F-actin and accumulates onto the cleavage furrow and the midbody. Expression of a phosphatase-inactive SSH1 induces aberrant accumulation of F-actin and phospho-cofilin near the midbody in the final stage of cytokinesis and frequently leads to the regression of the cleavage furrow and the formation of multinucleate cells. Co-expression of cofilin rescued the inhibitory effect of phosphatase-inactive SSH1 on cytokinesis. These results suggest that SSH1 plays a critical role in cytokinesis by dephosphorylating and reactivating cofilin in later stages of mitosis.

Actin Depolymerizing Factors↗