Occludin regulates actin cytoskeleton in endothelial cells

Occludin is a major membrane component of tight junctions of endothelial cells, though the role of this molecule is not fully understood. RLE cells, derived from rat lung endothelial cells, express a negligible level of occludin with clear expression of E-cadherin and ZO-1 at cell junctions. Introduction of occludin by transfection induced clear junctional expression of occludin with few or no changes of expression of E-cadherin and ZO-1. The paracellular barrier function, as determined by transelectrical resistance and flux of non-ionic small molecules, was not detectably upregulated. When cells expressing occludin were cocultured with RLE cells null for occludin, clear junctional expression of occludin was observed irrespective of the expression of occludin on the apposing cells. Cortical actin was developed at the site of these occludin positive cell junctions. Treatment of cells with an actin depolymerizing agent, mycalolide B, abolished junctional expression of occludin together with E-cadherin and circumferential actin. ZO-1 showed relative resistance to this actin depolymerizing treatment and was maintained at the cell junctions, though fragmentation of immunoreactivity was detectable. Collectively, junctional expression of occludin was not associated with paracellular barrier function in this cell line. There was, however, a close correlation of occludin with the actin cytoskeleton, indicating a role of occludin as an important molecule in the regulation of the actin cytoskeleton in endothelial cells.     

Disruption of the actin cytoskeleton regulates cytokine-induced iNOS expression

Interleukin-1 (IL-1) induces the induciblenitric oxide synthase (iNOS), resulting in the release of nitric oxide(NO) from glomerular mesangial cells. In this study, we demonstratedthat disruption of F-actin formation by sequestration of G-actin with the toxin latrunculin B (LatB) dramatically potentiated IL-1-induced iNOS protein expression in a dose-dependent manner. LatB by itself hadlittle or no effect on iNOS expression. Staining of F-actin withnitrobenzoxadiazole (NBD)-phallacidin demonstrated that LatB significantly impaired F-actin stress fiber formation. Jasplakinolide (Jasp), which binds to and stabilizes F-actin, suppressed iNOS expression enhanced by LatB. These data strongly suggest that actincytoskeletal dynamics regulates IL-1-induced iNOS expression. Wedemonstrated that LatB decreases serum response factor (SRF) activityas determined by reporter gene assays, whereas Jasp increases SRFactivity. The negative correlation between SRF activity and iNOSexpression suggests a negative regulatory role for SRF in iNOSexpression. Overexpression of a dominant negative mutant of SRFincreases the IL-1-induced iNOS expression, providing directevidence that SRF inhibits iNOS expression.

     

TrkB-T1 regulates the RhoA signaling and actin cytoskeleton in glioma cells

Recently, the truncated TrkB receptor, T1, has been reported to be involved in the control of cell morphology via the regulation of Rho proteins, through which T1 binds Rho guanine nucleotide dissociation inhibitor (Rho GDI) 1 and dissociates it in a brain-derived neurotrophic factor (BDNF)-dependent manner. However, it is unclear whether T1 signaling regulates the downstream of Rho signaling and the actin cytoskeleton. In this study, we investigated this question using C6 rat glioma cells, which express T1 endogenously. Rho GDI1 was dissociated from T1 in a BDNF-dependent manner, which also causes decreases in the activities of Rho-signaling molecules such as RhoA, Rho-associated kinase, p21-activated kinase, and extracellular-signal regulated kinase1/2. Moreover, BDNF treatment resulted in the disappearance of stress fibers in the cells treated with lysophosphatidic acid, an activator of RhoA, and in morphological changes in cells. Furthermore, a competitive assay with cyan fluorescent protein fusion proteins of T1-specific sequences reduced the effects of BDNF. These results suggest that T1 regulates the Rho-signaling pathways and the actin cytoskeleton.     

Glucocorticoid receptor-mediated expression of caldesmon regulates cell migration via the reorganization of the actin cytoskeleton

Glucocorticoids (GCs) play important roles in numerous cellular processes, including growth, development, homeostasis, inhibition of inflammation, and immunosuppression. Here we found that GC-treated human lung carcinoma A549 cells exhibited the enhanced formation of the thick stress fibers and focal adhesions, resulting in suppression of cell migration. In a screen for GC-responsive genes encoding actin-interacting proteins, we identified caldesmon (CaD), which is specifically up-regulated in response to GCs. CaD is a regulatory protein involved in actomyosin-based contraction and the stability of actin filaments. We further demonstrated that the up-regulation of CaD expression was controlled by glucocorticoid receptor (GR). An activated form of GR directly bound to the two glucocorticoid-response element-like sequences in the human CALD1 promoter and transactivated the CALD1 gene, thereby up-regulating the CaD protein. Forced expression of CaD, without GC treatment, also enhanced the formation of thick stress fibers and focal adhesions and suppressed cell migration. Conversely, depletion of CaD abrogated the GC-induced phenotypes. The results of this study suggest that the GR-dependent up-regulation of CaD plays a pivotal role in regulating cell migration via the reorganization of the actin cytoskeleton.     

Caldesmon regulates the motility of vascular smooth muscle cells by modulating the actin cytoskeleton stability

Background  

Migration of vascular smooth muscle cells (SMCs) from the media to intima constitutes a critical step in the development of proliferative vascular diseases. To elucidate the regulatory mechanism of vacular SMC motility, the roles of caldesmon (CaD) and its phosphorylation were investigated.     

Regulation of the actin cytoskeleton in living cells

The dynamic behavior of pure actin in vitro is more complex than that of most simple polymers, due to the energy input from the irreversible nucleotide hydrolysis associated with polymerization. However, the dynamic behavior of actin is vastly more complicated inside cells, where dozens of different types of actin-binding proteins alter every rate constant for actin polymerization and the chemical environment is inhomogeneous both temporally and spatially. Actin dynamics in cells are tightly regulated, so that rapid filament polymerization can occur in response to external signals or at the front of an active lamellipodium, while rapid depolymerization occurs simultaneously elsewhere in the cell. Although more direct observations of actin dynamics in vivo are accumulating, it is not yet clear how to reconcile the behavior of actin in cells with its well-documented in vitro properties.     

Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton

We have previously shown that tyrosine phosphorylation of the actin-regulatory protein villin is accompanied by the redistribution of phosphorylated villin and a concomitant decrease in the F-actin content of intestinal epithelial cells. The temporal and spatial correlation of these two events suggested that tyrosine phosphorylation of villin may be involved in the rearrangement of the microvillar cytoskeleton. This hypothesis was investigated by analyzing the effects of tyrosine phosphorylation of villin on the kinetics of actin polymerization by reconstituting in vitro the tyrosine phosphorylation of villin and its association with actin. Full-length recombinant human villin was phosphorylated in vitro by expression in the TKX1-competent cells that carry an inducible tyrosine kinase gene. The actin-binding properties of villin were examined using a co-sedimentation assay. Phosphorylation of villin did not change the stoichiometry (1:2) but decreased the binding affinity (4.4 microm for unphosphorylated versus 0.6 microm for phosphorylated) of villin for actin. Using a pyrene-actin-based fluorescence assay, we demonstrated that tyrosine phosphorylation had a negative effect on actin nucleation by villin. In contrast, tyrosine phosphorylation enhanced actin severing by villin. Electron microscopic analysis showed complementary morphological changes. Phosphorylation inhibited the actin bundling and enhanced the actin severing functions of villin. Taken together our data show that tyrosine phosphorylation of villin decreases the amount of villin bound to actin filaments, inhibits the actin-polymerizing properties of villin, and promotes the actin-depolymerizing functions instead. These observations suggest a role for tyrosine phosphorylation in modulating the microvillar cytoskeleton in vivo by villin in response to specific physiological stimuli.     

Regulation of actin cytoskeleton dynamics in cells

The dynamic remolding of the actin cytoskeleton is a critical part of most cellular activities, and malfunction of cytoskeletal proteins results in various human diseases. The transition between two forms of actin, monomeric or G-actin and filamentous or F-actin, is tightly regulated in time and space by a large number of signaling, scaffolding and actin-binding proteins (ABPs). New ABPs are constantly being discovered in the post-genomic era. Most of these proteins are modular, integrating actin binding, protein-protein interaction, membrane-binding, and signaling domains. In response to extracellular signals, often mediated by Rho family GTPases, ABPs control different steps of actin cytoskeleton assembly, including filament nucleation, elongation, severing, capping, and depolymerization. This review summarizes structure-function relationships among ABPs in the regulation of actin cytoskeleton assembly.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Association of villin with phosphatidylinositol 4,5-bisphosphate regulates the actin cytoskeleton

Villin, an epithelial cell actin-binding protein, severs actin in vitro and in vivo. Previous studies report that phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates actin severing by villin, presumably by interaction with villin. However, direct association of villin with PIP(2) has never been characterized. In this report, we presented mutational analysis to identify the PIP(2)-binding sites in villin. Villin (human) binds PIP(2) with a K(d) of 39.5 microm, a stoichiometry of 3.3, and a Hill coefficient of 1. We generated deletion mutants of villin lacking putative PIP(2)-binding sites and examined the impact of these mutations on PIP(2) binding and actin dynamics. Our analysis revealed the presence of three PIP(2)-binding sites, two in the amino-terminal core and one in the carboxyl-terminal headpiece of human villin. Synthetic peptides analogous with these sites confirmed the binding domains. Circular dichroism and quenching of intrinsic tryptophan fluorescence revealed a significant conformational change in these peptides ensuing in their association with PIP(2). By using site-directed mutagenesis (arginine 138 to alanine), we demonstrated the presence of an identical F-actin and PIP(2)-binding site in the capping and severing domain of villin. In contrast, the mutants lysine 822 and 824 to alanine demonstrated the presence of an overlapping F-actin and PIP(2)-binding site in the actin cross-linking domain of villin. Consistent with this observation, association of villin with PIP(2) inhibited the actin capping and severing functions of villin and enhanced the actin bundling function of villin. Our studies revealed that structural changes induced by association with PIP(2) could regulate the actin-modifying functions of villin. This study provided biochemical proof of the functional significance of villin association with PIP(2) and identified the molecular mechanisms involved in the regulation of actin dynamics by villin and PIP(2).     

The actin cytoskeleton regulates LFA-1 ligand binding through avidity rather than affinity changes.

To elucidate the role of the cytoskeleton regulating avidity or affinity changes in the leukocyte adhesion receptor lymphocyte function-associated antigen-1 (LFA-1) (alpha(L)beta(2)), we generated mutant cytoplasmic LFA-1 receptors and expressed these into the erythroleukemic cell line K562. We determined whether intercellular adhesion molecule-1 (ICAM-1)-mediated adhesion of LFA-1, lacking parts of its cytoplasmic tails, is regulated through receptor diffusion/clustering and/or by altered ligand binding affinity. All cytoplasmic deletion mutants that lack the complete beta(2) cytoplasmic tail and/or the conserved KVGFFKR sequence in the alpha(L) cytoplasmic tail were constitutively active and expressed high levels of the activation epitopes NKI-L16 and M24. Surprisingly, whereas these mutants showed a clustered cell surface distribution of LFA-1, the ligand-binding affinity as measured by titration of soluble ligand ICAM-1 remained unaltered. The notion that redistribution of LFA-1 does not alter ligand-binding affinity is further supported by the finding that disruption of the cytoskeleton by cytochalasin D did not alter the binding affinity nor adhesion to ICAM-1 of these mutants. Most cytoplasmic deletion mutants that spontaneously bound ICAM-1 were not capable to spread on ICAM-1, demonstrating that on these mutants LFA-1 is not coupled to the actin cytoskeleton. From these data we conclude that LFA-1-mediated cell adhesion to ICAM-1 is predominantly regulated by receptor clustering and that affinity alterations do not necessarily coincide with strong ICAM-1 binding.     

Disorders of the actin cytoskeleton in transformed epithelial cells

The actin cytoskeleton of 8 transformed epithelial cell lines was studied using electron microscopy of platinum replicas. Seven of these lines belonged to the IAR series of rat liver epithelial cells, being at different stages of neoplastic progression. One cell line (FBT) was derived from the epithelium of bovine fetal trachea. The extent of actin cytoskeleton alteration in cell lines studied has been shown to correlate with other signs of neoplastic transformation. Among various actin-containing cell structures (microfilament bundles, actin meshwork at active edges, cell-cell adherence junctions, and endoplasmic microfilament sheath) the latter was the most sensitive to transformation. The loosening of the sheath and the alteration of its fine structure were observed in all the cell lines. The degree of these changes increased in the following order: FBT; non-tumorigenic IAR lines; IAR lines transformed in vitro; IAR lines obtained from the latter by single or double selection in vivo. The alteration of sheath was the only disturbance of actin cytoskeleton in FBT cells, whereas in other groups of epithelial cell lines some other changes occurred. These involved disruption of actin-containing intercellular junctions, the cell polarization accompanied by progressive shortening of length of the cell active edge containing actin meshwork, and disappearance or reorganization of microfilament bundles.     

The actin cytoskeleton regulates exocytosis of all neutrophil granule subsets

A comprehensive analysis of the role of the actin cytoskeleton in exocytosis of the four different neutrophil granule subsets had not been performed previously. Immunoblot analysis showed that, compared with plasma membrane, there was less actin associated with secretory vesicles (SV, 75%), gelatinase granules (GG, 40%), specific granules (SG, 10%), and azurophil granules (AG, 5%). Exocytosis of SV, SG, and AG was measured as increased plasma membrane expression of CD35, CD66b, and CD63, respectively, with flow cytometry, and GG exocytosis was measured as gelatinase release with an ELISA. N-formylmethionyl-leucyl-phenylalanine (FMLP) stimulated exocytosis of SV, GG, and SG with an ED₅₀ of 15, 31, and 28 nM, respectively, with maximal response at 10^–7 M FMLP by 5 min, while no exocytosis of AG was detected. Disruption of the actin cytoskeleton by latrunculin A and cytochalasin D induced a decrease in FMLP-stimulated CD35 expression after an initial increase. Both drugs enhanced the rate and extent of FMLP-stimulated GG, SG, and AG exocytosis, while the EC₅₀ for FMLP was not altered. We conclude that the actin cytoskeleton controls access of neutrophil granules to the plasma membrane, thereby limiting the rate and extent of exocytosis of all granule subsets. Differential association of actin with the four granule subsets was not associated with graded exocytosis. human; cell activation     

p120 catenin regulates the actin cytoskeleton via Rho family GTPases

Cadherins are calcium-dependent adhesion molecules responsible for the establishment of tight cell-cell contacts. p120 catenin (p120ctn) binds to the cytoplasmic domain of cadherins in the juxtamembrane region, which has been implicated in regulating cell motility. It has previously been shown that overexpression of p120ctn induces a dendritic morphology in fibroblasts (Reynolds, A.B. , J. Daniel, Y. Mo, J. Wu, and Z. Zhang. 1996. Exp. Cell Res. 225:328-337.). We show here that this phenotype is suppressed by coexpression of cadherin constructs that contain the juxtamembrane region, but not by constructs lacking this domain. Overexpression of p120ctn disrupts stress fibers and focal adhesions and results in a decrease in RhoA activity. The p120ctn-induced phenotype is blocked by dominant negative Cdc42 and Rac1 and by constitutively active Rho-kinase, but is enhanced by dominant negative RhoA. p120ctn overexpression increased the activity of endogenous Cdc42 and Rac1. Exploring how p120ctn may regulate Rho family GTPases, we find that p120ctn binds the Rho family exchange factor Vav2. The behavior of p120ctn suggests that it is a vehicle for cross-talk between cell-cell junctions and the motile machinery of cells. We propose a model in which p120ctn can shuttle between a cadherin-bound state and a cytoplasmic pool in which it can interact with regulators of Rho family GTPases. Factors that perturb cell-cell junctions, such that the cytoplasmic pool of p120ctn is increased, are predicted to decrease RhoA activity but to elevate active Rac1 and Cdc42, thereby promoting cell migration.     

An endostatin-derived peptide interacts with integrins and regulates actin cytoskeleton and migration of endothelial cells

Endostatin, the C-terminal fragment of collagen XVIII, is a potent inhibitor of angiogenesis and endothelial cell migration. To define its critical cell interaction domains we used endostatin-derived synthetic peptides containing surface-exposed sequences. We observed that, when immobilized, an arginine-rich peptide of 11 amino acids from its N terminus efficiently promoted endothelial cell adhesion through beta(1) integrin- and heparin-dependent mechanisms. In addition, the peptide induced the formation of membrane ruffles and focal contacts. In the soluble form, the peptide inhibited basic fibroblast growth factor-induced directional migration and tubular morphogenesis of microvascular endothelial cells. Accordingly, the peptide induced the loss of focal adhesions and actin stress fibers in these cells. Substitution of the arginine residues with alanines resulted in the loss of these properties. In the current study we describe a putative integrin-binding sequence with anti-migratory activity within endostatin.     

Benzamide-induced changes in the actin cytoskeleton in human skin fibroblasts.

The incubation of human skin fibroblasts in the presence of 10 mM benzamide in Joklik's modification of Eagle's Minimal Essential Medium caused an extensive reorganization of actin filaments. The disappearance of stress fibers and changes in cell morphology were observed, whereas no changes in the microtubule architecture were noticed. The observed effects appeared fully reversible within 3 hours after the removal of benzamide. The results are discussed in relation to the two known activities of benzamide as an anaesthetic and an inhibitor of ADP-ribosylation.     

Glycine-rich region regulates cysteine-rich protein 1 binding to actin cytoskeleton

Cysteine-rich protein 1 (CRP1) has a unique structure with two well separated LIM domains, each followed by a glycine-rich region. Although CRP1 has been shown to interact with actin-binding proteins and actin filaments, the mechanism regulating localization to the actin cytoskeleton in cells is not clear. Experiments using truncated forms showed that the first LIM domain and glycine-rich region are necessary for CRP1 bundling of actin filaments and localization to the actin cytoskeleton. Furthermore, domain swapping experiments replacing the first glycine-rich region with the second resulted in the loss of CRP1 bundling activity and localization to the actin cytoskeleton, identifying seven critical amino acid residues. These results highlight the importance of the first glycine-rich region for CRP1 bundling activity and localization to the actin cytoskeleton. In addition, this work identifies the first LIM domain and glycine-rich region as a distinct actin filament bundling module.