Reorganization Of Stress Fibers Research Articles

Analysis of senescence-responsive stress fiber proteome reveals reorganization of stress fibers mediated by elongation factor eEF2 in HFF-1 cells.

Stress fibers (SFs), which are actomyosin structures, reorganize in response to various cues to maintain cellular homeostasis. Currently, the protein components of SFs are only partially identified, limiting our understanding of their responses. Here we isolate SFs from human fibroblasts HFF-1 to determine with proteomic analysis the whole protein components and how they change with replicative senescence (RS), a state where cells decline in the ability to replicate after repeated divisions. We found that at least 135 proteins are associated with SFs, and 63 of them are up-regulated with RS, by which SFs become larger in size. Among them, we focused on eEF2 (eukaryotic translation elongation factor 2) as it exhibited on RS the most significant increase in abundance. We show that eEF2 is critical to the reorganization and stabilization of SFs in senescent fibroblasts. Our findings provide a novel molecular basis for SFs to be reinforced to resist cellular senescence.

Roles of RAGE/ROCK1 Pathway in HMGB1-Induced Early Changes in Barrier Permeability of Human Pulmonary Microvascular Endothelial Cell.

BackgroundHigh mobility group box 1 (HMGB1) causes microvascular endothelial cell barrier dysfunction during acute lung injury (ALI) in sepsis, but the mechanisms have not been well understood. We studied the roles of RAGE and Rho kinase 1 (ROCK1) in HMGB1-induced human pulmonary endothelial barrier disruption.MethodsIn the present study, the recombinant human high mobility group box 1 (rhHMGB1) was used to stimulate human pulmonary microvascular endothelial cells (HPMECs). The endothelial cell (EC) barrier permeability was examined by detecting FITC-dextran flux. CCK-8 assay was used to detect cell viability under rhHMGB1 treatments. The expression of related molecules involved in RhoA/ROCK1 pathway, phosphorylation of myosin light chain (MLC), F-actin, VE-cadherin and ZO-1 of different treated groups were measured by pull-down assay, western blot and immunofluorescence. Furthermore, we studied the effects of Rho kinase inhibitor (Y-27632), ROCK1/2 siRNA, RAGE-specific blocker (FPS-ZM1) and RAGE siRNA on endothelial barrier properties to elucidate the related mechanisms.ResultsIn the present study, we demonstrated that rhHMGB1 induced EC barrier hyperpermeability in a dose-dependent and time-dependent manner by measuring FITC-dextran flux, a reflection of the loss of EC barrier integrity. Moreover, rhHMGB1 induced a dose-dependent and time-dependent increases in paracellular gap formation accompanied by the development of stress fiber rearrangement and disruption of VE-cadherin and ZO-1, a phenotypic change related to increased endothelial contractility and endothelial barrier permeability. Using inhibitors and siRNAs directed against RAGE and ROCK1/2, we systematically determined that RAGE mediated the rhHMGB1-induced stress fiber reorganization via RhoA/ROCK1 signaling activation and the subsequent MLC phosphorylation in ECs.ConclusionHMGB1 is capable of disrupting the endothelial barrier integrity. This study demonstrates that HMGB1 activates RhoA/ROCK1 pathway via RAGE, which phosphorylates MLC inducing stress fiber formation at short time, and HMGB1/RAGE reduces AJ/TJ expression at long term independently of RhoA/ROCK1 signaling pathway.

3D mechanical analysis of a self-contractile cell with stress fibers reorganization

Contractile stress generated by a cell itself is crucial in sensing its surrounding microenvironment and regulating cell adhesion, differentiation and cytokinesis. However, the precise mechanisms underlying mechanotransduction remain unknown. In this paper, based on the Eshelby inclusion problem, we develop a theoretical model to characterize quantitatively the mechanical environment around a self-contractile cell experiencing stress fibers reorganization. We divide the contractile stress into two parts: the constant contractile stress and the perturbed contractile stress due to stress fibers reorganization, for internal stress fibers have enough time to reorganize actively during long-term deformation, leading to changes of contractile stress in both magnitude and direction. Obtained results suggest that stress fibers reorganization may cause significant changes in the mechanical environment of the cell, helpful for exploring the mechanisms behind cell mechanotransduction.

Mechanoadaptive organization of stress fiber subtypes in epithelial cells under cyclic stretches and stretch release

Cyclic stretch applied to cells induces the reorganization of stress fibers. However, the correlation between the reorganization of stress fiber subtypes and strain-dependent responses of the cytoplasm and nucleus has remained unclear. Here, we investigated the dynamic involvement of stress fiber subtypes in the orientation and elongation of cyclically stretched epithelial cells. We applied uniaxial cyclic stretches at 5%, 10%, and 15% strains to cells followed by the release of the mechanical stretch. Dorsal, transverse arcs, and peripheral stress fibers were mainly involved in the cytoplasm responses whereas perinuclear cap fibers were associated with the reorientation and elongation of the nucleus. Dorsal stress fibers and transverse arcs rapidly responded within 15 min regardless of the strain magnitude to facilitate the subsequent changes in the orientation and elongation of the cytoplasm. The cyclic stretches induced the additional formation of perinuclear cap fibers and their increased number was almost maintained with a slight decline after 2-h-long stretch release. The slow formation and high stability of perinuclear cap fibers were linked to the slow reorientation kinetics and partial morphology recovery of nucleus in the presence or absence of cyclic stretches. The reorganization of stress fiber subtypes occurred in accordance with the reversible distribution of myosin II. These findings allowed us to propose a model for stretch-induced responses of the cytoplasm and nucleus in epithelial cells based on different mechanoadaptive properties of stress fiber subtypes.

Calponin-3 deficiency augments contractile activity, plasticity, fibrogenic response and Yap/Taz transcriptional activation in lens epithelial cells and explants

The transparent ocular lens plays a crucial role in vision by focusing light on to the retina with loss of lens transparency leading to impairment of vision. While maintenance of epithelial phenotype is recognized to be essential for lens development and function, knowledge of the identity of different molecular mechanisms regulating lens epithelial characteristics remains incomplete. This study reports that CNN-3, the acidic isoform of calponin, an actin binding contractile protein, is expressed preferentially and abundantly relative to the basic and neutral isoforms of calponin in the ocular lens, and distributes predominantly to the epithelium in both mouse and human lenses. Expression and MEKK1-mediated threonine 288 phosphorylation of CNN-3 is induced by extracellular cues including TGF-β2 and lysophosphatidic acid. Importantly, siRNA-induced deficiency of CNN3 in lens epithelial cell cultures and explants results in actin stress fiber reorganization, stimulation of focal adhesion formation, Yap activation, increases in the levels of α-smooth muscle actin, connective tissue growth factor and fibronectin, and decreases in E-cadherin expression. These results reveal that CNN3 plays a crucial role in regulating lens epithelial contractile activity and provide supporting evidence that CNN-3 deficiency is associated with the induction of epithelial plasticity, fibrogenic activity and mechanosensitive Yap/Taz transcriptional activation.

Cerebral ischemia induces TRPC6 via HIF1\u03b1/ZEB2 axis in the glomerular podocytes and contributes to proteinuria

Podocytes are specialized cells of the glomerulus and key component of the glomerular filtration apparatus (GFA). GFA regulates the permselectivity and ultrafiltration of blood. The mechanism by which the integrity of the GFA is compromised and manifest in proteinuria during ischemic stroke remains enigmatic. We investigated the mechanism of ischemic hypoxia-induced proteinuria in a middle cerebral artery occlusion (MCAO) model. Ischemic hypoxia resulted in the accumulation of HIF1α in the podocytes that resulted in the increased expression of ZEB2 (Zinc finger E-box-binding homeobox 2). ZEB2, in turn, induced TRPC6 (transient receptor potential cation channel, subfamily C, member 6), which has increased selectivity for calcium. Elevated expression of TRPC6 elicited increased calcium influx and aberrant activation of focal adhesion kinase (FAK) in podocytes. FAK activation resulted in the stress fibers reorganization and podocyte foot process effacement. Our study suggests overactive HIF1α/ZEB2 axis during ischemic-hypoxia raises intracellular calcium levels via TRPC6 and consequently altered podocyte structure and function thus contributes to proteinuria.

HIV glycoprotein gp120 enhances mesenchymal stem cell migration by upregulating CXCR4 expression

BackgroundHIV infection and/or the direct pathogenic effects of circulating HIV proteins impairs the physiological function of mesenchymal stem cells (MSCs), and contribute to the pathogenesis of age-related clinical comorbidities in people living with HIV. The SDF-1/CXCR4 pathway is vital for modulating MSC proliferation, migration and differentiation. HIV glycoprotein gp120 inhibits SDF-1 induced chemotaxis by downregulating the expression and function of CXCR4 in monocytes, B and T cells. The influence of gp120 on CXCR4 expression and migration in MSCs is unknown. MethodsWe investigated CXCR4 expression and SDF-1/CXCR4-mediated MSC migration in response to gp120, and its effect on downstream signaling pathways: focal adhesion kinase (FAK)/Paxillin and extracellular signal-regulated kinase (ERK). ResultsGp120 upregulated MSC CXCR4 expression. This potentiated the effects of SDF-1 in inducing chemotaxis; FAK/Paxillin and ERK pathways were over-activated, thereby facilitating actin stress fiber reorganization. CXCR4 blockage or depletion abrogated the observed effects. ConclusionGp120 from both T- and M- tropic HIV strains upregulated CXCR4 expression in MSCs, resulting in enhanced MSC chemotaxis in response to SDF-1. General significanceHIV infection and its proteins are known to disrupt physiological differentiation of MSC; increased gp120-driven migration amplifies the total MSC population destined for ineffective and inappropriate differentiation, thus contributing to the pathogenesis of HIV-related comorbidities. Additionally, given that MSCs are permissive to HIV infection, initial cellular priming by gp120 results in increased expression of CXCR4 and could lead to co-receptor switching and cell tropism changes in chronic HIV infection and may have implications against CCR5-knockout based HIV cure strategies.

MYH9 E1841K Mutation Augments Proteinuria and Podocyte Injury and Migration.

Intronic variants of the MYH9 gene that encodes the nonmuscle myosin heavy chain IIA are associated with diabetic nephropathy in European Americans and with sickle cell disease-associated nephropathy. However, the causal functional variants of MYH9 have remained elusive. Rare missense mutations in MYH9 cause macrothrombocytopenia and are occasionally associated with development of nephropathy. The E1841K mutation is among the common MYH9 missense mutations and has been associated with nephropathy in some carriers. To determine the contribution of the E1841K mutation in kidney disease, we studied the effects of the E1841K mutation in mice subjected to high salt or angiotensin II (Ang II) as models of hypertension and in mice subjected to renal mass reduction as a model of CKD. Despite similar levels of BP among wild-type (MYH9+/+ ) mice and mice heterozygous (MYH9+/E1841K ) and homozygous (MYH9E1841K/E1841K ) for the mutation in each model, MYH9E1841K/E1841K mice exhibited mildly increased albuminuria in response to high salt; severe albuminuria, nephrinuria, FSGS, and podocyte foot effacement in Ang II-induced hypertension; and early mortality in the renal mass reduction model. Treatment with candesartan during Ang II-induced hypertension attenuated kidney disease development in MYH9E1841K/E1841K mice. In vitro, isolated primary podocytes from MYH9E1841K/E1841K mice exhibited increased lamellipodia formation and reorganization of F-actin stress fibers. Wound healing assays revealed that MYH9+/+ podocytes had the lowest migration rate, followed by MYH9+/E1841K then MYH9E1841K/E1841K podocytes. In conclusion, the MYH9 E1841K variant alters podocyte cytoskeletal structure and renders podocytes more susceptible to injury after a damaging stimulus.

Uni-axial stretch induces actin stress fiber reorganization and activates c-Jun NH2 terminal kinase via RhoA and Rho kinase in human bladder smooth muscle cells.

BackgroundExcessive mechanical overload may be involved in bladder wall remodelling. Since the activity of Rho kinase is known to be upregulated in the obstructed bladder, we investigate the roles of the RhoA/Rho kinase pathway in mechanical overloaded bladder smooth muscle cells.MethodsHuman bladder smooth muscle cells were stimulated on silicon culture plates by 15 % elongated uni-axial cyclic stretch at 1 Hz. The activity of c-Jun NH2-terminal kinase was measured by western blotting and actin stress fibers were observed by stained with phallotoxin conjugated with Alexa-Fluor 594.ResultsThe activity of c-Jun NH2-terminal kinase 1 peaked at 30 min (4.7-fold increase vs. before stretch) and this activity was partially abrogated by the RhoA inhibitor, C3 exoenzoyme or by the Rho kinase inhibitor, Y-27632. Stretch induced the strong formation of actin stress fibers and these fibers re-orientated in a direction that was perpendicular to the stretch direction. The average angle of the fibers from the perpendicular to the direction of stretch was significantly different between before, and 4 h after, stretch. Actin stress fibers reorganization was also suppressed by the C3 exoenzyme or Y-27632.ConclusionsBladder smooth muscle cells appear to have elaborate mechanisms for sensing mechanical stress and for adapting to mechanical stress overload by cytoskeletal remodeling and by activating cell growth signals such as c-Jun NH2-terminal kinase via RhoA/Rho kinase pathways.

Actin Fusion Proteins Alter the Dynamics of Mechanically Induced Cytoskeleton Rearrangement

Mechanical forces can regulate various functions in living cells. The cytoskeleton is a crucial element for the transduction of forces in cell-internal signals and subsequent biological responses. Accordingly, many studies in cellular biomechanics have been focused on the role of the contractile acto-myosin system in such processes. A widely used method to observe the dynamic actin network in living cells is the transgenic expression of fluorescent proteins fused to actin. However, adverse effects of GFP-actin fusion proteins on cell spreading, migration and cell adhesion strength have been reported. These shortcomings were shown to be partly overcome by fusions of actin binding peptides to fluorescent proteins. Nevertheless, it is not understood whether direct labeling by actin fusion proteins or indirect labeling via these chimaeras alters biomechanical responses of cells and the cytoskeleton to forces. We investigated the dynamic reorganization of actin stress fibers in cells under cyclic mechanical loading by transiently expressing either egfp-Lifeact or eyfp-actin in rat embryonic fibroblasts and observing them by means of live cell microscopy. Our results demonstrate that mechanically-induced actin stress fiber reorganization exhibits very different kinetics in EYFP-actin cells and EGFP-Lifeact cells, the latter showing a remarkable agreement with the reorganization kinetics of non-transfected cells under the same experimental conditions.

Continuous Blood Purification Ameliorates RhoA-Mediated Endothelial Permeability in Severe Acute Pancreatitis Patients with Lung Injury

In the early phase of severe acute pancreatitis (SAP), serious pulmonary complications which are directly correlated with mortality are very common. Endothelial injury has been shown to play a key role in the pathogenesis of ALI/ARDS. Continuous blood purification (CBP) has been widely used in treating patients with multiple organ dysfunction syndrome (MODS) including ARDS. However, the impact of CBP on endothelial function has been little studied. Human umbilical vein endothelial cells (HUVECs) were exposed to serum samples or replacement fluid taken from patients at specific time points during CBP, or pretreated with Y-27632 followed by treatment with serum, then, changes in cytoskeletal configuration, endothelial monolayer permeability, and RhoA activation were studied. Endothelial permeability, RhoA activity, and stress fiber reorganization increased in HUVECs treated with serum from patients before CBP initiation, and lessened in HUVECs treated with serum from patients after CBP initiation. Endothelial hyperpermeability and stress fiber reorganization reduced in HUVECs pretreated with Rho-kinase inhibitor, Y-27632, and in a dose-dependent fashion. Endothelial permeability and RhoA activity increased in HUVECs treated with waste replacement fluid collected 2 h after CBP initiation. After CBP treatment, endothelial hyperpermeability induced by serum from SAP patients with lung injury was reduced. The inhibition of RhoA-mediated F-actin remodeling might be the mechanism.

Sarcomeric model of stretch-induced stress fiber reorganization

Sarcomeric model of stretch-induced stress fiber reorganization Roland Kaunas1, Hui-Ju Hsu1, Shinji Deguchi21Department of Biomedical Engineering, Texas A&M University, College Station, TX, USA; 2Department of Biomedical Engineering, Tohoku University, Sendai, JapanAbstract: Actin stress fibers (SFs) are mechanosensitive structural elements that respond to applied stress and strain to regulate cell morphology, signal transduction, and cell function. Results from various studies indicate that SFs tend to maintain stress or strain at a constant level. We developed a simple quantitative sarcomeric model of SFs to predict the role of actomyosin crossbridge cycling in SF tension regulation and reorientation in response to cyclic stretching. Under static conditions, the steady-state levels of SF tension were determined by the fiber passive stiffness and the stall force of the constituent myosin II filaments. When subject to cyclic changes in length at low frequencies, SFs change their unloaded reference length levels through myosin sliding to maintain tension at the original level. At high stretch frequencies, myosin cannot respond quickly enough and the SF behaves elastically. Myosin sliding also contributes to SF turnover, resulting in SF reorientation away from the direction of stretching at high, but not low, stretch frequencies. Using model parameters extracted from the literature, our model describes the dependence of cyclic stretch-induced SF alignment on stretch frequency and pattern consistent with experimental findings. This analysis predicts that myosin II plays multiple roles in regulating the ability of SFs to adapt to a dynamic mechanical environment.Keywords: mechanical stretch, cytoskeletal dynamics, myosin, mechanotransduction

Stretch-induced stress fiber remodeling and the activations of JNK and ERK depend on mechanical strain rate, but not FAK.

BackgroundCells within tissues are subjected to mechanical forces caused by extracellular matrix deformation. Cells sense and dynamically respond to stretching of the matrix by reorienting their actin stress fibers and by activating intracellular signaling proteins, including focal adhesion kinase (FAK) and the mitogen-activated proteins kinases (MAPKs). Theoretical analyses predict that stress fibers can relax perturbations in tension depending on the rate of matrix strain. Thus, we hypothesized stress fiber organization and MAPK activities are altered to an extent dependent on stretch frequency.Principal FindingsBovine aortic endothelial cells and human osteosarcoma cells expressing GFP-actin were cultured on elastic membranes and subjected to various patterns of stretch. Cyclic stretching resulted in strain rate-dependent increases in stress fiber alignment, cell retraction, and the phosphorylation of the MAPKs JNK, ERK and p38. Transient step changes in strain rate caused proportional transient changes in the levels of JNK and ERK phosphorylations without affecting stress fiber organization. Disrupting stress fiber contractile function with cytochalasin D or Y27632 decreased the levels of JNK and ERK phosphorylation. Previous studies indicate that FAK is required for stretch-induced cell alignment and MAPK activations. However, cyclic uniaxial stretching induced stress fiber alignment and the phosphorylation of JNK, ERK and p38 to comparable levels in FAK-null and FAK-expressing mouse embryonic fibroblasts.ConclusionsThese results indicate that cyclic stretch-induced stress fiber alignment, cell retraction, and MAPK activations occur as a consequence of perturbations in fiber strain. These findings thus shed new light into the roles of stress fiber relaxation and reorganization in maintenance of tensional homeostasis in a dynamic mechanical environment.

Group B Streptococcus induces tyrosine phosphorylation of annexin V and glutathione S-transferase in human umbilical vein endothelial cells

Group B Streptococcus (GBS), a human pathogen that causes infection and invasive diseases in newborns, pregnant women and immunocompromised adults, has been shown to invade human umbilical vein endothelial cells (HUVECs). The objective of this study was to investigate the molecular mechanisms underlying GBS-HUVEC interaction, focusing specifically on the responsiveness of host protein tyrosine kinase (PTK). We found that GBS serotypes III and V induced actin reorganization and formation of stress fibers into HUVECs. Since rearrangements of the actin cytoskeleton into eukaryotic cells are usually associated with the activation of PTK, we decided to follow the expression of this class of kinases in the course of the interaction. Unexpectedly, treatment of HUVECs with genistein greatly increased both cytoadherence and intracellular viability, for all GBS strains studied. GBS increased tyrosine phosphorylation of two proteins with an apparent molecular mass of 35 and 23 kDa in HUVECs as demonstrated by Western blot analysis with anti-phosphotyrosine antibodies. Mass spectra analysis identified these proteins as annexin V and glutathione S-transferase. Studies are in progress to identify the role of these two proteins on GBS-HUVEC interaction.

Nonmuscle myosin II is responsible for maintaining endothelial cell basal tone and stress fiber integrity

Cultured confluent endothelial cells exhibit stable basal isometric tone associated with constitutive myosin II regulatory light chain (RLC) phosphorylation. Thrombin treatment causes a rapid increase in isometric tension concomitant with myosin II RLC phosphorylation, actin polymerization, and stress fiber reorganization while inhibitors of myosin light chain kinase (MLCK) and Rho-kinase prevent these responses. These findings suggest a central role for myosin II in the regulation of endothelial cell tension. The present studies examine the effects of blebbistatin, a specific inhibitor of myosin II activity, on basal tone and thrombin-induced tension development. Although blebbistatin treatment abolished basal tension, this was accompanied by an increase in myosin II RLC phosphorylation. The increase in RLC phosphorylation was Ca(2+) dependent and mediated by MLCK. Similarly, blebbistatin inhibited thrombin-induced tension without interfering with the increase in RLC phosphorylation or in F-actin polymerization. Blebbistatin did prevent myosin II filament incorporation and association with polymerizing or reorganized actin filaments leading to the disappearance of stress fibers. Thus the inhibitory effects of blebbistatin on basal tone and induced tension are consistent with a requirement for myosin II activity to maintain stress fiber integrity.

Morphological Responses of Vascular Endothelial Cells Induced by Local Stretch Transmitted Through Intercellular Junctions

It has been well established that mechanical stimuli including fluid shear stress and cyclic stretch play a key role in endothelial cell (EC) remodeling. However, in contrast to global remodeling to these mechanical stimuli, little is known of how local mechanical forces are transmitted through cells to induce cell remodeling leading to alteration in cell functions. In this study, we demonstrated that EC remodeling can be exerted by local tension generated in a neighboring EC. In this technique, a glass microneedle was used to apply local stretch in an EC in confluent monolayer and the resulting tension is transmitted to a neighboring EC across intercellular junctions. Local stretch induced reorientation and elongation of ECs parallel to the direction of stretch associated with reorganization of stress fibers. In addition, recruitment of Src homology 2-containing tyrosine phosphatase-2, binding to intercellular adhesion molecules platelet-endothelial cellular adhesion molecules-1, was selectively observed at the force-transmitted intercellular junctions after application of local stretch. These findings suggest that intercellular junctions can not only transmit but also sense local forces, and are potentially involved in EC mechanotransduction pathways.

Regulation of Endothelial Cell Cytoskeletal Reorganization by a Secreted Frizzled-Related Protein-1 and Frizzled 4- and Frizzled 7-Dependent Pathway: Role in Neovessel Formation

Consistent with findings of Wnt pathway members involved in vascular cells, a role for Wnt/Frizzled signaling has recently emerged in vascular cell development. Among the few Wnt family members implicated in vessel formation in adult, Wnt7b and Frizzled 4 have been shown as involved in vessel formation in the lung and in the retina, respectively. Our previous work has shown a role for secreted Frizzled-related protein-1 (sFRP-1), a proposed Wnt signaling inhibitor, in neovascularization after an ischemic event and demonstrated its role as a potent angiogenic factor. However the mechanisms involved have not been investigated. Here, we show that sFRP-1 treatment increases endothelial cell spreading on extracellular matrix as revealed by actin stress fiber reorganization in an integrin-dependent manner. We demonstrate that sFRP-1 can interact with Wnt receptors Frizzled 4 and 7 on endothelial cells to transduce downstream to cellular machineries requiring Rac-1 activity in cooperation with GSK-3beta. sFRP-1 overexpression in endothelium specifically reversed the inactivation of GSK-3 beta and increased neovascularization in ischemia-induced angiogenesis in mouse hindlimb. This study illustrates a regulated pathway by sFRP-1 involving GSK-3beta and Rac-1 in endothelial cell cytoskeletal reorganization and in neovessel formation.

A Peptide Derived from Tenascin-C Induces β1 Integrin Activation through Syndecan-4

Tenascin-C (TN-C) is unique for its cell adhesion modulatory function. We have shown that TNIIIA2, a synthetic 22-mer peptide derived from TN-C, stimulated beta1 integrin-mediated cell adhesion of nonadherent and adherent cell types, by inducing activation of beta1 integrin. The active site of TNIIIA2 appeared cryptic in the TN-C molecule but was exposed by MMP-2 processing of TN-C. The following results suggest that cell surface heparan sulfate (HS) proteoglycan (HSPG), including syndecan-4, participated in TNIIIA2-induced beta1 integrin activation: 1) TNIIIA2 bound to cell surface HSPG via its HS chains, as examined by photoaffinity labeling; 2) heparitinase I treatment of cells abrogated beta1 integrin activation induced by TNIIIA2; 3) syndecan-4 was isolated by affinity chromatography using TNIIIA2-immobilized beads; 4) small interfering RNA-based down-regulation of syndecan-4 expression reduced TNIIIA2-induced beta1 integrin activation, and consequent cell adhesion to fibronectin; 5) overexpression of syndecan-4 core protein enhanced TNIIIA2-induced activation of beta1 integrin. However, treatments that targeted the cytoplasmic region of syndecan-4, including ectopic expression of its mutant truncated with the cytoplasmic domains and treatment with protein kinase Calpha inhibitor Gö6976, did not influence the TNIIIA2 activity. These results suggest that a TNIIIA2-related matricryptic site of the TN-C molecule, exposed by MMP-2 processing, may have bound to syndecan-4 via its HS chains and then induced conformational change in beta1 integrin necessary for its functional activation. A lateral interaction of beta1 integrin with the extracellular region of the syndecan-4 molecule may be involved in this conformation change.

Substrate stretching and reorganization of stress fibers as a finite elasticity problem

It is well known that substrate stretching reorganizes the actin cytoskeleton of an adherent cell. Experiments have proved that the stress fibers are reoriented into one or two distinct directions. It is further pointed out that reorientation of the stress fibers phenomena are observed with quite high strains, where linear elasticity theory is not valid. Therefore, the existing linear theories for mechanics interpretation of those phenomena are not satisfactory. The experimental evidence of the existence of two distinct directions of the reorientation indicates a co-existence of phases non-linear elastic phenomenon. Further that observation is quite strong evidence that the strain energy density function should be non-convex. The reorganization of the stress fibers under uniaxial substrate stretching is studied as a non-linear elastic stability problem. Adapting the global (Maxwell’s) criterion for stability, various phenomena, concerning the reorientation of the stress fibers, are interpreted into the context of finite elasticity. The experiments demonstrate co-existence of phases phenomena that are justified in the present study. The influence of various factors like contractility and extracellular stretching is demonstrated. Furthermore, the non-convexity factor is attributed to the influence of the small GTPase Rho regulating the formation of the actin stress fibers. The predominant final stress fibers placement that is transverse to the extracellular stress direction and appears in long time after the co-existence of phases placement is also justified.

Roles of Rho/ROCK and MLCK in TNF‐α‐induced changes in endothelial morphology and permeability

Tumor necrosis factor-alpha (TNF-alpha) is known to induce changes in endothelial cell morphology and permeability, but the mechanisms have not been extensively characterized. TNF-alpha rapidly induced RhoA activation and myosin light chain phosphorylation, but caused only small changes to cortical F-actin, without significantly increasing paracellular permeability up to 30 min after stimulation. TNF-alpha subsequently caused a progressive increase in permeability and in stress fiber reorganization, cell elongation, and intercellular gap formation over 8-24 h. Consistent with the increased permeability, Occludin and JAM-A were removed from tight junctions and ZO-1 was partially redistributed. Rho/ROCK but not MLCK inhibition prevented the long-term TNF-alpha-induced changes in F-actin and cell morphology, but ROCK inhibition did not affect permeability. These results suggest that the gradual increase in permeability induced by TNF-alpha does not reflect contractile mechanisms mediated by Rho, ROCK, and MLCK, but involves long-term reorganization of tight junction proteins.