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Stiffness responses of fibroblasts were measured by scanning probe microscopy, following elongation or compression by deformation of an elastic substrate by 8%. The cellular stiffness, reflecting intracellular tension acting along stress fibers, decreased or increased instantly in response to the elongating or compressing stimuli, respectively. After this rapid change, the fibroblasts gradually recovered to their initial stiffness during the following 2 h, and then stabilized. The cells did not show conspicuous changes in shape after the 8% deformation during the SPM measurements. Fluorescence examination for GFP-actin demonstrated that the structure of the stress fibers was not altered noticeably by this small degree of deformation. Treatment with Y-27632, to inhibit myosin phosphorylation and abrogate cellular contractility, eliminated the change in stiffness after the mechanical elongation. These results indicate that fibroblasts possess a mechanism that regulates intracellular tension along stress fibers to maintain the cellular stiffness in a constant equilibrium state., (2004 Wiley-Liss, Inc.)

Hemoglobin (Hb)-based O2 carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (PDH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing alpha-human and beta-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)n (molecular mass: approximately 1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 x 10(-7) vs. 2.47 x 10(-6) cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of PDH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.

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.

Cell adhesion to the extracellular matrix triggers the formation of integrin-mediated contact and reorganization of the actin cytoskeleton. Examination of nascent adhesions, formed during early stages of fibroblast spreading, reveals a variety of forms of actin-associated matrix adhesions. These include: (1). small ( approximately 1 microm), dot-like, integrin-, vinculin-, paxillin-, and phosphotyrosine-rich structures, with an F-actin core, broadly distributed over the ventral surfaces of the cells; (2). integrin-, vinculin-, and paxillin-containing "doublets" interconnected by short actin bundles; (3). arrays of actin-vinculin complexes. Such structures were formed by freshly plated cells, as well as by cells recovering from latrunculin treatment. Time-lapse video microscopy of such cells, expressing GFP-actin, indicated that long actin cables are formed by an end-to-end lining-up and apparent fusion of short actin bundles. All these structures were prominent during cell spreading, and persisted for up to 30-60 min after plating. Upon longer incubation, they were gradually replaced by stress fibers, associated with focal adhesions at the cell periphery. Direct examination of paxillin and actin reorganization in live cells revealed alignment of paxillin doublets, forming long and highly dynamic actin bundles, undergoing translocation, shortening, splitting, and convergence. The mechanisms underlying the assembly and reorganization of actin-associated focal adhesions and the involvement of mechanical forces in regulating their dynamic properties are discussed., (Copyright 2004 Wiley-Liss, Inc.)

To study the dynamics of stress fiber components in cultured fibroblasts, we expressed alpha-actinin and the myosin II regulatory myosin light chain (MLC) as fusion proteins with green fluorescent protein. Myosin activation was stimulated by treatment with calyculin A, a serine/threonine phosphatase inhibitor that elevates MLC phosphorylation, or with LPA, another agent that ultimately stimulates phosphorylation of MLC via a RhoA-mediated pathway. The resulting contraction caused stress fiber shortening and allowed observation of changes in the spacing of stress fiber components. We have observed that stress fibers, unlike muscle myofibrils, do not contract uniformly along their lengths. Although peripheral regions shortened, more central regions stretched. We detected higher levels of MLC and phosphorylated MLC in the peripheral region of stress fibers. Fluorescence recovery after photobleaching revealed more rapid exchange of myosin and alpha-actinin in the middle of stress fibers, compared with the periphery. Surprisingly, the widths of the myosin and alpha-actinin bands in stress fibers also varied in different regions. In the periphery, the banding patterns for both proteins were shorter, whereas in central regions, where stretching occurred, the bands were wider.

We have examined the role of endogenous 70-kDa S6 kinase (p70(S6K)) in actin cytoskeletal organization and cell migration in Swiss 3T3 fibroblasts. Association of p70(S6K) with the actin cytoskeleton was demonstrated by cosedimentation of p70(S6K) with F-actin and by subcellular fractionation in which p70(S6K) activity was measured in the F-actin cytoskeletal fraction. Immunocytochemical studies showed that p70(S6K), Akt1, PDK1, and p85 phosphoinositide 3-kinase (PI 3-kinase) were localized to the actin arc, a caveolin-enriched cytoskeletal structure located at the leading edge of migrating cells. Using a phospho-specific antibody to mammalian target of rapamycin (mTOR), we find that activated mTOR is enriched at the actin arc, suggesting that activation of the p70(S6K) signaling pathway is important to cell migration. Using the actin arc to assess migration, epidermal growth factor (EGF) stimulation was found to induce actin arc formation, an effect that was blocked by rapamycin treatment. We show further that actin stress fibers may function to down-regulate p70(S6K). Fibronectin stimulated stress fiber formation in the absence of growth factors and caused an inactivation of p70(S6K). Conversely, cytochalasin D and the Rho kinase inhibitor Y-27632, both of which cause stress fiber disruption, increased p70(S6K) activity. These studies provide evidence that the p70(S6K) pathway is important for signaling at two F-actin microdomains in cells and regulates cell migration.

Objectives: To investigate the time-dependent effects of serum from burned rats on cytoskeletal filamentous actin (F-actin) reorganization by visualizing their distribution in human umbilical vein endothelial cell line ECV-304 and evaluate the role of myosin light-chain kinase (MLCK) in this process., Methods: The serum-starved ECV-304 cells were incubated with the serum from burned rats for 30 min, 1, 2, 4, and 6 h, respectively, and 30 min before or after the incubation, the cells were treated with 5 micromol/L ML-7 for 30 min. F-actin was stained with rhodamine-phalloidin and observed under fluorescence microscope., Results: Under normal condition, F-actin was distributed mainly in the cortical area of the endothelial cells. After stimulation with the burn serum, stress fiber formation could be clearly seen in the endothelial cells, exhibiting a time-dependent enhancement in a time course ranging from 30 min to 6 h. Such an effect could be significantly inhibited by a 30-min pretreatment of the cells with MLCK-specific inhibitor ML-7. Inhibition of MLCK also reversed actin reorganization in the endothelial cells pretreated with the burn serum., Conclusion: Serum from burned rats induces characteristic morphological changes in the endothelial cell actin cytoskeleton mainly due to the MLCK activation, an effect that can be reversed by the inhibition of MLCK.

Endothelial cells undergo profound morphologic changes in response to alterations in shear stresses that are imposed on them by blood flow, and these responses have important implications for physiologic and pathophysiologic function of blood vessels. Shear-induced changes in cell morphology represent a unique mode of cell motility: elongation of the cells in the direction of shear stress is achieved by a reorientation and assembly of F-actin stress fibers at the basal cell surface that ultimately protrudes the upstream and downstream limits of the plasma membrane. Shape change is also accommodated by dramatic reorganization of cell-substrate and cell-cell junctional complexes. Both of these structures are sites of mechanotransduction, which raises intriguing questions concerning how shear-induced regulation of endothelial cell physiology is integrated with cell morphologic responses.

The interactions between Notch (N) receptors and their transmembrane ligands, Jagged1 (JI) and Delta1 (Dl1), mediate signaling events between neighboring cells that are crucial during embryonal development and in adults. Since the non-transmembrane extracellular form of J1 acts as an antagonist of N activation in NIH 3T3 mouse fibroblast cells and induces fibroblast growth factor 1 (FGF1)-dependent transformation (Small, D., Kovalenko, D., Soldi, R., Mandinova, A., Kolev, V., Trifonova, R., Bagala, C., Kacer, D., Battelli, C., Liaw, L., Prudovsky, I., and Maciag, T. (2003) J. Biol. Chem. 278, 16405-16413), we examined the potential redundant functions of the two subfamilies of Notch ligands and report that while the soluble (s) forms of both Dl1 and J1 act as N signaling antagonists in NIH 3T3 cells, they do display disparate functions. While sJ1 induced an attenuation of cell motility which is accompanied by a decrease in actin stress fibers and an increase in adherence junctions, sDl1 does not. However, sJ1, like sDl1, induces a NIH 3T3 cell tranformed phenotype mediated by FGF signaling. Because the inhibition of N signaling by sJ1 and sDl1 is rescued by dominant-negative Src expression, we suggest that there may be cooperation between the Notch and Src signaling pathways.

Fluid shear stress greatly influences the biology of vascular endothelial cells and the pathogenesis of atherosclerosis. Endothelial cells undergo profound shape change and reorientation in response to physiological levels of fluid shear stress. These morphological changes influence cell function; however, the processes that produce them are poorly understood. We have examined how actin assembly is related to shear-induced endothelial cell shape change. To do so, we imposed physiological levels of shear stress on cultured endothelium for up to 96 hours and then permeabilized the cells and exposed them briefly to fluorescently labeled monomeric actin at various time points to assess actin assembly. Alternatively, monomeric actin was microinjected into cells to allow continuous monitoring of actin distribution. Actin assembly occurred primarily at the ends of stress fibers, which simultaneously reoriented to the shear axis, frequently fused with neighboring stress fibers, and ultimately drove the poles of the cells in the upstream and/or downstream directions. Actin polymerization occurred where stress fibers inserted into focal adhesion complexes, but usually only at one end of the stress fiber. Neither the upstream nor downstream focal adhesion complex was preferred. Changes in actin organization were accompanied by translocation and remodeling of cell-substrate adhesion complexes and transient formation of punctate cell-cell adherens junctions. These findings indicate that stress fiber assembly and realignment provide a novel mode by which cell morphology is altered by mechanical signals.

Cell movement is driven by the coordinated regulation of cytoskeletal reorganization through Rho GTPases downstream of integrin and growth-factor receptor signaling. We have reported that mDia, a target protein of Rho, interacts with Src and DIP. Here we show that DIP binds to p190RhoGAP and Vav2, and that DIP is phosphorylated by Src and mediates the phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation. When endogenous DIP was inhibited by expressing dominant-negative mutants of DIP or siRNA, phosphorylation of p190RhoGAP and Vav2 upon EGF stimulation was diminished, and EGF-induced actin organization, distribution of p190RhoGAP and Vav2, and cell movement were affected. Therefore, DIP seems to transfer the complex of the three proteins from cytosol to beneath the membrane, and the three proteins, in turn, can be phosphorylated by Src. DIP inactivated Rho and activated Rac following EGF stimulation in the membrane fraction. Thus, DIP acts as a regulatory molecule causing Src kinase-dependent feedback modulation of Rho GTPases downstream of Rho-mDia upon EGF stimulation, and plays an important role in cell motility.

Accumulating evidence suggests that p21(Cip1) located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21(Cip1), which uncouples Rho-GTP from stress fiber formation by inhibiting Rho kinase (ROCK). Concomitant with the loss of stress fibers in Ras-transformed cells, there is a decrease in the phosphorylation level of cofilin, which is indicative of a compromised ROCK/LIMK/cofilin pathway. Inhibition of MEK in Ras-transformed NIH3T3 results in restoration of actin stress fibers accompanied by a loss of cytoplasmic p21(Cip1), and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21(Cip1) in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21(Cip1) was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21(Cip1) expression accompanied by restoration of phosphocofilin, which was not accompanied by stress fiber formation. These results suggest that restoration of cofilin phosphorylation in Ras-transformed cells is necessary but not sufficient for stress fiber formation. Our findings define a novel mechanism for coupling cytoplasmic p21(Cip1) to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21(Cip1) to the cytoplasm in transformed cells contributes to pathways that favor not only cell proliferation, but also cell motility thereby contributing to invasion and metastasis.

It is presently accepted that fibroblast/myofibroblast modulation represents a crucial step in granulation tissue contraction and in the production of the connective tissue deformations typical of fibrocontractive diseases. In addition to synthesizing extracellular matrix (ECM) components, myofibroblasts can develop tensile force through the neoformation of alpha-smooth muscle actin (alpha-SMA) containing cytoplasmic stress fibers. Tension has been shown to be a crucial regulator of connective tissue remodeling. In order to coordinate tension distribution within connective tissue, cell-matrix and cell-cell contacts appear essential. This review addresses the formation, molecular structure and function of such structures that are characterized by their association with intracytoplasmic actin filaments. Actin associated cell-matrix adhesions appear to provide the interface between ECM components and intracellular stress fibers, thus contributing to the transmission of force to the substrate and to the detection of stress level in the matrix. Cell-cell adherens junctions appear to synchronize myofibroblast contractile activity. Further studies investigating the functions of these structures will be important for the understanding of the mechanisms of granulation tissue evolution and for the planification of strategies in view of influencing connective tissue deformations.

Organizational relationships between endothelial stress fibers and fibronectin fibrils in the developing chick abdominal aorta, from 5th day embryos to 3rd day young chicks, were studied with immunofluorescence and electron microscopy. Stress fibers, axially aligned parallel to the longitudinal cell axis, were expressed in the largely elongated endothelial cells, in embryos older than 8th day of incubation. Fibronectin fibrils in the aortic basal lamina, changed its organizational pattern from the network-like form to the straight bundles arranged parallel to the vessel's longitudinal axis after 9th day of incubation. Such axial alignment was dominant in the matrix beneath the elongated cells containing stress fibers, suggesting the existence of stress fibers may possibly modify the fibronectin's organizational pattern. The vinculin-containing dense plaque, which shaped like as the adhesion plaque in the cultured cells, was located at the ends of or lateral associating sites of stress fibers in embryos older than 8th day stage. The expression of stress fibers, as well as the formation of stress fiber's end plaques, may closely relate to the alignment between the stress fiber and fibronectin fibrils in the extracellular matrix.

Background: Reorientation of the cell axis induced by cyclic stretching is an early response to mechanical forces in vitro. However, quantitative assay for this phenomenon has been difficult due to lack of robust methods. We hypothesized that cell orientation may be redefined by the orientation of actin fibers. We developed image processing methods to quantitate the orientation and density of actin fibers., Methods: A convolution filter using Sobel kernels was adapted to determine the orientation and density of actin fibers in human endothelial cells. Unidirectional stretching (10%, 0.5 Hz) was applied to induce cytoskeletal remodeling by varying the duration of stimulation (control, 0.5, 1, 2, 5, 10, and 20 h). Actin fibers were visualized by fluorescent phalloidin. The image processing method was compared with the manual method for reproducibility. Both confluent and subconfluent cells were tested to assess the efficacy of the methods., Results: Cyclic stretch-induced dense and uninterrupted actin cabling formed across the cell body and, later, the actin fibers became aligned perpendicular to the stretch direction. The variance of actin fiber orientation became smaller after 2 h of stretch (F < 0.01). The actin fiber density index, a derived parameter related to the density of actin fibers, increased as early as 30 min of stretching (P < 0.05) and decreased after 10 h of stretching. Reproducibility of our method was extremely good. Applicability of the method was not compromised by cell density., Conclusions: Our method is reliable for quantifying cytoskeletal remodeling induced by mechanical force., (Copyright 2003 Wiley-Liss, Inc.)

Fibroblast migration is an integral component of the processes resulting in the formation of restrictive adhesions in the injured tendon, especially in Zone II. Pre-requisites for cell migration are an intact cytoskeleton and an ability to biochemically degrade the extra-cellular matrix. The relative characteristics of fibroblasts from the fibro-osseus sheath (SC), the tissue surrounding the tendon in Zone II, and the endotenon (TC) with respect to morphology, cytoskeletal structure and ability to produce matrix metalloproteinases (MMPs) 2 and 9 were compared in vitro. It was found that SCs were larger in size and demonstrated greater amounts of intra-cellular alpha-smooth muscle actin (alpha-SMA) and intra-membranous vinculin. Filamentous actin (F-actin) fibres in SCs were more densely packed and concentrated, resulting in stress fibres. The SCs also produce greater amounts of MMP-2 and MMP-9 compared to TCs. These observations imply that SCs play an active role in adhesion formation and should be specifically targeted to inhibit or treat tendon adhesions.

Eph kinases and their ephrin ligands are widely expressed in epithelial cells in vitro and in vivo. Our results show that activation of endogenous EphA kinases in Madin-Darby canine kidney (MDCK) cells negatively regulates hepatocyte growth factor/scatter factor (HGF)-induced branching morphogenesis in collagen gel. Cotreatment with HGF and ephrin-A1 reduced sprouting of cell protrusions, an early step in branching morphogenesis. Moreover, addition of ephrin-A1 after HGF stimulation resulted in collapse and retraction of preexisting cell protrusions. In a newly developed assay that simulates the localized interactions between Ephs and ephrins in vivo, immobilized ephrin-A1 suppressed HGF-induced MDCK cell scattering. Ephrin-A1 inhibited basal ERK1/2 mitogen-activated protein kinase activity; however, the ephrin-A1 effect on cell protrusion was independent of the mitogen-activated protein kinase pathway. Ephrin-A1 suppressed HGF-induced activation of Rac1 and p21-activated kinase, whereas RhoA activation was retained, leading to the preservation of stress fibers. Moreover, dominant-negative RhoA or inhibitor of Rho-associated kinase (Y27632) substantially negated the inhibitory effects of ephrin-A1. These data suggest that interfering with c-Met signaling to Rho GTPases represents a major mechanism by which EphA kinase activation inhibits HGF-induced MDCK branching morphogenesis.

We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4-5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4-5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10+FNIII4-5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.

Thrombin receptors couple to G(i/o), G(q), and G(12/13) proteins to regulate a variety of signal transduction pathways that underlie the physiological role of endothelial cells in wound healing or inflammation. Whereas the involvement of G(i), G(q), G(12), or G(13) proteins in thrombin signaling has been investigated extensively, the role of G(o) proteins has largely been ignored. To determine whether G(o) proteins could contribute to thrombin-mediated signaling in endothelial cells, we have developed minigenes that encode an 11-amino acid C-terminal peptide of G(o1) proteins. Previously, we have shown that use of the C-terminal minigenes can specifically block receptor activation of G protein families (). In this study, we demonstrate that G(o) proteins are present in human microvascular endothelial cells (HMECs). Moreover, we show that thrombin receptors can stimulate [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to G(o) proteins when co-expressed in Sf9 membranes. The potential coupling of thrombin receptors to G(o) proteins was substantiated by transfection of the G(o1) minigene into HMECs, which led to a blockade of thrombin-stimulated release of [Ca(2+)](i) from intracellular stores. Transfection of the beta-adrenergic kinase C terminus blocked the [Ca(2+)](i) response to the same extent as with G(o1) minigene peptide, suggesting that this G(o)-mediated [Ca(2+)](i) transient was caused by Gbetagamma stimulation of PLCbeta. Transfection of a G(i1/2) minigene had no effect on thrombin-stimulated [Ca(2+)](i) signaling in HMEC, suggesting that Gbetagamma derived from G(o) but not G(i) could activate PLCbeta. The involvement of G(o) proteins on events downstream from calcium signaling was further evidenced by investigating the effect of G(o1) minigenes on thrombin-stimulated stress fiber formation and endothelial barrier permeability. Both of these effects were sensitive to pertussis toxin treatment and could be blocked by transfection of G(o1) minigenes but not G(i1/2) minigenes. We conclude that the G(o) proteins play a role in thrombin signaling distinct from G(i1/2) proteins, which are mediated through their Gbetagamma subunits and involve coupling to calcium signaling and cytoskeletal rearrangements.

Beta-thymosins are structurally related, highly conserved acidic polypeptides, originally isolated from calf thymus. We have recently shown that the TB10 gene is overexpressed in human thyroid carcinoma cell lines and tissues, particularly in undifferentiated thyroid carcinomas, but expressed at an undetectable level in normal thyroid cells. The precise role of thymosin beta-10 (TB10) activity in maintaining the malignant phenotype of thyroid cell lines is unknown. To investigate TB10 function and relevance in a model system we used an antisense methodology to suppress TB10 protein synthesis in two human thyroid carcinoma cell lines (NPA and ARO). The growth in soft agar of NPA and ARO cells carrying a TB10 construct in an antisense orientation was significantly reduced. Conversely, anchorage-dependent growth was unchanged in NPA and ARO cells carrying the TB10 construct in a sense orientation or carrying the backbone vector. TB10 expression also affected actin organization. In fact, stress fibers were long and thick in ARO cells in which TB10 expression was suppressed by the antisense construct. Conversely, they were scarce and short in the vector-transfected ARO cells. These data suggest that TB10 plays a critical role in the regulation of anchorage-independent growth and assembly of actin filaments.

E-cadherin-mediated adherens junction formation and maintenance are thought to involve actin filament rearrangements through the action of small GTPases. Recently, we demonstrated that microtubule disruption in normal human epidermal keratinocytes grown in low calcium media conditions induces cell-cell adhesion by redistribution of endogenous E-cadherin, and it promotes stress fiber formation. This actin rearrangement was apparently mediated by RhoA activation. This model system therefore provides a tool with which to dissect relationships between cell-cell adhesion and Rho-mediated stress fiber formation. In this study, we have demonstrated in normal human epidermal keratinocytes that disruption of actin structures including stress fibers does not interfere with E-cadherin redistribution during microtubule-induced cell-cell adhesion. Moreover, this cell-cell adhesion could not be blocked by RhoA inactivation at the level for inhibition of stress fiber formation. Additionally, in the immortalized HaCaT keratinocyte cell line, which does not undergo cell-cell adhesion after microtubule disruption in low calcium conditions, expression of dominant-active RhoA could induce stress fiber formation without inducing adhesion. On the other hand, a variant of the HaCaT cell line, HC-R1, showed microtubule-disruption-induced cell-cell adhesion without stress fiber formation. Together, our results suggest that, in keratinocytes, the process of cell adhesion can occur independently of RhoA-mediated stress fiber formation.

Tendons consist of parallel longitudinal rows of cells separated by collagen fibres. The cells are in intimate contact longitudinally within rows, and laterally via sheet-like lateral cell processes between rows. At points of contact, they are linked by gap junctions. Since tendons stretch under load, such cell contacts require protection. Here we describe the organisation of the actin cytoskeleton and actin-based cell-cell interactions in vivo and examine the effect of cyclic tensile loading on tendon cells in vitro. Cells within longitudinal rows contained short longitudinally running actin stress fibres. Each fibre was aligned with similar fibres in the cells longitudinally on either side, and fibres appeared to be linked via adherens junctions. Overall, these formed long oriented rows of stress fibres running along the rows of tendon cells. In culture, junctional components n-cadherin and vinculin and the stress fibre component tropomyosin increased in strained cultures, whereas actin levels remained constant. These results suggest that: (1) cells are linked via actin-associated adherens junctions along the line of principal strain; and (2) under load, cells appear to attach themselves more strongly together, and assemble more of their cytoplasmic actin into stress fibres with tropomyosin. Taken together, this suggests that cell-cell contacts are protected during stretch, and also that the stress fibres, which are contractile, may provide an active mechanism for recovery from stretch. In addition, stress fibres are ideally oriented to monitor tensile load and thus may be important in mechanotransduction and the generation of signals passed via the gap junction network.

Sequential images of the local stiffness distribution of living fibroblasts (NIH3T3) were captured under a culture condition using scanning probe microscopy in a force modulation mode. We found a clear relation between cell migration and local stiffness distribution on the cell: When cells were stationary at one position, the stiffness distribution of their cellular surface was quite stable. On the other hand, once the cells started to move, the stiffness in their nuclear regions drastically decreased. Possible explanations for the correlation between the cell migration and the cell stiffness are proposed., (Copyright 2001 Wiley-Liss, Inc.)

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine that activates several signaling cascades. We determined the extent to which ceramide is a second messenger for TNF-alpha-induced signaling leading to cytoskeletal rearrangement in Rat2 fibroblasts. TNF-alpha, sphingomyelinase, or C(2)-ceramide induced tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin, and stress fiber formation. Ly 294002, a phosphatidylinositol 3-kinase (PI 3-K) inhibitor, or expression of dominant/negative Ras (N17) completely blocked C(2)-ceramide- and sphingomyelinase-induced tyrosine phosphorylation of FAK and paxillin and severely decreased stress fiber formation. The TNF-alpha effects were only partially inhibited. Dimethylsphingosine, a sphingosine kinase (SK) inhibitor, blocked stress fiber formation by TNF-alpha and C(2)-ceramide. TNF-alpha, sphingomyelinase, and C(2)-ceramide translocated Cdc42, Rac, and RhoA to membranes, and stimulated p21-activated protein kinase downstream of Ras-GTP, PI 3-K, and SK. Transfection with inactive RhoA inhibited the TNF-alpha- and C(2)-ceramide-induced stress fiber formation. Our results demonstrate that stimulation by TNF-alpha, which increases sphingomyelinase activity and ceramide formation, activates sphingosine kinase, Rho family GTPases, focal adhesion kinase, and paxillin. This novel pathway of ceramide signaling can account for approximately 70% of TNF-alpha-induced stress fiber formation and cytoskeletal reorganization.

Thyroid papillary carcinomas are characterized by RET/PTC rearrangements that cause the tyrosine kinase domain of the RET receptor to fuse with N-terminal sequences encoded by heterologous genes. This results in the aberrant expression of a ligand-independent and constitutively active RET kinase. We analysed actin reorganization induced by the RET/PTC1 oncogene in PC Cl 3 rat thyroid epithelial cells. Differently from oncogenes Src, Ras and Raf, RET/PTC1 caused actin filaments to form prominent stress fibers. Moreover, stress fibers were identified in human thyroid papillary carcinoma cell lines harboring RET/PTC1 rearrangements but not in thyroid carcinoma cells negative for RET/PTC rearrangements. RET/MEN 2A, a constitutively active but unrearranged membrane-bound RET oncoprotein, did not induce stress fibers in PC Cl 3 cells. Induction of stress fibers by RET/PTC1 was restricted to thyroid cells; it did not occur in NIH3T3 fibroblasts or MCF7 mammary cells. RET/PTC1-mediated stress fiber formation depended on Rho but not Rac small GTPase activity. In addition, inhibition of Rho, but not of Rac, caused apoptosis of RET/PTC1-expressing thyroid cells. We conclude that Rho is implicated in the actin reorganization and cell survival mediated by the chimeric RET/PTC1 oncogene in thyroid epithelial cells, both phenotypes being cell type- and oncogene type-specific.

Myofibroblasts, characterised by high expression of alpha-smooth muscle actin (alpha-SMA), are important and transient cells in normal wound healing but are found in increased number in various pathological conditions of the lung including asthma and pulmonary fibrosis. The mechanisms that regulate the myofibroblast phenotype are unknown but are likely to involve signals from the extracellular matrix transmitted via specific integrins. Vitronectin is a glycoprotein released during inflammation and has been shown to regulate the phenotype of vascular smooth muscle cells via alpha v and beta 1 integrins. In the current study we have examined whether vitronectin influences the phenotype and function of normal human lung fibroblasts (HFL-1). Incubation of HFL-1 cells with vitronectin induced a concentration-dependent reduction in alpha-SMA expression. By contrast, function-blocking monoclonal antibodies to the vitronectin integrins alpha v, beta 1, alpha v beta 3 and alpha v beta 5 induced the expression of alpha-SMA and its organization into stress fibers. Expression of alpha-SMA induced by all function-blocking monoclonal antibodies was abrogated by inhibition of protein kinase C and phosphatidylinositol-3 kinase, but the effects of inhibition of other signalling pathways was integrin dependent. Exposure to other extracellular matrix proteins such as fibronectin, collagen or their integrins did not influence expression of alpha-SMA. The expression and organization of alpha-SMA induced by exposure to function-blocking antibodies was translated into an augmented capacity of HFL-1 cells to contract fibroblast populated collagen gels. By contrast, contraction of collagen gels following incubation with vitronectin was not significantly different to control. This study has shown that vitronectin influences the phenotype and behaviour of HFL-1 cells by downregulating the expression of alpha-SMA and reducing their contractile ability. By contrast, occupancy of specific integrins by function-blocking antibodies upregulated the expression of alpha-SMA and induced the formation of functional stress fibers capable of contracting collagen gels. These results suggest that vitronectin modulates the fibroblast-myofibroblast phenotype, implying an important role in the remodelling process during lung development or response to injury.

Formation of actin stress fibers and the focal adhesion complex between cell and the substratum are crucial for nonmalignant cells to achieve anchorage-dependent growth. We show here that the adhesion complex formed in normal human mammary epithelial (HME) cells which adhered to type IV collagen, involved the EGF receptor (EGFR) and phospholipase Cgamma (PLCgamma) as signaling molecules, in addition to integrin beta1, alpha-actinin, and actin even before stimulation of the cells with EGF. Stimulation of cells with EGF induced tyrosine phosphorylation of EGFR and activation of PLCgamma, as assessed by the production of a second messenger diacylglycerol (DAG), without any significant increase in the amount of EGFR-bound PLCgamma. Disruption of either actin filaments by cytochalasin D (CD) or actin-myosin contractility by ML-7, an inhibitor of myosin light chain kinase (MLCK), altered the flattened morphology of quiescent cells to a retracted one, without affecting the association between EGFR and PLCgamma. Stimulation of CD- or ML-7-treated cells with EGF failed to inhibit tyrosine phosphorylation of EGFR and its association and colocalization with PLCgamma, but inhibited the PLCgamma activation. Phosphatidylinositol 4,5-bisphosphate (PtdInsP2), substrate of PLCgamma, was tightly associated with alpha-actinin and the content of alpha-actinin-bound PtdInsP2 was reduced by treatment of cells with ML-7 but not with CD. The amount of PtdInsP2 bound to alpha-actinin was increased by the addition of EGF and this EGF-induced increase was blocked by either CD or ML-7. The present results suggest that anchorage-dependent EGF signaling in HME cells may require both actin filament assembly and actin-myosin contractility for the PLCgamma activation., (Copyright 2001 Wiley-Liss, Inc.)

1. The role of intracellular Ca(2+) mobilization in the mechanism of increased endothelial permeability was studied. Human umbilical vein endothelial cells (HUVECs) were exposed to thapsigargin or thrombin at concentrations that resulted in similar increases in intracellular Ca(2+) concentration ([Ca(2+)](i)). The rise in [Ca(2+)](i) in both cases was due to release of Ca(2+) from intracellular stores and influx of extracellular Ca(2+). 2. Both agents decreased endothelial cell monolayer electrical resistance (a measure of endothelial cell shape change) and increased transendothelial (125)I-albumin permeability. Thapsigargin induced activation of PKCalpha and discontinuities in VE-cadherin junctions without formation of actin stress fibres. Thrombin also induced PKCalpha activation and similar alterations in VE-cadherin junctions, but in association with actin stress fibre formation. 3. Thapsigargin failed to promote phosphorylation of the 20 kDa myosin light chain (MLC(20)), whereas thrombin induced MLC(20) phosphorylation consistent with formation of actin stress fibres. 4. Calphostin C pretreatment prevented the disruption of VE-cadherin junctions and the decrease in transendothelial electrical resistance caused by both agents. Thus, the increased [Ca(2+)](i) elicited by thapsigargin and thrombin may activate a calphostin C-sensitive PKC pathway that signals VE-cadherin junctional disassembly and increased endothelial permeability. 5. Results suggest a critical role for Ca(2+) signalling and activation of PKCalpha in mediating the disruption of VE-cadherin junctions, and thereby in the mechanism of increased endothelial permeability.

It is widely accepted that actin filaments and the conventional double-headed myosin interact to generate force for many types of nonmuscle cell motility, and that this interaction occurs when the myosin regulatory light chain (MLC) is phosphorylated by MLC kinase (MLCK) together with calmodulin and Ca(2+). However, recent studies indicate that Rho-kinase is also involved in regulating the smooth muscle and nonmuscle cell contractility. We have recently isolated reactivatable stress fibers from cultured cells and established them as a model system for actomyosin-based contraction in nonmuscle cells. Here, using isolated stress fibers, we show that Rho-kinase mediates MLC phosphorylation and their contraction in the absence of Ca(2+). More rapid and extensive stress fiber contraction was induced by MLCK than was by Rho-kinase. When the activity of Rho-kinase but not MLCK was inhibited, cells not only lost their stress fibers and focal adhesions but also appeared to lose cytoplasmic tension. Our study suggests that actomyosin-based nonmuscle contractility is regulated by two kinase systems: the Ca(2+)-dependent MLCK and the Rho-kinase systems. We propose that Ca(2+) is used to generate rapid contraction, whereas Rho-kinase plays a major role in maintaining sustained contraction in cells.

Expression of voltage-gated K(+) (Kv) channel genes is regulated by polyamines in intestinal epithelial cells (IEC-6 line), and Kv channel activity is involved in the regulation of cell migration during early restitution by controlling membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). This study tests the hypothesis that RhoA of small GTPases is a downstream target of elevated ([Ca2+](cyt)) following activation of K(+) channels by increased polyamines in IEC-6 cells. Depletion of cellular polyamines by alpha-difluoromethylornithine (DFMO) reduced whole cell K+ currents [I(K(v))] through Kv channels and caused membrane depolarization, which was associated with decreases in ([Ca2+](cyt)), RhoA protein, and cell migration. Exogenous polyamine spermidine reversed the effects of DFMO on I(K(v)), E(m), ([Ca2+](cyt)), and RhoA protein and restored cell migration to normal. Elevation of ([Ca2+](cyt)) induced by the Ca2+ ionophore ionomycin increased RhoA protein synthesis and stimulated cell migration, while removal of extracellular Ca2+ decreased RhoA protein synthesis, reduced protein stability, and inhibited cell motility. Decreased RhoA activity due to Clostridium botulinum exoenzyme C(3) transferase inhibited formation of myosin II stress fibers and prevented restoration of cell migration by exogenous spermidine in polyamine-deficient cells. These findings suggest that polyamine-dependent cell migration is partially initiated by the formation of myosin II stress fibers as a result of Ca2+-activated RhoA activity.

Corneal wound healing fibroblasts (myofibroblasts) develop a muscle-like contractile apparatus composed of prominent microfilament bundles (stress fibers) and express alpha-smooth muscle actin (alpha-SMA). In this study, gelsolin, an actin filament-severing protein, was overexpressed in a alpha-SMA-expressing corneal myofibroblast cell line (TRK43) to assess whether intact stress fibers are required for in vitro matrix organization and wound contraction. Stably integrated gelsolin was introduced by electroporation of an expression construct (pREPCG8) into cultured cells. Thirty-seven clones were isolated with half of the clones showing a fibroblastic phenotype while the remaining half appeared epithelioid. One fibroblastic clone, GS56, and one epithelioid clone, GS44, were selected for detailed characterization. The GS56 cells appeared highly elongated and spindle-shaped and had prominent stress fibers and focal adhesions. GS44 cells showed disruption of stress fibers and a cortical f-actin organization as well as the down regulation of alpha-SMA expression by immunocytochemistry and Western blotting. Both phenotypes showed enhanced gelsolin expression; however, fractionation of cell extracts demonstrated differences in the subcellular distribution of gelsolin with GS44 cells having markedly reduced and GS56 cells having markedly increased cytoskeletal gelsolin. In an in vitro wound contraction assay, epithelioid GS44 cells showed a significantly impaired ability to contract a collagen matrix compared to that of TRK43 cells, CT9 or GS56 transfectants. Loss of stress fibers in GS44 cells also correlated with enhanced cell motility. Together, these results demonstrate that the ability to form microfilament bundles or stress fibers is required for matrix organization and contraction by corneal myofibroblasts. Although no clear explanation is available, we suspect that differences in gene insertion of the gelsolin overexpression vector may have led to differential intercellular localization of gelsolin and its effect on stress fiber formation in the two cell lines.

Background: Glomerular distention is associated with cellular mechanical strain. In addition, glomerular distention/contraction is assumed to influence the filtration rate through changes in filtration surface area. A contractile cytoskeleton in podocytes and mesangial cells, formed by F-actin-containing stress fibers, maintains structural integrity and modulates glomerular expansion. In this study, the glomerular cell distribution of F-actin and vimentin filaments was studied in normal control and nine-month streptozotocin-diabetic rats. Results in situ were compared with observations in tissue culture., Methods: Microdissected rat glomeruli were perfused to obtain a physiological 25% glomerular expansion over the basal value. Fixation was done without change in glomerular volume. Dual fluorescent labeling of F-actin and vimentin was carried out, and samples were examined by confocal laser scanning microscopy., Results: The podocyte cell bodies and their cytoplasmic projections, including the foot processes, contained bundles of vimentin-containing fibers. Except for a thin layer at the base of foot processes, they did not demonstrate F-actin. While mesangial cells in culture had F-actin as long stress fibers resembling tense cables, mesangial cells stretched in situ contained a maze of short tortuous F-actin fibers organized in bundles that often encircled vascular spaces. This fibrillar organization was disrupted in diabetic glomeruli., Conclusion: Mesangial cells, but not podocytes, contain a cytoskeleton capable of contraction that is disorganized in long-term diabetes. Together with previous observations, the distribution of this cytoskeleton suggests that mesangial cell contraction may be involved in the redistribution of glomerular capillary blood flow, but not substantially in the modulation of glomerular distention. Disorganization of stress fibers may be a cause of hyperfiltration in diabetes.

We studied stress fiber orientation under a wide range of uniaxial cyclic deformations. We devised and validated a hypothesis consisting of two parts, as follows: (1) a stress fiber aligns to avoid a mechanical stimulus in the fiber direction under cyclic deformation. This means that, among all allowable directions, a stress fiber aligns in the direction which minimizes the stimulus, i. e., the summation of the changes in length of the stress fiber over one stretch cycle; and (2) there is a limit in the sensitivity of the cellular response to the mechanical stimulus. Due to this sensing limit, the orientation angle in stress fibers is distributed around the angle corresponding to the minimum stimulus. To validate this hypothesis, we approximated an anisotropic deformation of the membrane on which cells were to be cultured. We then obtained the relationships between the stretch range and the fiber angle in the undeformed state which minimize the mechanical stimuli, assuming that the membrane on which stress fibers and cells adhered was homogeneous and incompressible. Numerical simulation results showed that the proposed hypothesis described our previous experimental results well and was consistent with the experimental results in the literature. The simulation results, taking account of the second part of the hypothesis with a small value for the limit in sensitivity to the mechanical stimulus, could explain why cell orientation is distributed so widely with cyclic stretch ranges of <10%. The proposed hypothesis can be applied to various types of deformation because the mechanical stimulus is always sensed and accumulates under cyclic deformation without the necessity of a reference state to measure the stimulus.