Rho and Rab Small G Proteins Coordinately Reorganize Stress Fibers and Focal Adhesions in MDCK Cells

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.

Morphogenetic Effects of Neuregulin (Neu Differentiation Factor) in Cultured Epithelial Cells

Neuregulin, or neu differentiation factor, induces cell proliferation or differentiation through interaction with members of the ErbB family of receptor tyrosine kinases. We report that neuregulin can also induce profound morphogenic responses in cultured epithelial cells of different origins. These effects include scattering of small epithelial islands and rearrangement of larger cell islands into ordered ring-shaped arrays with internal lumens. The ring-forming cells are interconnected by cadherin- and β-catenin-containing adherens junctions. In confluent cultures, neuregulin treatment induces formation of circular lumenlike gaps in the monolayer. Both cell scattering and ring formation are accompanied by a marked increase in cell motility that is independent of hepatocyte growth factor/scatter factor and its receptor (c-Met). Affinity-labeling experiments implied that a combination of ErbB-2 with ErbB-3 mediates the morphogenic signal of neuregulin in gastric cells. Indeed, a similar morphogenic effect could be reconstituted in nonresponsive cells by coexpression of ErbB-2 and -3. We conclude that a heterodimer between the kinase-defective neuregulin receptor, ErbB-3, and the coreceptor, ErbB-2, mediates the morphogenetic action of neuregulin.

Keratocytes Generate Traction Forces in Two PhasesV⃞

Forces generated by goldfish keratocytes and Swiss 3T3 fibroblasts have been measured with nanonewton precision and submicrometer spatial resolution. Differential interference contrast microscopy was used to visualize deformations produced by traction forces in elastic substrata, and interference reflection microscopy revealed sites of cell-substratum adhesions. Force ranged from a few nanonewtons at submicrometer spots under the lamellipodium to several hundred nanonewtons under the cell body. As cells moved forward, centripetal forces were applied by lamellipodia at sites that remained stationary on the substratum. Force increased and abruptly became lateral at the boundary of the lamellipodium and the cell body. When the cell retracted at its posterior margin, cell-substratum contact area decreased more rapidly than force, so that stress (force divided by area) increased as the cell pulled away. An increase in lateral force was associated with widening of the cell body. These mechanical data suggest an integrated, two-phase mechanism of cell motility: (1) low forces in the lamellipodium are applied in the direction of cortical flow and cause the cell body to be pulled forward; and (2) a component of force at the flanks pulls the rear margins forward toward the advancing cell body, whereas a large lateral component contributes to detachment of adhesions without greatly perturbing forward movement.

Characterization of Cell–Matrix Adhesion Requirements for the Formation of Fascin Microspikes

Cell adhesion to thrombospondin-1 (TSP-1) correlates with assembly of cell–substratum contact structures that contain fascin microspikes. In this analysis, cell-matrix requirements for assembly of fascin microspikes were examined in detail. In six cell lines, cell spreading on a TSP-1 substratum correlated with expression of fascin protein and formation of fascin microspikes. Microspikes were not formed by H9c2 cells adherent on fibronectin, vitronectin, collagen IV, or platelet factor 4. However, both fascin microspikes and focal contacts were assembled by cells adherent on laminin-1. Using mixed substrata containing different proportions of TSP-1, and fibronectin, fascin microspike formation by H9c2 and C2C12 cells was found to be reduced on substrata containing 25% fibronectin and abolished on substrata containing 75% fibronectin. Adhesion to intermediate mixtures of TSP-1 and fibronectin resulted in coassembly of fascin microspikes and focal contacts, colocalization of fascin with actin stress fiber bundles and altered distributions of β1 integrins, cortical α-actinin, and tropomyosin. In cells adherent on 50% TSP-1:50% fibronectin, GRGDSP peptide treatment decreased focal contact assembly and altered cytoskeletal organization but did not inhibit microspike assembly. Treatment with chondroitin sulfate A or p-nitrophenol β-d-xylopyranoside decreased microspike formation and modified cytoskeletal organization but did not inhibit focal contact formation. In polarized migratory and postmitotic C2C12 cells, fascin microspikes and ruffles were localized at leading edges and TSP matrix deposition was also concentrated in this region. Depletion of matrix TSP by heparin treatment correlated with decreased microspike formation and cell motility. Thus, the balance of adhesive receptors ligated at the cell surface during initial cell–matrix attachment serves to regulate the type of substratum adhesion contact assembled and subsequent cytoskeletal organization. A role for fascin microspikes in cell motile behavior is indicated.

Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells

We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B61L or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B61L and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B17N do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.

Protrusive growth from giant liposomes driven by actin polymerization

Development of protrusions in the cell is indispensable in the process of cell motility. Membrane protrusion has long been suggested to occur as a result of actin polymerization immediately beneath the cell membrane at the leading edge, but elucidation of the mechanism is insufficient because of the complexity of the cell. To study the mechanism, we prepared giant liposomes containing monomeric actin (100 or 200 μM) and introduced KCl into individual liposomes by an electroporation technique. On the electroporation, the giant liposomes deformed. Most importantly, protrusive structure grew from the liposomes containing 200 μM actin at rates (ranging from 0.3 to 0.7 μm/s) similar to those obtained in the cell. The deformation occurred in a time range (30 ∼ 100 s) similar to that of actin polymerization monitored in a cuvette (ca. 50 s). Concomitant with deformation, Brownian motion of micron-sized particles entrapped in the liposomes almost ceased. From these observations, we conclude that actin polymerization in the liposomes caused the protrusive formation.

A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis in vivo in combination with cisplatin

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of Å6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. Å6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells in vitro. In contrast, Å6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth in vivo by 48% and 53%, respectively, and, more strikingly, the combination of Å6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or Å6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving Å6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.

Extracellular Matrix Assembly in Diatoms (Bacillariophyceae)1 : III. Organization of Fucoglucuronogalactans within the Adhesive Stalks of Achnanthes longipes

Achnanthes longipes is a marine, biofouling diatom that adheres to surfaces via adhesive polymers extruded during motility or organized into structures called stalks that contain three distinct regions: the pad, shaft, and collar. Four monoclonal antibodies (AL.C1–AL.C4) and antibodies from two uncloned hybridomas (AL.E1 and AL.E2) were raised against the extracellular adhesives of A. longipes. Antibodies were screened against a hot-water-insoluble/hot-bicarbonate-soluble-fraction. The hot-water-insoluble/hot-bicarbonate-soluble fraction was fractionated to yield polymers in three size ranges: F1, ≥ 20,000,000 Mr; F2, ≅100,000 Mr; and F3, <10,000 Mr relative to dextran standards. The ≅100,000-Mr fraction consisted of highly sulfated (approximately 11%) fucoglucuronogalactans (FGGs) and low-sulfate (approximately 2%) FGGs, whereas F1 was composed of O-linked FGG (F2)-polypeptide (F3) complexes. AL.C1, AL.C2, AL.C4, AL.E1, and AL.E2 recognized carbohydrate complementary regions on FGGs, with antigenicity dependent on fucosyl-containing side chains. AL.C3 was unique in that it had a lower affinity for FGGs and did not label any portion of the shaft. Enzyme-linked immunosorbent assay and immunocytochemistry indicated that low-sulfate FGGs are expelled from pores surrounding the raphe terminus, creating the cylindrical outer layers of the shaft, and that highly sulfated FGGs are extruded from the raphe, forming the central core. Antibody-labeling patterns and other evidence indicated that the shaft central-core region is related to material exuded from the raphe during cell motility.

Dynamics of active lamellae in cultured epithelial cells: effects of expression of exogenous N-ras oncogene.

We examined the functional consequences of cellular transformation of rat IAR-2 epithelial cells, by a mutant N-ras oncogene, on the dynamics of active lamellae, structures that play an important role in cell motility, adhesion, and surface-receptor capping. Lamellar activity was assessed by measuring the rate of outer-edge pseudopodial activity and by analyzing the motility of Con A-coated beads placed on lamellar surfaces with optical tweezers. Although transformation dramatically affected the shape and size of active cellular lamellae, there was little detectable effect on either pseudopodial activity or bead movement. To investigate the potential relationship between functional lamellar activity and the microtubule cytoskeleton, lamellar activity was examined in nontransformed and transformed cells treated with the microtubule-disrupting drug nocodazole. In the absence of microtubules, transformed cells were less polarized and possessed decreased rates of pseudopodial and bead motility. On the basis of these observations, it is suggested that ras-induced transformation of epithelial cells consists of two cytoskeletal modifications: overall diminished actin cytoskeletal dynamics in lamellae and reorganization of the microtubule cytoskeleton that directs pseudopodial activity to smaller polarized lamellae.

Mutation of juxtamembrane tyrosine residue 1001 suppresses loss-of-function mutations of the met receptor in epithelial cells.

Signals transduced by the met tyrosine kinase, which is the receptor for scatter factor/hepatocyte growth factor, are of major importance for the regulation of epithelial cell motility, morphogenesis, and proliferation. We report here that different sets of tyrosine residues in the cytoplasmic domain of the met receptor affect signal transduction in epithelial cells in a positive or negative fashion: mutation of the C-terminal tyrosine residues 13-16 (Y1311, Y1347, Y1354, and Y1363) reduced or abolished ligand-induced cell motility and branching morphogenesis. In contrast, mutation of the juxtamembrane tyrosine residue 2 (Y1001) produced constitutively mobile, fibroblastoid cells. Furthermore, the gain-of-function mutation of tyrosine residue 2 suppressed the loss-of-function mutations of tyrosine residue 15 or 16. The opposite roles of the juxtamembrane and C-terminal tyrosine residues may explain the suggested dual function of the met receptor in both epithelial-mesenchymal interactions and tumor progression.

Bifurcation Analysis of Reaction Diffusion Systems on Arbitrary Surfaces

In this article we present a computational framework for isolating spatial patterns arising in the steady states of reaction-diffusion systems. Such systems have been used to model many different phenomena in areas such as developmental and cancer biology, cell motility and material science. Often one is interested in identifying parameters which will lead to a particular pattern. To attempt to answer this, we compute eigenpairs of the Laplacian on a variety of domains and use linear stability analysis to determine parameter values for the system that will lead to spatially inhomogeneous steady states whose patterns correspond to particular eigenfunctions. This method has previously been used on domains and surfaces where the eigenvalues and eigenfunctions are found analytically in closed form. Our contribution to this methodology is that we numerically compute eigenpairs on arbitrary domains and surfaces. Here we present various examples and demonstrate that mode isolation is straightforward especially for low eigenvalues. Additionally we see that if two or more eigenvalues are in a permissible range then the inhomogeneous steady state can be a linear combination of the respective eigenfunctions. Finally we show an example which suggests that pattern formation is robust on similar surfaces in cases that the surface either has or does not have a boundary.

S100A4 downregulates filopodia formation through increased dynamic instability

Cell migration requires the initial formation of cell protrusions, lamellipodia and/or filopodia, the attachment of the leading lamella to extracellular cues and the formation and efficient recycling of focal contacts at the leading edge. The small calcium binding EF-hand protein S100A4 has been shown to promote cell motility but the direct molecular mechanisms responsible remain to be elucidated. In this work, we provide new evidences indicating that elevated levels of S100A4 affect the stability of filopodia and prevent the maturation of focal complexes. Increasing the levels of S100A4 in a rat mammary benign tumor derived cell line results in acquired cellular migration on the wound healing scratch assay. At the cellular levels, we found that high levels of S100A4 induce the formation of many nascent filopodia, but that only a very small and limited number of those can stably adhere and mature, as opposed to control cells, which generate fewer protrusions but are able to maintain these into more mature projections. This observation was paralleled by the fact that S100A4 overexpressing cells were unable to establish stable focal adhesions. Using different truncated forms of the S100A4 proteins that are unable to bind to myosin IIA, our data suggests that this newly identified functions of S100A4 is myosin-dependent, providing new understanding on the regulatory functions of S100A4 on cellular migration.

Plasma membrane calcium ATPase isoform 4 inhibits vascular endothelial growth factor-mediated angiogenesis through interaction with calcineurin

Approach and Results - Using in vitro and in vivo assays, we here demonstrate that the interaction between PMCA4 and calcineurin in VEGF-stimulated endothelial cells leads to downregulation of the calcineurin/NFAT pathway and to a significant reduction in the subsequent expression of the NFAT-dependent, VEGF-activated, proangiogenic genes RCAN1.4 and Cox-2. PMCA4-dependent inhibition of calcineurin signaling translates into a reduction in endothelial cell motility and blood vessel formation that ultimately impairs in vivo angiogenesis by VEGF. Objective - Vascular endothelial growth factor (VEGF) has been identified as a crucial regulator of physiological and pathological angiogenesis. Among the intracellular signaling pathways triggered by VEGF, activation of the calcineurin/ nuclear factor of activated T cells (NFAT) signaling axis has emerged as a critical mediator of angiogenic processes. We and others previously reported a novel role for the plasma membrane calcium ATPase (PMCA) as an endogenous inhibitor of the calcineurin/NFAT pathway, via interaction with calcineurin, in cardiomyocytes and breast cancer cells. However, the functional significance of the PMCA/calcineurin interaction in endothelial pathophysiology has not been addressed thus far. Conclusions - Given the importance of the calcineurin/NFAT pathway in the regulation of pathological angiogenesis, targeted modulation of PMCA4 functionality might open novel therapeutic avenues to promote or attenuate new vessel formation in diseases that occur with angiogenesis.

Development of potent and selective tissue transglutaminase inhibitors:their effect on TG2 function and application in pathological conditions

Potent-selective peptidomimetic inhibitors of tissue transglutaminase (TG2) were developed through a combination of protein-ligand docking and molecular dynamic techniques. Derivatives of these inhibitors were made with the aim of specific TG2 targeting to the intra- and extracellular space. A cell-permeable fluorescently labeled derivative enabled detection of in situ cellular TG2 activity in human umbilical cord endothelial cells and TG2-transduced NIH3T3 cells, which could be enhanced by treatment of cells with ionomycin. Reaction of TG2 with this fluorescent inhibitor in NIH3T3 cells resulted in loss of binding of TG2 to cell surface syndecan-4 and inhibition of translocation of the enzyme into the extracellular matrix, with a parallel reduction in fibronectin deposition. In human umbilical cord endothelial cells, this same fluorescent inhibitor also demonstrated a reduction in fibronectin deposition, cell motility, and cord formation in Matrigel. Use of the same inhibitor in a mouse model of hypertensive nephrosclerosis showed over a 40% reduction in collagen deposition.

Regulating the expression of eEF1A to decipher its non canonical functions in eukaryotic cells

The canonical function of eEF1A is delivery of the aminoacylated tRNA to the A site of the ribosome during protein translation, however, it is also known to be an actin binding protein. As well as this actin binding function, eEF1A has been shown to be involved in other cellular processes such as cell proliferation and apoptosis. It has long been thought that the actin cytoskeleton and protein synthesis are linked and eEF1A has been suggested to be a candidate protein to form this link, though very little is understood about the relationship between its two functions. Overexpression of eEF1A has also been shown to be implicated in many different types of cancers, especially cancers that are metastatic, therefore it is important to further understand how eEF1A can affect both translation and the organisation of the actin cytoskeleton. To this end, we aimed to determine the effects of reduced expression of eEF1A on both translation and its non canonical functions in CHO cells. We have shown that reduced expression of eEF1A in this cell system results in no change in protein synthesis, however results in an increased number of actin stress fibres and other proteins associated with these fibres such as myosin IIA, paxillin and vinculin. Cell motility and attachment are also affected by this reduction in eEF1A protein expression. The organisational and motility phenotypes were found to be specific to eEF1A by transforming the cells with plasmids containing either human eEF1A1 or eEF1A2. Though the mechanisms by which these effects are regulated have not yet been established, this data provides evidence to show that the translation and actin binding functions of eEF1A are independent of each other as well as being suggestive of a role for eEF1A in cell motility as supported by the observation that overexpression of eEF1A protein tends to be associated with the cancer cells that are metastatic.