Hepatocyte adhesion to carbohydrate-derivatized surfaces. I. Surface topography of the rat hepatic lectin

The rat hepatic lectins, galactose- and N-acetylgalactosamine-binding proteins found on the hepatocyte cell surface, mediate adhesion of isolated primary rat hepatocytes to artificial galactose-derivatized polyacrylamide gels. Biochemical and immunohistochemical techniques were used to examine the topographical redistribution of the rat hepatic lectins in response to galactose-mediated cell adhesion. Hepatocytes isolated from rat liver by collagenase perfusion had an average of 7 x 10(5) cell surface lectin molecules per cell, representing 30-50% of the total lectin molecules per cell, the remainder residing in intracellular pools. Hepatocytes incubated on galactose-derivatized surfaces, whether at 0-4 degrees C or 37 degrees C, rapidly lost greater than 80% of their accessible cell surface lectin binding sites into an adhesive patch of characteristic morphology. The kinetics of rat hepatic lectin disappearance were used to estimate a lateral diffusion coefficient greater than 9 x 10(-9) cm2/s at 37 degrees C, suggesting rapid and unimpeded lectin diffusion in the plane of the membrane. Indirect immunofluorescence labeling of adherent cells using antihepatic lectin antibody revealed a structured ring of receptors surrounding an area of exclusion (patch) of reproducible size and shape which represented approximately 8% of the hepatocyte cell surface. Notably, adherent cells, which had lost greater than 80% of their accessible surface binding sites, still endocytosed soluble galactose-terminated radioligand at greater than 50% of the rate of nonadherent control cells. No net movement of rat hepatic lectin from intracellular pools to the cell surface was found on cells recovered after adhesion to galactose-derivatized surfaces at 37 degrees C, suggesting that the physical size and/or lectin density of the patch was restricted by kinetic or topological constraints.     

Regulation of cytoskeletal organization by syndecan transmembrane proteoglycans.

Syndecans, a family of transmembrane proteoglycans, interact with numerous extracellular ligands through specific sequences in their heparan sulfate chains and have been considered to be co-receptors for matrix molecules and growth factors. In addition to their roles as co-receptors, many studies have recently suggested that signaling through core protein of syndecans can regulate cytoskeletal organization through their clustering, association with cytoskeletal structures, binding to cytoplasmic binding proteins, and intracellular phosphorylation. Here we will review current understanding of signaling through syndecans in cytoskeletal organization.     

Regulation of cell adhesion by protein-tyrosine phosphatases: II. Cell-cell adhesion

Cell-cell adhesion is critical to the development and maintenance of multicellular organisms. The stability of many adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly. The level of tyrosine phosphorylation reflects the balance between protein-tyrosine kinase and protein-tyrosine phosphatase activity. Many protein-tyrosine phosphatases associate with the cadherin-catenin complex, directly regulating the phosphorylation of these proteins, thereby affecting their interactions and the integrity of cell-cell junctions. Tyrosine phosphatases can also affect cell-cell adhesions indirectly by regulating the signaling pathways that control the activities of Rho family G proteins. In addition, receptor-type tyrosine phosphatases can mediate outside-in signaling through both ligand binding and dimerization of their extracellular domains. This review will discuss the role of protein-tyrosine phosphatases in cell-cell interactions, with an emphasis on cadherin-mediated adhesions.     

Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion

The morphology and cytoskeletal structure of fibroblasts, endothelial cells, and neutrophils are documented for cells cultured on surfaces with stiffness ranging from 2 to 55,000 Pa that have been laminated with fibronectin or collagen as adhesive ligand. When grown in sparse culture with no cell-cell contacts, fibroblasts and endothelial cells show an abrupt change in spread area that occurs at a stiffness range around 3,000 Pa. No actin stress fibers are seen in fibroblasts on soft surfaces, and the appearance of stress fibers is abrupt and complete at a stiffness range coincident with that at which they spread. Upregulation of alpha5 integrin also occurs in the same stiffness range, but exogenous expression of alpha5 integrin is not sufficient to cause cell spreading on soft surfaces. Neutrophils, in contrast, show no dependence of either resting shape or ability to spread after activation when cultured on surfaces as soft as 2 Pa compared to glass. The shape and cytoskeletal differences evident in single cells on soft compared to hard substrates are eliminated when fibroblasts or endothelial cells make cell-cell contact. These results support the hypothesis that mechanical factors impact different cell types in fundamentally different ways, and can trigger specific changes similar to those stimulated by soluble ligands.     

Mobility and cytoskeletal interactions of cell adhesion receptors.

Clustering of cell adhesion receptors and their interactions with the cytoskeleton are key events in the formation and function of cell adhesion structures. On the free cell surface, cadherin molecules interact with the cytoskeleton/membrane skeleton by being bound or corralled, and such interactions are greatly enhanced by the formation of cadherin oligomers. Corralled cadherin molecules undergo hop diffusion from one compartment to an adjacent one (membrane skeleton fence model), which prompts the initial formation of small adhesion clusters at cell-cell contact sites, but larger-scale assemblies of cadherin and actin filaments might require a further co-ordinated recruitment of these molecules.     

Studies on the function of cell surface glycoproteins. II. Possible role of surface glycoproteins in the control of cytoskeletal organization and surface morphology

Immunoglobulin from goat antiserum directed against purified surface membranes from transformed BHK21/C13 cells (anti-M) has been shown to cause both control and transformed hamster cells to round and detach from the substrate (see accompanying paper). This paper documents the effects of the antiserum on the cytoskeletal organization and cell surface morphology of control BHK21/C13 cells examined by scanning and transmission electron microscopy. As a result of antiserum-induced rounding, the normally smooth cell surface becomes covered with filopodia and blebs, and the organization of all three components of the filamentous cytoskeleton is altered. In terms of cell surface morphology and cytoskeletal organization, the cells resemble rounded, postmitotic or trypsinized BHK cells rather than cells treated with either anticytoskeletal drugs or lectins. Immunocytochemical and radioimmune assay experiments support the suggestion that the rounding reaction induced by anti-M serum results from the specific interaction of antibodies with molecules on the cell surface. It is suggested that anti-M serum induces alterations in cytoskeletal organization via a transmembrane signal and that cytoskeletal reorganization is a fundamental part of the rounding and detachment process.     

Ubiquitin links to cytoskeletal dynamics, cell adhesion and migration

Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin. The protein ubiquitylation machinery is remarkably complex, comprising more than 15 Ubls (ubiquitin-like proteins) and several hundreds of ubiquitin-conjugating enzymes. Ubiquitin is best known for its role as a tag that induces protein destruction either by the proteasome or through targeting to lysosomes. However, addition of one or more Ubls also affects vesicular traffic, protein-protein interactions and signal transduction. It is by now well established that ubiquitylation is a component of most, if not all, cellular signalling pathways. Owing to its abundance in controlling cellular functions, ubiquitylation is also of key relevance to human pathologies, including cancer and inflammation. In the present review, we focus on its role in the control of cell adhesion, polarity and directional migration. It will become clear that protein modification by Ubls occurs at every level from the receptors at the plasma membrane down to cytoskeletal components such as actin, with differential consequences for the pathway's final output. Since ubiquitylation is fast as well as reversible, it represents a bona fide signalling event, which is used to fine-tune a cell's responses to receptor agonists.     

Photoreversible surfaces to regulate cell adhesion

We report the development of a photoreversible cell culture substrate. We demonstrate the capacity to modify the adhesivity of the substrate using light, altering its capacity to support cell growth. Polyelectrolyte multilayers (PEMs) were used to produce tunable substrates of different thickness and matrix stiffness, which have different intrinsic capacities to support cell adhesion and survival. Surfaces were top-coated with a poly(acrylic acid)-poly(allylamine hydrochloride) polyelectrolyte bilayer functionalized with a small fraction (<1%) of an azobenzene-based photoswitchable sidegroup, which included the cell-adhesive three-amino-acid peptide RGD. Irradiation with light-induced geometric switching of the azo bond, resulting in changes to RGD exposure and consequently to cell adhesion and survival, was investigated on a variety of surfaces of different thickness and stiffness. Substrate stiffness, as modified by the thickness, had a significant influence on the adhesion of NIH 3T3 cells, consistent with previous studies. However, by disrupting the isomerization state of the azobenzene-linked RGD and exposing it to the surface, cell adhesion and survival could be enhanced up to 40% when the positioning of the RGD peptide was manipulated on the softest substrates. These findings identify permissive, yet less-than-optimal, cell culture substrate conditions that can be substantially enhanced using noninvasive modification of the substrate triggered by light. Indeed, where cell adhesion was tuned to be suboptimal under baseline conditions, the light-induced triggers displayed the most enhanced effect, and identification of this 'Goldilocks zone' was key to enabling light triggering.     

Chemically induced changes in the morphology, dynamic activity and cytoskeletal organization of Physarum cell fragments

Spherical cell fragments derived from Physarum polycephalum by caffeine-treatment were used as an experimental system to investigate the influence of 15 externally applied substances on the general morphology, motile behavior and cytoskeletal organization of the acellular slime mold. In comparison to controls, the most obvious changes observed after chemical stimulation proved to be cytokinetic activities, ameboid-like movement phenomena, intense cell surface dynamics and formation of cytoplasmic actin fibrils. The results demonstrate the high adaptability of the microfilament system in Physarum even when subjected to extreme conditions in the external environment.     

A dynamical model for receptor-mediated cell adhesion to surfaces.

We present a dynamical model for receptor-mediated adhesion of cells in a shear field of viscous fluid to surfaces coated with ligand molecules complementary to receptors in the cell membrane. We refer to this model as the "point attachment model" because it considers the contact area between the cell and the surface to be a small, homogeneous region that mediates the initial attachment of the cell to the surface. Using a phase plane analysis of a system of nonlinear ordinary differential equations which govern the changes in free receptor density and bond density within the contact area with time, we can predict the conditions for which adhesion between the cell and the surface will take place. Whether adhesion occurs depends on values of dimensionless quantities that characterize the interaction of the cell and its receptors with the surface and its ligand, such as the bond formation rate, the receptor-ligand affinity, the fluid mechanical force, the receptor mobility, and the contact area. A key result is that there are two regimes in which different chemical and physical forces dominate: a rate-controlled high affinity regime and an affinity-controlled low-affinity regime. Many experimental observations can be explained by understanding which of these regimes is appropriate. We also provide simple approximate analytical solutions, relating adhesiveness to cell and surface properties as well as fluid forces, which allow convenient testing of model predictions by experiment.     

Helicobacter pylori VacA cytotoxin interacts with fibronectin and alters HeLa cell adhesion and cytoskeletal organization in vitro

Helicobacter pylori vacuolating cytotoxin VacA causes multiple effects on epithelial cell function and morphology, but the effects of VacA on signal transduction pathways and the cytoskeleton have not been investigated in detail. In this study, we analyzed the effects of native VacA on HeLa and AGS cell adhesion to fibronectin and laminin under serum-free conditions. Confocal microscopic examination revealed increased number of cells with rounded morphology and inhibition of actin fiber formation, in the presence of VacA. VacA binds to fibronectin in vitro in a dose-dependent manner. This interaction was partly inhibited by a peptide containing an arginine-glycine-aspartic acid motif. The adhesion of HeLa cells to fibronectin, but not to laminin, was decreased in the presence of VacA. Thus, VacA may interact with fibronectin and influence integrin receptor-induced cell signaling and cytoskeleton-dependent cell functions.     

Immature erythroid cells with novel morphology and cytoskeletal organization in adultXenopus

Summary Nucleated erythrocytes of non-mammalian vertebrates are a useful model system for studying the correlation between changes in cell shape and cytoskeletal organization during cellular morphogenesis. They are believed to transform from spheres to flattened discs to ellipsoids. Our previous work on developing erythroblasts suggested that pointed cells containing incomplete, pointed marginal bands (MBs) of microtubules might be intermediate stages in the larval axolotl. To test whether the occurrence of such pointed cells was characteristic of amphibian erythrogenesis, we have utilized phenylhydrazine (PHZ)-induced anemia in adultXenopus. In this system, circulating erythrocytes are destroyed and replaced by erythroblasts that differentiate in the blood, making them experimentally accessible. Thus, we followed the time-course of morphological and cytoskeletal changes in the new erythroid population during recovery. During days 7–9 post-PHZ, pointed cells did indeed begin to appear, as did spherical and discoidal cells. The percentage of pointed cells peaked at days 11–13 in different animals, subsequently declining as the percentage of elliptical cells increased. Since degenerating old erythrocytes were still present when pointed cells appeared, we tested directly whether pointed ones were old or new cells. Blood was removed via the dorsal tarsus vein, and the erythrocytes washed, fluorescently tagged, and re-injected. In different animals, 2–8% of circulating erythrocytes were labeled. Subsequent to induction of anemia in these frogs, time-course sampling showed that no pointed cells were labeled, identifying them as new cells. Use of propidium iodide revealed large nuclei and cytoplasmic staining indicative of immaturity, and video-enhanced phase contrast and anti-tubulin immunofluorescence showed that the pointed cells contained pointed MBs. The results show that pointed cells, containing incomplete, pointed MBs are a consistent feature of amphibian erythrogenesis. These cells may represent intermediate stages in the formation of elliptical erythrocytes.Abbreviations MB marginal band - MS membrane skeleton - PHZ phenylhydrazine     

The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion.

Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlated with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in cell morphology and cytoskeletal organization are induced by human mitotic checkpoint gene, Bub1

The mitotic spindle checkpoint prevents the onset of anaphase and subsequent cell division until chromosomes are properly aligned on a bipolar spindle. Thus, it regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. The budding uninhibited by benzimidazole (Bub1) is a key component of mitotic checkpoint. Bub1 encodes a serine/threonine kinase required for mitotic spindle checkpoint function. The regulation of cell morphology in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). Here we show that Bub1 directly affects the structural integrity of IFs. Constitutive expression of Bub1 caused disappearance of filamentous vimentin, a type III IF, and consequently changed cell morphology. Expression of kinase domain—deleted Bub1 induced neither morphological change nor disappearance of vimentin. These observations suggest that Bub1 not only regulates the cell cycle, but also may be involved in the cytoskeletal control in interphase cells.     

Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization

Associated with the metastatic progression of epithelial tumors is the dynamic regulation of cadherins. Whereas E-cadherin is expressed in most epithelium and carcinomas, recent studies suggest that the up-regulation of other cadherin subtypes in carcinomas, such as N-cadherin, may function in cancer progression. We demonstrate that a signal transduction cascade links the N-cadherin.catenin adhesion complex to up-regulation of the anti-apoptotic protein Bcl-2. In suspension, aggregates of DU-145 cells, an E-cadherin expressing human prostate carcinoma line, survive loss of integrin-dependent adhesion by a different anti-apoptotic signaling pathway than the N-cadherin expressing lines PC3 and PC3N. N-cadherin intercellular adhesion mediates a 3.5-fold increase in Bcl-2 protein expression, whereas the level of the proapoptotic protein Bax remains constant. Only N-cadherin ligation in PC3 cells, which express both N-cadherin and E-cadherin, is sufficient to induce activation of Akt/protein kinase B. N-cadherin homophilic ligation initiates phosphatidylinositol 3-kinase-dependent activation of Akt resulting in Akt phosphorylation of Bad on serine 136. Following N-cadherin homophilic adhesion phosphatidylinositol 3-kinase was identified in immunoprecipitates of the N-cadherin.catenin complex. The recruitment of phosphatidylinositol 3-kinase to the adhesion complex is dependent on ligation of N-cadherin and an organized actin cytoskeleton because cytochalasin D blocks the recruitment. We propose that N-cadherin homophilic adhesion can initiate anti-apoptotic signaling, which enhances the Akt cell survival pathway in metastatic cancer.     

Regulation of cell adhesion by protein-tyrosine phosphatases. I. Cell-matrix adhesion

Protein-tyrosine phosphatases are key regulators of protein tyrosine phosphorylation. More than merely terminating the pathways initiated by protein-tyrosine kinases, phosphatases are active participants in many signaling pathways. Signals involving tyrosine phosphorylation are frequently generated in response to cell-matrix adhesion. In addition, high levels of protein tyrosine phosphorylation generally promote disassembly or turnover of adhesions. In this brief review, we will discuss the role of protein-tyrosine phosphatases in cell-matrix adhesions.     

Effects of local anesthetics on cell morphology and membrane-associated cytoskeletal organization in BALB/3T3 cells

Tertiary amine local anesthetics (dibucaine, tetracaine, procaine) reversibly affect the morphology of untransformed BALB/3T3 cells and the organization of membrane-associated cytoskeletal elements. In the presence of these drugs cells contract and become rounded in shape with the appearance of numerous surface "blebs." Electron microscope examination of anesthetic-treated cells revealed significant reductions in plasma membrane-associated microtubules and microfilaments and/or their plasma membrane attachment. The relationship of the findings on local anesthetic-induced changes in cellular cytoskeletal systems is discussed in relation to previous proposals on plasma membrane organization and control of cell surface receptor topography and mobility.     

B-RAF regulation of Rnd3 participates in actin cytoskeletal and focal adhesion organization

The actin cytoskeleton controls multiple cellular functions, including cell morphology, movement, and growth. Accumulating evidence indicates that oncogenic activation of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) pathway is accompanied by actin cytoskeletal reorganization. However, the signaling events contributing to actin cytoskeleton remodeling mediated by aberrant ERK1/2 activation are largely unknown. Mutant B-RAF is found in a variety of cancers, including melanoma, and it enhances activation of the MEK/ERK1/2 pathway. We show that targeted knockdown of B-RAF with small interfering RNA or pharmacological inhibition of MEK increased actin stress fiber formation and stabilized focal adhesion dynamics in human melanoma cells. These effects were due to stimulation of the Rho/Rho kinase (ROCK)/LIM kinase-2 signaling pathway, cumulating in the inactivation of the actin depolymerizing/severing protein cofilin. The expression of Rnd3, a Rho antagonist, was attenuated after B-RAF knockdown or MEK inhibition, but it was enhanced in melanocytes expressing active B-RAF. Constitutive expression of Rnd3 suppressed the actin cytoskeletal and focal adhesion effects mediated by B-RAF knockdown. Depletion of Rnd3 elevated cofilin phosphorylation and stress fiber formation and reduced cell invasion. Together, our results identify Rnd3 as a regulator of cross talk between the RAF/MEK/ERK and Rho/ROCK signaling pathways, and a key contributor to oncogene-mediated reorganization of the actin cytoskeleton and focal adhesions.