Alphav-integrin utilization in human beta-cell adhesion, spreading, and motility

The role of individual integrins in human beta-cell development and function is largely unknown. This study describes the contribution of alpha(v)-integrins to human beta-cell adhesion, spreading, and motility. Developmental differences in alpha(v)-integrin utilization are addressed by comparing the responses of adult and fetal beta-cells, and vitronectin is used as a substrate based on its unique pattern of expression in the developing pancreas. Fetal and adult beta-cells attached equally to vitronectin and integrin alpha(v)beta(5) was found to support the adhesion of both mature and immature beta-cell populations. Fetal beta-cells were also observed to spread and migrate on vitronectin, and integrin alpha(v)beta(1) was found to be essential for these responses. In contrast to their fetal counterparts, adult beta-cells failed to either spread or migrate and this deficit was associated with a marked down-regulation of alpha(v)beta(1) expression in adult islet preparations. The integrin alpha(v)beta(3) was not found to support significant beta-cell attachment or migration. Based on our findings, we conclude that integrins alpha(v)beta(5) and alpha(v)beta(1) are important mediators of human beta-cell adhesion and motility, respectively. By supporting fetal beta-cell migration, alpha(v)beta(1) could play an important role in early motile processes required for islet neogenesis.     

Multivalent presentation of a hydrolytically stable GM3 lactone mimetic as modulator of melanoma cells motility and adhesion

A hydrolytically stable mimetic of the tumour antigen GM₃ lactone is used to decorate multivalent scaffolds. Two of them positively interfere on melanoma cell adhesion, migration and resistance to apoptosis (anoikis). Notably, their ability to hamper melanoma-cells adhesion and reduce the metastatic potential is enhanced when the two scaffolds, presenting a different shape, are used in combination.     

Cell adhesion - spreading frontiers, intricate insights

Cell-cell and cell-matrix adhesions play important roles in determining the structural organization and behaviour of cells in tissues. These adhesions are mediated by specific cell-surface receptors that are linked to the actin cytoskeleton through submembranous multiprotein complexes that also serve to generate intracellular signals. The molecular mechanisms by which regulation of cell adhesiveness is coordinated with other aspects of cell behaviour are now under study and some aspects of this are highlighted in this short review. New scope for analysis of the roles of individual adhesion molecules in vivo is being provided by mouse gene knockouts.     

Cell spreading and motility: A model lamellipod

Cells moving in vitro do so by means of a motile appendage, the lamellipod. This is a broad, flat sheet of cytogel which spreads in front of the cell and pulls the cell forward. We present here a mathematical model for lamellipodial motion based on the physical chemistry of actomyosin gels.     

Cell adhesion molecule T-cadherin regulates vascular cell adhesion, phenotype and motility

T-cadherin (T-cad), an unusual glycosylphosphatidylinositol (GPI)-anchored member of the cadherin family of cell adhesion molecules, is widely expressed in the cardiovascular system. The expression profile of T-cad within diseased (atherosclerotic and restenotic) vessels indicates some relationship between expression of T-cad and the phenotypic status of resident cells. Using cultures of human aortic smooth muscle cells (SMC) and human umbilical vein endothelial cells (HUVEC) we investigate the hypothesis that T-cad may function in modulating adhesive properties of vascular cells. Coating of culture plates with recombinant T-cad protein or with antibody against the first amino-terminal domain of T-cad (anti-EC1) significantly decreased adhesion and spreading of SMC and HUVEC. HUVECs adherent on T-cad or anti-EC1 substratum exhibited an elongated morphology and associated redistribution of the cytoskeleton and focal adhesions to a distinctly peripheral location. These changes are characteristic of the less-adhesive, motile or pro-migratory, pro-angiogenic phenotype. Boyden chamber migration assay demonstrated that the deadhesion induced by T-cad facilitates cell migration towards a serum gradient. Overexpression of T-cad in vascular cells using adenoviral vectors does not influence cell adhesion or motility per se, but increases the detachment and migratory responses induced by T-cad substratum. The data suggest that T-cad acts as an anti-adhesive signal for vascular cells, thus modulating vascular cell phenotype and migration properties.     

Arrestins regulate cell spreading and motility via focal adhesion dynamics

Focal adhesions (FAs) play a key role in cell attachment, and their timely disassembly is required for cell motility. Both microtubule-dependent targeting and recruitment of clathrin are critical for FA disassembly. Here we identify nonvisual arrestins as molecular links between microtubules and clathrin. Cells lacking both nonvisual arrestins showed excessive spreading on fibronectin and poly-d-lysine, increased adhesion, and reduced motility. The absence of arrestins greatly increases the size and lifespan of FAs, indicating that arrestins are necessary for rapid FA turnover. In nocodazole washout assays, FAs in arrestin-deficient cells were unresponsive to disassociation or regrowth of microtubules, suggesting that arrestins are necessary for microtubule targeting–dependent FA disassembly. Clathrin exhibited decreased dynamics near FA in arrestin-deficient cells. In contrast to wild-type arrestins, mutants deficient in clathrin binding did not rescue the phenotype. Collectively the data indicate that arrestins are key regulators of FA disassembly linking microtubules and clathrin.     

Cell adhesion and spreading factor. Chemical modification studies

The purified fetal calf serum factor that promotes cell adhesion and spreading of baby hamster kidney cells on tissue culture substrata has been subjected to a variety of chemical modifications and then tested for activity. These studies have shown that modification of the carbohydrate portions of the factor by glycosidic enzymes or by periodate oxidation did not alter its ability to promote cell spreading. On the other hand, modification of some protein portions of the factor by proteolytic enzymes or by specific modification of —COOH groups, tyrosine residues, or tryptophan residues resulted in a marked inhibition of factor activity. Modification of protein —SH groups, —NH₂ groups, or methionine residues did not affect factor activity. Control experiments indicate that the various modifications were directed at the activity of the factor and not its adsorption onto the substrata.     

Kinetics of adhesion and spreading of animal cells

The adhesion and spreading of cells at surfaces are well established phenomena which have been extensively observed using light or electron microscopy. The kinetics of both processes can be quantitatively measured by allowing the cells to attach themselves to the surface of a planar waveguide allows the number of cells per unit area and a parameter uniquely characterizing their shape, such as the area in contact with the surface, to be determined. Cells suspended in nutrient medium or pure phosphate buffer were allowed to attach to and spread on a metal oxide surface or a layer of fibronectin, and the kinetics of attachment and spreading have been measured. Attachment kinetics are the same in all cases, but spreading on the metal oxide requires nutrient medium, without which it does not occur. Spreading of cells attached to a layer of fibronectin in the absence of nutrient medium occurs at about a tenth of the rate of cells spreading on metal oxide in the presence of nutrient. (c) 1994 John Wiley & Sons, Inc.     

The interaction between ADAM 22 and 14-3-3zeta: regulation of cell adhesion and spreading

The ADAM family consists of a number of transmembrane proteins that contain disintegrin-like and metalloproteinase-like domains. Therefore, ADAMs potentially have cell adhesion and protease activities. 14-3-3 proteins are a highly conserved family of cytoplasmic proteins that associate with several intracellular signaling molecules in the regulation of various cellular functions. Here we report the identification of a novel interaction between the ADAM 22 cytoplasmic tail and the 14-3-3zeta isoform by a yeast two-hybrid screen. The interaction between the ADAM 22 cytoplasmic tail and 14-3-3zeta was confirmed by an in vitro protein pull-down assay as well as by co-immunoprecipitation, and the binding sites were mapped to the 28 amino acid residues of the C-terminus of the ADAM 22 cytoplasmic tail. Furthermore, we found that overexpression of the ADAM 22 cytoplasmic tail in human SGH44 cells inhibited cell adhesion and spreading and that deletion or mutation of the binding site for 14-3-3zeta within the ADAM 22 cytoplasmic tail abolished the ability of the overexpressed cytoplasmic tail to alter cell adhesion and spreading. Taken together, these results for the first time demonstrate an association between ADAM 22 and a 14-3-3 protein and suggest a potential role for the 14-3-3zeta/ADAM 22 association in the regulation of cell adhesion and related signaling events.     

Characterization of a synthetic peptide from type IV collagen that promotes melanoma cell adhesion, spreading, and motility

The adhesion and motility of tumor cells on basement membranes is a central consideration in tumor cell invasion and metastasis. Basement membrane type IV collagen directly promotes the adhesion and migration of various tumor cell types in vitro. Our previous studies demonstrated that tumor cells adhered and spread on surfaces coated with intact type IV collagen or either of the two major enzymatically purified domains of this protein. Only one of these major domains, the pepsin-generated major triple helical fragment, also supported tumor cell motility in vitro, implicating the involvement of the major triple helical region in type IV collagen-mediated tumor cell invasion in vivo. The present studies extend our previous observations using a synthetic peptide approach. A peptide, designated IV-H1, was derived from a continuous collagenous region of the major triple helical domain of the human alpha 1(IV) chain. This peptide, which has the sequence GVKGDKGNPGWPGAP, directly supported the adhesion, spreading, and motility of the highly metastatic K1735 M4 murine melanoma cell line, as well as the adhesion and spreading of other cell types, in a concentration-dependent manner in vitro. Furthermore, excess soluble peptide IV-H1, or polyclonal antibodies directed against peptide IV-H1, inhibited type IV collagen-mediated melanoma cell adhesion, spreading, and motility, but had no effect on these cellular responses to type I collagen. The full complement of cell adhesion, spreading, and motility promoting activities was dependent upon the preservation of the three prolyl residues in the peptide IV-H1 sequence. These studies indicate that peptide IV-H1 represents a cell-specific adhesion, spreading, and motility promoting domain that is active within the type IV collagen molecule.     

Further studies on cell adhesion based on Lex-Lex interaction,with new approaches: embryoglycan aggregation of F9 teratocarcinoma cells,and adhesion of various tumour cells based on Lex expression

We previously proposed specific interaction of Le^x (Gal1 4[Fuc1 3]-GlcNAc1 3Gal) with Le^x as a basis of cell adhesion in pre-implantation embryos and in aggregation of F9 teratocarcinoma cells, based on several lines of evidence (Eggenset al., J Biol Chem (1989)264:9476–9484). We now present additional evidence for this concept, based on autoaggregation studies of plastic beads coated with glycosphingolipids (GSLs) bearing Le^x or other epitopes, and affinity chromatography on Le^x-columns of multivalent lactofucopentaose III (Le^x oligosaccharide) conjugated with lysyllysine. Comparative adhesion studies of Le^x-expressing tumour cellsvs their Le^x-non-expressing variants showed that only Le^x-expressing cells adhere to Le^x-coated plates and are involved in tumour cell aggregation, in analogy to F9 cell aggregation. The major carrier of Le^x determinant in F9 cells is not GSL but rather polylactosaminoglycan (embryoglycan), and we demonstrated autoaggregation of purified embryoglycan in the presence of Ca²⁺, and reversible dissociation in the absence of Ca²⁺ (addition of EDTA). Defucosylated embryoglycan did not show autoaggregation under the same conditions. Thus, Le^x-Le^x interaction has been demonstrated on a lactosaminoglycan basis as well as a GSL basis. A molecular model of Le^x-Le^x interaction based on minimum energy conformation with involvement of Ca²⁺ is presented.Abbreviations BSA bovine serum albumin - CHO carbohydrate - DMEM Dulbecco's modified Eagle's medium - EDTA ethylenediaminetetraacetic acid - GP glycopeptide - GSL glycosphingolipid - LAG lactosaminoglycan - Le^x Gal1 4[Fuc-1 3]GlcNAc1 R - LFP lacto-N-fucopentaose - LysLys-OH lysyllysinol - Mr relative molecular weight - PBS phosphate-buffered saline - PG paragloboside (Gal1 4GlcNAc1 3Gal1 4Glc1 1Cer) - TBS Tris-buffered saline (10mM Tris-HCl, pH 7.4, containing 0.15M NaCl) - TC tumour cell     

A novel GRK2/HDAC6 interaction modulates cell spreading and motility

Cell motility and adhesion involves dynamic microtubule (MT) acetylation/deacetylation, a process regulated by enzymes as HDAC6, a major cytoplasmic α-tubulin deacetylase. We identify G protein-coupled receptor kinase 2 (GRK2) as a key novel stimulator of HDAC6. GRK2, which levels inversely correlate with the extent of α-tubulin acetylation in epithelial cells and fibroblasts, directly associates with and phosphorylates HDAC6 to stimulate α-tubulin deacetylase activity. Remarkably, phosphorylation of GRK2 itself at S670 specifically potentiates its ability to regulate HDAC6. GRK2 and HDAC6 colocalize in the lamellipodia of migrating cells, leading to local tubulin deacetylation and enhanced motility. Consistently, cells expressing GRK2-K220R or GRK2-S670A mutants, unable to phosphorylate HDAC6, exhibit highly acetylated cortical MTs and display impaired migration and protrusive activity. Finally, we find that a balanced, GRK2/HDAC6-mediated regulation of tubulin acetylation differentially modulates the early and late stages of cellular spreading. This novel GRK2/HDAC6 functional interaction may have important implications in pathological contexts.     

Angiopoietin-related growth factor (AGF) supports adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells through interaction with RGD-binding integrins

Angiopoietin-related growth factor (AGF) is a newly identified member of angiopoietin-related proteins (ARPs)/angiopoietin-like proteins (Angptls). AGF has been considered as a novel growth factor in accelerating cutaneous wound healing, as it is capable of stimulating keratinocytes proliferation as well as angiogenesis. But in our paper, we demonstrate that AGF stimulates keratinocytes proliferation only at high protein concentration, however, it can potently promote adhesion, spreading, and migration of keratinocytes, fibroblasts, and endothelial cells. Furthermore, we confirm that the adhesion and migration cellular events are mediated by RGD-binding integrins, most possibly the alpha(v)-containing integrins, by in vitro inhibition assays using synthetic competitive peptides. Our results strongly suggest that AGF is an integrin ligand as well as a mitogenic growth factor and theoretically participates in cutaneous wound healing in a more complex mechanism.     

Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro

Human umbilical vein endothelial cells (ECs) have been shown to attach to a substratum of fibrinogen (fg). Later, ECs undergo spreading, organization of thick microfilament bundles of the stress fiber type, and formation of focal contacts (adhesion plaques) that correspond to accumulation of vinculin at the cytoplasmic aspect of the ventral membrane. The rate of attachment to fg and the type of spreading is virtually identical to that obtained on substrata coated with fibronectin (FN). Antibodies to fg, but not to FN, prevent EC adhesion to fg; conversely, antibodies to FN, but not to fg, prevent adhesion of ECs to a FN-coated substratum. The removal of residual FN contamination from fg preparations by means of DEAE-cellulose chromatography does not result in any difference in EC adhesion on fg. Moreover, pretreatment of cells with inhibitors of synthesis and release of proteins does not impair their adhesion capacity on an fg-coated substratum. In contrast, human arterial smooth muscle cells do not adhere and spread on fg substrata but do so on FN. The synthetic peptides (Gly-Arg-Gly-Asp[GRGD] and Gly-Arg-Gly-Asp-Ser-Pro[GRGDSP]) containing the tripeptide Arg-Gly-Asp (RGD), originally found to be responsible for the cell binding activity of FN, have been found to inhibit EC spreading and the redistribution of their cytoskeleton, including the formation of stress fibers and the localization of vinculin either on fg or on FN. Conversely, the synthetic peptide Arg-Gly-Gly (RGG) was completely uneffective in inhibiting the adhesion and the sequence of events leading to spreading and cytoskeletal organization. These results indicate that ECs, but not smooth muscle cells, specifically adhere and spread on an fg substratum and this occurs by recognition mechanisms similar to those reported for FN.     

EDI3 links choline metabolism to integrin expression,cell adhesion and spreading

Endometrial carcinoma differential 3 (EDI3) was the first member of the glycerophosphodiesterase (GDE) protein family shown to be associated with cancer. Our initial work demonstrated that endometrial and ovarian cancer patients with primary tumors overexpressing EDI3 had a higher risk of developing metastasis and decreased survival. Further analysis indicated that EDI3 cleaves glycerophosphocholine to choline and glycerol-3-phosphate, increases the levels of active PKC, and enhances the migratory activity of tumor cells. Despite these initial findings, EDI3 remained mainly uncharacterized. Therefore, to obtain an overview of processes in which EDI3 may be involved, gene array analysis was performed using MCF-7 breast cancer cells after EDI3 knockdown compared with a non-targeting control siRNA. Several biological motifs were altered, including an enrichment of genes involved in integrin-mediated signaling. More specifically, silencing of EDI3 in MCF-7 and OVCAR-3 cells was associated with reduced expression of the key receptor subunit integrin β1, leading to decreased cell attachment and spreading accompanied by delayed formation of cell protrusions. To confirm these results, we stably overexpressed EDI3 in MCF-7 cells which led to elevated integrin β1 expression associated with enhanced cell attachment and spreading - two processes critical for metastasis. In conclusion, our data provide further insight into the role of EDI3 during cancer progression.     

EDI3 links choline metabolism to integrin expression,cell adhesion and spreading

Endometrial carcinoma differential 3 (EDI3) was the first member of the glycerophosphodiesterase (GDE) protein family shown to be associated with cancer. Our initial work demonstrated that endometrial and ovarian cancer patients with primary tumors overexpressing EDI3 had a higher risk of developing metastasis and decreased survival. Further analysis indicated that EDI3 cleaves glycerophosphocholine to choline and glycerol-3-phosphate, increases the levels of active PKC, and enhances the migratory activity of tumor cells. Despite these initial findings, EDI3 remained mainly uncharacterized. Therefore, to obtain an overview of processes in which EDI3 may be involved, gene array analysis was performed using MCF-7 breast cancer cells after EDI3 knockdown compared with a non-targeting control siRNA. Several biological motifs were altered, including an enrichment of genes involved in integrin-mediated signaling. More specifically, silencing of EDI3 in MCF-7 and OVCAR-3 cells was associated with reduced expression of the key receptor subunit integrin β1, leading to decreased cell attachment and spreading accompanied by delayed formation of cell protrusions. To confirm these results, we stably overexpressed EDI3 in MCF-7 cells which led to elevated integrin β1 expression associated with enhanced cell attachment and spreading - two processes critical for metastasis. In conclusion, our data provide further insight into the role of EDI3 during cancer progression.     

Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands

Integrin-mediated adhesion is regulated by multiple features of the adhesive surface, including its chemical composition, topography, and physical properties. In this study we investigated integrin lateral clustering, as a mechanism to control integrin functions, by characterizing the effect of nanoscale variations in the spacing between adhesive RGD ligands on cell spreading, migration, and focal adhesion dynamics. For this purpose, we used nanopatterned surfaces, containing RGD-biofunctionalized gold dots, surrounded by passivated gaps. By varying the spacing between the dots, we modulated the clustering of the associated integrins. We show that cell-surface attachment is not sensitive to pattern density, whereas the formation of stable focal adhesions and persistent spreading is. Thus cells plated on a 108-nm-spaced pattern exhibit delayed spreading with repeated protrusion-retraction cycles compared to cells growing on a 58-nm pattern. Cell motility on these surfaces is erratic and nonpersistent, leaving thin membrane tethers bound to the RGD pattern. Dynamic molecular profiling indicated that the adhesion sites formed with the 108-nm pattern undergo rapid turnover and contain reduced levels of zyxin. These findings indicate that a critical RGD density is essential for the establishment of mature and stable integrin adhesions, which, in turn, induce efficient cell spreading and formation of focal adhesions.     

Change in the topographical distribution of GM3 during cell spreading and growth: immunostaining with monoclonal antibody against GM3

A monoclonal antibody, M2590, that recognizes hematoside (GM3) was used to analyze the immunostaining localization of GM3 of the surface of transformed and non-transformed hamster embryo fibroblasts and B16 melanoma cells. The reactivity of GM3 with the antibody changed markedly depending on the cell density. At the sparse density cells were clearly made visible by the antibody, but at the confluency the accessibility of the antibody to GM3 was greatly decreased. This density dependent change in the reactivity of GM3 was found for both normal and transformed cells. The staining pattern of GM3 was examined in relation to the actin fibers made visible with NBD-Phallacidin during cell spreading. When the cells were still round, the GM3 on microspikes or blebs was highly reactive with the antibody, and by the time cells showed circumferential staining of their actin fibers, GM3 had been distributed over the entire cell surface as punctuated spots. GM3 also was visible in substrate attachment materials (SAM). Trypsin treatment of SAM diminished the reactivity of GM3 with the antibody. The antibody did not inhibit cell attachment or spreading on a substratum coated with fibronectin or laminin.     

Low Mr phosphotyrosine protein phosphatase associates and dephosphorylates p125 focal adhesion kinase,interfering with cell motility and spreading

Low M(r) phosphotyrosine protein phosphatase interferes in vivo with the activation of several growth factor receptors and is transiently redistributed, following cell stimulation with platelet-derived growth factor, from the cytosol to the cytoskeleton. We demonstrate here that this phosphatase also participates in the regulation of cell spreading and migration, pointing to its involvement in cytoskeleton organization. Low M(r) phosphotyrosine protein phosphatase-overexpressing fibroblasts are, indeed, less spread than controls and display a significantly decreased number of focal adhesions and increased cell motility. Furthermore, p125 focal adhesion kinase is associated to, and dephosphorylated by, low M(r) phosphotyrosine protein phosphatase both in vitro and in vivo. This event is consistent with an altered association of pp60(src) with focal adhesion kinase. The activation of extracellular signal-regulated kinase, another well known event downstream of the focal adhesion kinase, is also affected. On the other hand, cells overexpressing the dominant-negative form of low M(r) phosphotyrosine protein phosphatase exhibit hyperphosphorylated focal adhesion kinase, reduced motility, and an increased number of focal adhesions, which are distributed all over the ventral cell surface. Taken together, the results reported here are in keeping with low M(r) phosphotyrosine protein phosphatase participation in FAK-mediated focal adhesion remodeling.     

Interplay of RhoA and motility in the programmed spreading of daughter cells postmitosis

Upon cortical retraction in mitosis, mammalian cells have a dramatically decreased physical association with their environment. Hence, mechanisms that prevent mitotic detachment and ensure appropriate positioning of the resulting daughter cells are critical for effective tissue morphogenesis and repair, and are the subject of this study. We find that, unlike low-motility cells, highly motile cells spread isotropically upon division and do not typically reoccupy their mother-cell footprint, and often even disseminate their mitotic cells. To elucidate these different motility-based phenotypes, we investigated their partial recapitulation and rescue using defined molecular perturbations. We show that activated RhoA is localized at the mitotic cell cortex, and Rho-associated kinase inhibition increases the degree of reoccupation of the mother-cell outline in highly motile cells. Conversely, we show that induction of motility in low-motility cells by RasV12 overexpression results in increased isotropic daughter-cell spreading. We thus propose that a balance between cortical retraction forces, which depend in part on RhoA activation, and substrate adhesion forces, which diminish with increasing motility rates, governs the integrity of mitotic actin retraction fibers and influences subsequent daughter-cell spreading. This balance of forces during mitosis has implications for cancer metastasis.