Trimers of the fibronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation

Previous studies have shown that small beads coated with FN7-10, a four-domain cell adhesion fragment of fibronectin, bind to cell surfaces and translocate rearward. Here we investigate whether soluble constructs containing two to five FN7-10 units might be sufficient for activity. We have produced a monomer, three forms of dimers, a trimer and a pentamer of FN7-10,on the end of spacer arms. These oligomers could bind small clusters of up to five integrins. Fluorescence microscopy showed that the trimer and pentamer bound strongly to the cell surface, and within 5 minutes were prominently localized to actin fiber bundles. Monomers and dimers showed only diffuse localization. Beads coated with a low concentration (probably one complex per bead) of trimer or pentamer showed prolonged binding and rearward translocation, presumably with the translocating actin cytskeleton. Beads containing monomer or dimer showed only brief binding and diffusive movements. We conclude that clusters of three integrin-binding ligands are necessary and sufficient for coupling to and translocating with the actin cytoskeleton.

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Functional partitioning of beta1 integrins revealed by activating and inhibitory mAbs

Journal of Cell Science ◽

10.1242/jcs.110.21.2647 ◽

1997 ◽

Vol 110 (21) ◽

pp. 2647-2659 ◽

Cited By ~ 1

Author(s):

M.T. Cruz ◽

C.L. Dalgard ◽

M.J. Ignatius

Keyword(s):

Cell Surface ◽

Actin Cytoskeleton ◽

Dermal Fibroblasts ◽

The Other ◽

Embryonic Chick ◽

Cell Surfaces ◽

Double Labeling ◽

Focal Contacts ◽

Functional Partitioning ◽

Discrete Populations

Integrins exist in different activation states on the surfaces of cells. Addition of the proper signal, ligand, or antibody can alter the activation state of these molecules. We report here the identification of two immunocytochemically distinct populations of beta1 integrins on fixed embryonic chick dermal fibroblasts. One population, recognized by the integrin activating mAb TASC, localizes to discrete regions of the cell, most likely focal contacts. These integrins co-localize with other proteins, such as vinculin and F-actin, and their retention at these sites is dependent on the actin cytoskeleton. The other population, identified with the inhibitory mAb W1B10, is more evenly distributed throughout the cell surface, and its pattern remains unchanged after disruption of the actin cytoskeleton. Double labeling experiments using Fab fragments of TASC alongside whole W1B10 IgG revealed non-overlapping staining patterns. These results show that it is possible to visualize and study discrete populations of integrins on cell surfaces using two different antibodies. We hypothesize that these antibodies report differences in the distribution of receptors in two different states. A model is proposed describing the ligand independent recruitment of integrins based on these findings and results from other labs.

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Dual role of the actin cytoskeleton in regulating cell adhesion mediated by the integrin lymphocyte function-associated molecule-1.

Molecular Biology of the Cell ◽

10.1091/mbc.8.2.341 ◽

1997 ◽

Vol 8 (2) ◽

pp. 341-351 ◽

Cited By ~ 122

Author(s):

M Lub ◽

Y van Kooyk ◽

S J van Vliet ◽

C G Figdor

Keyword(s):

Cell Adhesion ◽

Cell Surface ◽

Ligand Binding ◽

Actin Cytoskeleton ◽

Interleukin 2 ◽

Peripheral Blood Lymphocytes ◽

Lymphocyte Function ◽

Intracellular Signals ◽

Cytoskeletal Elements

Intracellular signals are required to activate the leukocyte-specific adhesion receptor lymphocyte function-associated molecule-1 (LFA-1; CD11a/CD18) to bind its ligand, intracellular adhesion molecule-1 (ICAM-1). In this study, we investigated the role of the cytoskeleton in LFA-1 activation and demonstrate that filamentous actin (F-actin) can both enhance and inhibit LFA-1-mediated adhesion, depending on the distribution of LFA-1 on the cell surface. We observed that LFA-1 is already clustered on the cell surface of interleukin-2/phytohemagglutinin-activated lymphocytes. These cells bind strongly ICAM-1 and disruption of the actin cytoskeleton inhibits adhesion. In contrast to interleukin-2/phytohemagglutinin-activated peripheral blood lymphocytes, resting lymphocytes, which display a homogenous cell surface distribution of LFA-1, respond poorly to intracellular signals to bind ICAM-1, unless the actin cytoskeleton is disrupted. On resting peripheral blood lymphocytes, uncoupling of LFA-1 from the actin cytoskeleton induces clustering of LFA-1 and this, along with induction of a high-affinity form of LFA-1, via "inside-out" signaling, results in enhanced binding to ICAM-1, which is dependent on intact intermediate filaments, microtubules, and metabolic energy. We hypothesize that linkage of LFA-1 to cytoskeletal elements prevents movement of LFA-1 over the cell surface, thus inhibiting clustering and strong ligand binding. Release from these cytoskeletal elements allows lateral movement and activation of LFA-1, resulting in ligand binding and "outside-in" signaling, that subsequently stimulates actin polymerization and stabilizes cell adhesion.

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Cell adhesion and the actin cytoskeleton of the enveloping layer in the zebrafish embryo during epiboly

Biochemistry and Cell Biology ◽

10.1139/o99-058 ◽

1999 ◽

Vol 77 (6) ◽

pp. 527-542 ◽

Cited By ~ 40

Author(s):

Sara E Zalik ◽

Ewa Lewandowski ◽

Zvi Kam ◽

Benjamin Geiger

Keyword(s):

Cell Adhesion ◽

Cell Surface ◽

Actin Cytoskeleton ◽

Zebrafish Embryo ◽

Cultured Cells ◽

Kinase Inhibitor ◽

Embryonic Cells ◽

Cortical Actin ◽

Actin Cables

As the zebrafish embryo undergoes gastrulation and epiboly, the cells of the enveloping layer (EVL) expand, covering the entire yolk cell. During the epiboly process, the EVL cells move as a coherent layer, remaining tightly attached to each other and to the underlying yolk syncytial layer (YSL). In view of the central role of the actin cytoskeleton, in both cell motility and cell cell adhesion, we have labeled these cells in situ with fluorescent phalloidin and anti-actin antibodies. We show that, throughout their migration, the EVL cells retain a conspicuous cortical actin cytoskeletal belt coinciding with cell surface cadherins. At the margins approaching the YSL, the EVL cells extend, from their apicolateral domains, actin-rich filopodial protrusions devoid of detectable cadherin. We have studied the role of the actin cytoskeleton in the maintenance of EVL cohesion during epiboly. Cytochalasin treatment of embryos induces EVL dissociation accompanied by general detachment of the rest of the embryonic cells. In the dissociating EVL cells, the cortical actin belt undergoes fragmentation with the formation of actin aggregates; cadherins, on the other hand, remain evenly distributed at the junctional cell surface. Removal of Ca2+ by ethyleneglycolbis (amino-ethyl-ether)-tetraacetic acid (EGTA) treatment also induces cell dissociation without visible disruption of the cortical actin belt. The protein kinase inhibitor (1-isoquinolinylsulfonyl)-2-methyl-piperazine dihydrochloride (H-7), which blocks acto-myosin contractility and disrupts actin cables in cultured cells, also potentiates cytochalasin-induced dissociation and promotes the projection of numerous actin-rich lamellipodial extensions. The fact that EVL cells produce microspike-like structures towards the YSL and are capable of lamellipodial activity lend further support to the suggestion (R.W. Keller and J.P. Trinkaus. 1987. Dev. Biol. 120: 12-24) that the EVL cells are not passively mobilized on the expanding YSL but actively participate in epiboly.Key words: actin, adhesion, cadherin, cytochalasin, embryo, zebrafish.

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Cell adhesion in sponges: potentiation by a cell surface 68 kDa proteoglycan-binding protein

Journal of Cell Science ◽

10.1242/jcs.108.9.3119 ◽

1995 ◽

Vol 108 (9) ◽

pp. 3119-3126 ◽

Cited By ~ 3

Author(s):

J.A. Varner

Keyword(s):

Cell Adhesion ◽

Cell Surface ◽

Intercellular Adhesion ◽

Cell Surface Receptor ◽

Sponge Cell ◽

Cell Surfaces ◽

Aggregation Factor ◽

Surface Receptor ◽

A Cell ◽

Molecule Aggregation

Constitutive, stable intercellular adhesion is one of the distinguishing properties of metazoans, of which the sponges (Phylum Porifera) are the most primitive representatives. In sponges, intercellular adhesion is mediated by the large proteoglycan-like cell agglutinating molecule ‘aggregation factor’, which binds to cell surfaces via an oligosaccharide moiety. Previous studies indicated that this aggregation factor binds to two proteins associated with the surface of sponge cells. One of these, a 68 kDa peripheral membrane protein, was isolated by affinity chromatography on aggregation factor conjugated to Sepharose. This monomeric 68 kDa glycoprotein plays a key role in sponge cell adhesion since it potently inhibits the binding of aggregation factor to cell surfaces and completely prevents aggregation factor-mediated cell adhesion. The 68 kDa aggregation factor ligand binds with high affinity to both aggregation factor (KD = 2 × 10(−9) M) and cell surfaces (KD = 6 × 10(−8) M) providing evidence that it serves as an intramolecular bridge between the aggregation factor molecule and a cell surface receptor. Therefore, this early metazoan protein may represent one of the earliest extracellular matrix adhesion proteins to have arisen in the course of metazoan evolution.

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Integrating the Actin and Vimentin Cytoskeletons

The Journal of Cell Biology ◽

10.1083/jcb.146.4.831 ◽

1999 ◽

Vol 146 (4) ◽

pp. 831-842 ◽

Cited By ~ 127

Author(s):

Ivan Correia ◽

Donald Chu ◽

Ying-Hao Chou ◽

Robert D. Goldman ◽

Paul Matsudaira

Keyword(s):

Cell Adhesion ◽

Fluorescence Microscopy ◽

Actin Cytoskeleton ◽

Binding Site ◽

Intermediate Filament ◽

Adhesion Sites ◽

Specific Antisera ◽

Detergent Extraction ◽

Biochemical Fractionation ◽

Dependent Interaction

Cells adhere to the substratum through specialized structures that are linked to the actin cytoskeleton. Recent studies report that adhesion also involves the intermediate filament (IF) and microtubule cytoskeletons, although their mechanisms of interaction are unknown. Here we report evidence for a novel adhesion-dependent interaction between components of the actin and IF cytoskeletons. In biochemical fractionation experiments, fimbrin and vimentin coprecipitate from detergent extracts of macrophages using vimentin- or fimbrin-specific antisera. Fluorescence microscopy confirms the biochemical association. Both proteins colocalized to podosomes in the earliest stages of cell adhesion and spreading. The complex is also found in filopodia and retraction fibers. After detergent extraction, fimbrin and vimentin staining of podosomes, filopodia, and retraction fibers are lost, confirming that the complex is localized to these structures. A 1:4 stoichiometry of fimbrin binding to vimentin and a low percentage (1%) of the extracted vimentin suggest that fimbrin interacts with a vimentin subunit. A fimbrin-binding site was identified in the NH2-terminal domain of vimentin and the vimentin binding site at residues 143–188 in the CH1 domain of fimbrin. Based on these observations, we propose that a fimbrin–vimentin complex may be involved in directing the assembly of the vimentin cytoskeleton at cell adhesion sites.

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Spatial control of actin-based motility through plasmalemmal PtdIns(4,5)P2-rich raft assemblies.

Biochemical Society Symposium ◽

10.1042/bss0720119 ◽

2005 ◽

Vol 72 ◽

pp. 119-127 ◽

Cited By ~ 19

Author(s):

Tamara Golub ◽

Caroni Pico

Keyword(s):

Protein Kinase ◽

Cell Surface ◽

Actin Cytoskeleton ◽

Lipid Rafts ◽

Patch Clustering ◽

Pkc Protein Kinase C ◽

Membrane Bound ◽

And Function ◽

Cytoskeleton Dynamics ◽

Syndrome Protein

The interactions of cells with their environment involve regulated actin-based motility at defined positions along the cell surface. Sphingolipid- and cholesterol-dependent microdomains (rafts) order proteins at biological membranes, and have been implicated in most signalling processes at the cell surface. Many membrane-bound components that regulate actin cytoskeleton dynamics and cell-surface motility associate with PtdIns(4,5)P2-rich lipid rafts. Although raft integrity is not required for substrate-directed cell spreading, or to initiate signalling for motility, it is a prerequisite for sustained and organized motility. Plasmalemmal rafts redistribute rapidly in response to signals, triggering motility. This process involves the removal of rafts from sites that are not interacting with the substrate, apparently through endocytosis, and a local accumulation at sites of integrin-mediated substrate interactions. PtdIns(4,5)P2-rich lipid rafts can assemble into patches in a process depending on PtdIns(4,5)P2, Cdc42 (cell-division control 42), N-WASP (neural Wiskott-Aldrich syndrome protein) and actin cytoskeleton dynamics. The raft patches are sites of signal-induced actin assembly, and their accumulation locally promotes sustained motility. The patches capture microtubules, which promote patch clustering through PKA (protein kinase A), to steer motility. Raft accumulation at the cell surface, and its coupling to motility are influenced greatly by the expression of intrinsic raft-associated components that associate with the cytosolic leaflet of lipid rafts. Among them, GAP43 (growth-associated protein 43)-like proteins interact with PtdIns(4,5)P2 in a Ca2+/calmodulin and PKC (protein kinase C)-regulated manner, and function as intrinsic determinants of motility and anatomical plasticity. Plasmalemmal PtdIns(4,5)P2-rich raft assemblies thus provide powerful organizational principles for tight spatial and temporal control of signalling in motility.

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