Molecular diversity of cell-matrix adhesions

1999 ◽  
Vol 112 (11) ◽  
pp. 1655-1669 ◽  
Author(s):  
E. Zamir ◽  
B.Z. Katz ◽  
S. Aota ◽  
K.M. Yamada ◽  
B. Geiger ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Focal Adhesions ◽  
Automatic Identification ◽  
Molecular Heterogeneity ◽  
Cell Matrix ◽  
Adhesion Sites ◽  
Actomyosin Contractility ◽  
Focal Contacts ◽  
Quantitative Immunofluorescence ◽  
Ratio Imaging

In this study we have examined for molecular heterogeneity of cell-matrix adhesions and the involvement of actomyosin contractility in the selective recruitment of different plaque proteins. For this purpose, we have developed a novel microscopic approach for molecular morphometry, based on automatic identification of matrix adhesions, followed by quantitative immunofluorescence and morphometric analysis. Particularly informative was fluorescence ratio imaging, comparing the local labeling intensities of different plaque molecules, including vinculin, paxillin, tensin and phosphotyrosine-containing proteins. Ratio imaging revealed considerable molecular heterogeneity between and within adhesion sites. Most striking were the differences between focal contacts, which are vinculin- and paxillin-rich and contain high levels of phosphotyrosine, and fibrillar adhesions, which are tensin-rich and contain little or no phosphotyrosine. Ratio imaging also revealed considerable variability in the molecular substructure of individual focal contacts, pointing to a non-uniform distribution of phosphotyrosine and the different plaque constituents. Studying the quantitative relationships between the various components of the submembrane plaque indicated that the levels of vinculin, paxillin and phosphotyrosine in adhesion sites are positively correlated with each other and negatively correlated with the levels of tensin. Tyrosine phosphorylation of focal contacts was highly sensitive to cellular contractility, and was diminished within 5 minutes after treatment with the kinase inhibitor H-7, an inhibitor of actomyosin contractility. This was followed by the loss of paxillin and vinculin from the focal adhesions. Tensin-rich fibrillar adhesions were relatively insensitive to H-7 treatment. These findings suggest a role for contractility in the generation of matrix adhesion diversity.

scholarly journals Zasp is required for the assembly of functional integrin adhesion sites

2007 ◽  
Vol 179 (7) ◽  
pp. 1583-1597 ◽  
Author(s):  
Klodiana Jani ◽  
Frieder Schöck
Keyword(s):  
Focal Adhesions ◽  
Myotendinous Junction ◽  
Lim Domain ◽  
Transmembrane Receptors ◽  
Cell Matrix ◽  
Adhesion Sites ◽  
Alternatively Spliced ◽  
Dependent Cell ◽  
Cell Matrix Adhesion ◽  
Myotendinous Junctions

The integrin family of heterodimeric transmembrane receptors mediates cell–matrix adhesion. Integrins often localize in highly organized structures, such as focal adhesions in tissue culture and myotendinous junctions in muscles. Our RNA interference screen for genes that prevent integrin-dependent cell spreading identifies Z band alternatively spliced PDZ-motif protein (zasp), encoding the only known Drosophila melanogaster Alp/Enigma PDZ-LIM domain protein. Zasp localizes to integrin adhesion sites and its depletion disrupts integrin adhesion sites. In tissues, Zasp colocalizes with βPS integrin in myotendinous junctions and with α-actinin in muscle Z lines. Zasp also physically interacts with α-actinin. Fly larvae lacking Zasp do not form Z lines and fail to recruit α-actinin to the Z line. At the myotendinous junction, muscles detach in zasp mutants with the onset of contractility. Finally, Zasp interacts genetically with integrins, showing that it regulates integrin function. Our observations point to an important function for Zasp in the assembly of integrin adhesion sites both in cell culture and in tissues.

scholarly journals WT1-interacting protein (Wtip) regulates podocyte phenotype by cell-cell and cell-matrix contact reorganization

2012 ◽  
Vol 302 (1) ◽  
pp. F103-F115 ◽  
Author(s):  
Jane H. Kim ◽  
Amitava Mukherjee ◽  
Sethu M. Madhavan ◽  
Martha Konieczkowski ◽  
John R. Sedor
Keyword(s):  
Kinase Inhibitor ◽  
Adherens Junctions ◽  
Focal Adhesions ◽  
Stress Fibers ◽  
Actin Dynamics ◽  
Transcriptional Responses ◽  
Interacting Protein ◽  
Cell Matrix ◽  
Actin Stress Fibers ◽  
Cell Cell

Podocytes respond to environmental cues by remodeling their slit diaphragms and cell-matrix adhesive junctions. Wt1-interacting protein (Wtip), an Ajuba family LIM domain scaffold protein expressed in the podocyte, coordinates cell adhesion changes and transcriptional responses to regulate podocyte phenotypic plasticity. We evaluated effects of Wtip on podocyte cell-cell and cell-matrix contact organization using gain-of- and loss-of-function methods. Endogenous Wtip targeted to focal adhesions in adherent but isolated podocytes and then shifted to adherens junctions after cells made stable, homotypic contacts. Podocytes with Wtip knockdown (shWtip) adhered but failed to spread normally. Noncontacted shWtip podocytes did not assemble actin stress fibers, and their focal adhesions failed to mature. As shWtip podocytes established cell-cell contacts, stable adherens junctions failed to form and F-actin structures were disordered. In shWtip cells, cadherin and β-catenin clustered in irregularly distributed spots that failed to laterally expand. Cell surface biotinylation showed diminished plasma membrane cadherin, β-catenin, and α-catenin in shWtip podocytes, although protein expression was similar in shWtip and control cells. Since normal actin dynamics are required for organization of adherens junctions and focal adhesions, we determined whether Wtip regulates F-actin assembly. Undifferentiated podocytes did not elaborate F-actin stress fibers, but when induced to overexpress WTIP, formed abundant stress fibers, a process blocked by the RhoA inhibitor C3 toxin and a RhoA kinase inhibitor. WTIP directly interacted with Rho guanine nucleotide exchange factor (GEF) 12 (Arhgef12), a RhoA-specific GEF enriched in the glomerulus. In conclusion, stable assembly of podocyte adherens junctions and cell-matrix contacts requires Wtip, a process that may be mediated by spatiotemporal regulation of RhoA activity through appropriate targeting of Arhgef12.

scholarly journals Probing mechanical principles of focal contacts in cell–matrix adhesion with a coupled stochastic–elastic modelling framework

2011 ◽  
Vol 8 (62) ◽  
pp. 1217-1232 ◽  
Author(s):  
Huajian Gao ◽  
Jin Qian ◽  
Bin Chen
Keyword(s):  
Numerical Models ◽  
Focal Adhesions ◽  
Point Of View ◽  
Matrix Adhesion ◽  
Modelling Framework ◽  
Cell Matrix ◽  
Focal Contacts ◽  
Wide Range ◽  
Sense And Respond ◽  
Cell Matrix Adhesion

Cell–matrix adhesion depends on the collective behaviours of clusters of receptor–ligand bonds called focal contacts between cell and extracellular matrix. While the behaviour of a single molecular bond is governed by statistical mechanics at the molecular scale, continuum mechanics should be valid at a larger scale. This paper presents an overview of a series of recent theoretical studies aimed at probing the basic mechanical principles of focal contacts in cell–matrix adhesion via stochastic–elastic models in which stochastic descriptions of molecular bonds and elastic descriptions of interfacial traction–separation are unified in a single modelling framework. The intention here is to illustrate these principles using simple analytical and numerical models. The aim of the discussions is to provide possible clues to the following questions: why does the size of focal adhesions (FAs) fall into a narrow range around the micrometre scale? How can cells sense and respond to substrates of varied stiffness via FAs? How do the magnitude and orientation of mechanical forces affect the binding dynamics of FAs? The effects of cluster size, cell–matrix elastic modulus, loading direction and cytoskeletal pretension on the lifetime of FA clusters have been investigated by theoretical arguments as well as Monte Carlo numerical simulations, with results showing that intermediate adhesion size, stiff substrate, cytoskeleton stiffening, low-angle pulling and moderate cytoskeletal pretension are factors that contribute to stable FAs. From a mechanistic point of view, these results provide possible explanations for a wide range of experimental observations and suggest multiple mechanisms by which cells can actively control adhesion and de-adhesion via cytoskeletal contractile machinery in response to mechanical properties of their surroundings.

scholarly journals Physical State of the Extracellular Matrix Regulates the Structure and Molecular Composition of Cell-Matrix Adhesions

2000 ◽  
Vol 11 (3) ◽  
pp. 1047-1060 ◽  
Author(s):  
Ben-Zion Katz ◽  
Eli Zamir ◽  
Alexander Bershadsky ◽  
Zvi Kam ◽  
Kenneth M. Yamada ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Tyrosine Phosphorylation ◽  
Physical State ◽  
Critical Factor ◽  
Molecular Composition ◽  
Molecular Organization ◽  
Cell Contractility ◽  
Cell Matrix ◽  
Adhesion Sites ◽  
Focal Contacts

This study establishes that the physical state of the extracellular matrix can regulate integrin-mediated cytoskeletal assembly and tyrosine phosphorylation to generate two distinct types of cell-matrix adhesions. In primary fibroblasts, α5β1 integrin associates mainly with fibronectin fibrils and forms adhesions structurally distinct from focal contacts, independent of actomyosin-mediated cell contractility. These “fibrillar adhesions” are enriched in tensin, but contain low levels of the typical focal contact components paxillin, vinculin, and tyrosine-phosphorylated proteins. However, when the fibronectin is covalently linked to the substrate, α5β1integrin forms highly tyrosine-phosphorylated, “classical” focal contacts containing high levels of paxillin and vinculin. These experiments indicate that the physical state of the matrix, not just its molecular composition, is a critical factor in defining cytoskeletal organization and phosphorylation at adhesion sites. We propose that molecular organization of adhesion sites is controlled by at least two mechanisms: 1) specific integrins associate with their ligands in transmembrane complexes with appropriate cytoplasmic anchor proteins (e.g., fibronectin–α5β1integrin–tensin complexes), and 2) physical properties (e.g., rigidity) of the extracellular matrix regulate local tension at adhesion sites and activate local tyrosine phosphorylation, recruiting a variety of plaque molecules to these sites. These mechanisms generate structurally and functionally distinct types of matrix adhesions in fibroblasts.

scholarly journals pp60c-src and related tyrosine kinases: a role in the assembly and reorganization of matrix adhesions

2001 ◽  
Vol 114 (12) ◽  
pp. 2279-2289 ◽  
Author(s):  
Tova Volberg ◽  
Lewis Romer ◽  
Eli Zamir ◽  
Benjamin Geiger
Keyword(s):  
Tyrosine Kinases ◽  
Mutant Cell ◽  
Focal Adhesions ◽  
Src Family Kinases ◽  
Wild Type ◽  
Altered Expression ◽  
Cell Matrix ◽  
Focal Contacts ◽  
Cell Matrix Adhesion

Activation of tyrosine kinases during integrin-mediated cell-matrix adhesion is involved both in the regulation of focal contact assembly and in the initiation of signaling processes at the cell-matrix adhesive interface. In order to determine the role of pp60c-src and related kinases in these processes, we have compared the dynamic reorganization of phosphotyrosine, vinculin, focal adhesion kinase and tensin in cells with altered expression of Src-family kinases. Both null cells for pp60c-src and triple knockout cells for pp60c-src, pp59fyn, and pp62c-yes exhibited decreased phosphotyrosine levels in focal contacts when compared with wild-type cells. pp60c-src-null cells also exhibited faster assembly of cell-matrix adhesions and a more exuberant recruitment of FAK to these sites. Tensin, which normally segregates into fibrillar adhesions was localized in large focal contacts in the two mutant cell lines, suggesting involvement of pp60c-src in the segregation of focal contacts and fibrillar adhesions. Moreover, treatment of wild-type cells with tyrphostin AG1007, which inhibits both pp60c-src and FAK activity, induced accumulation of tensin in peripheral focal adhesions. These findings demonstrate that Src family kinases, and pp60c-src in particular, have a central role in regulating protein dynamics at cell-matrix interfaces, both during early stages of interaction and in mature focal contacts.

scholarly journals Symmetric exchange of multi-protein building blocks between stationary focal adhesions and the cytosol

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jan-Erik Hoffmann ◽  
Yessica Fermin ◽  
Ruth LO Stricker ◽  
Katja Ickstadt ◽  
Eli Zamir
Keyword(s):  
Self Assembly ◽  
Cross Correlation ◽  
Protein Complexes ◽  
Focal Adhesions ◽  
Building Blocks ◽  
Correlation Spectroscopy ◽  
Cell Matrix ◽  
Adhesion Sites ◽  
Dynamic Exchange ◽  
Cell Matrix Adhesion

How can the integrin adhesome get self-assembled locally, rapidly, and correctly as diverse cell-matrix adhesion sites? Here, we investigate this question by exploring the cytosolic state of integrin-adhesome components and their dynamic exchange between adhesion sites and cytosol. Using fluorescence cross-correlation spectroscopy (FCCS) and fluorescence recovery after photobleaching (FRAP) we found that the integrin adhesome is extensively pre-assembled already in the cytosol as multi-protein building blocks for adhesion sites. Stationary focal adhesions release symmetrically the same types of protein complexes that they recruit, thereby keeping the cytosolic pool of building blocks spatiotemporally uniform. We conclude a model in which multi-protein building blocks enable rapid and modular self-assembly of adhesion sites and symmetric exchange of these building blocks preserves their specifications and thus the assembly logic of the system.

Fibronexus-Like Adhesion Sites are Present at the Invasive Zones of Human Epidermoid Carcinomas

1982 ◽  
Vol 40 ◽  
pp. 106-109
Author(s):  
Irwin I. Singer
Keyword(s):  
Cell Cycle ◽  
Serum Concentration ◽  
Substrate Binding ◽  
High Serum ◽  
Adhesion Sites ◽  
Substrate Adhesion ◽  
Focal Contacts ◽  
Adhesion Complex ◽  
Low Serum

Our previous results indicate that two types of fibronectin-cytoskeletal associations may be formed at the fibroblast surface: dorsal matrixbinding fibronexuses generated in high serum (5% FBS) cultures, and ventral substrate-adhering units formed in low serum (0.3% FBS) cultures. The substrate-adhering fibronexus consists of at least vinculin (VN) and actin in its cytoplasmic leg, and fibronectin (FN) as one of its major extracellular components. This substrate-adhesion complex is localized in focal contacts, the sites of closest substratum approach visualized with interference reflection microscopy, which appear to be the major points of cell-tosubstrate adhesion. In fibroblasts, the latter substrate-binding complex is characteristic of cultures that are arrested at the G1 phase of the cell cycle due to the low serum concentration in their medium. These arrested fibroblasts are very well spread, flattened, and immobile.

scholarly journals Blockade of ROCK inhibits migration of human primary keratinocytes and malignant epithelial skin cells by regulating actomyosin contractility

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Srisathya Srinivasan ◽  
Sreya Das ◽  
Vishakha Surve ◽  
Ankita Srivastava ◽  
Sushant Kumar ◽  
...  
Keyword(s):  
Light Chain ◽  
Myosin Light Chain ◽  
Focal Adhesions ◽  
Primary Keratinocytes ◽  
Skin Cells ◽  
Physiological Processes ◽  
Actomyosin Contractility ◽  
Malignant Skin ◽  
And Migration ◽  
Mlc Phosphorylation

AbstractActomyosin contractility, crucial for several physiological processes including migration, is controlled by the phosphorylation of myosin light chain (MLC). Rho-associated protein kinase (ROCK) and Myosin light chain kinase (MLCK) are predominant kinases that phosphorylate MLC. However, the distinct roles of these kinases in regulating actomyosin contractility and their subsequent impact on the migration of healthy and malignant skin cells is poorly understood. We observed that blockade of ROCK in healthy primary keratinocytes (HPKs) and epidermal carcinoma cell line (A-431 cells) resulted in loss of migration, contractility, focal adhesions, stress fibres, and changes in morphology due to reduction in phosphorylated MLC levels. In contrast, blockade of MLCK reduced migration, contractile dynamics, focal adhesions and phosphorylated MLC levels of HPKs alone and had no effect on A-431 cells due to the negligible MLCK expression. Using genetically modified A-431 cells expressing phosphomimetic mutant of p-MLC, we show that ROCK dependent phosphorylated MLC controls the migration, focal adhesion, stress fibre organization and the morphology of the cells. In conclusion, our data indicate that ROCK is the major kinase of MLC phosphorylation in both HPKs and A-431 cells, and regulates the contractility and migration of healthy as well as malignant skin epithelial cells.

scholarly journals Lipid rafts mediate internalization of β1-integrin in migrating intestinal epithelial cells

2008 ◽  
Vol 295 (5) ◽  
pp. G965-G976 ◽  
Author(s):  
Elena V. Vassilieva ◽  
Kirsten Gerner-Smidt ◽  
Andrei I. Ivanov ◽  
Asma Nusrat
Keyword(s):  
Epithelial Cells ◽  
Lipid Rafts ◽  
Lipid Raft ◽  
Intestinal Epithelial Cells ◽  
Focal Adhesions ◽  
Endocytic Pathway ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Caveolin 1 ◽  
Cell Matrix

Intestinal mucosal inflammation is associated with epithelial wounds that rapidly reseal by migration of intestinal epithelial cells (IECs). Cell migration involves cycles of cell-matrix adhesion/deadhesion that is mediated by dynamic turnover (assembly and disassembly) of integrin-based focal adhesions. Integrin endocytosis appears to be critical for deadhesion of motile cells. However, mechanisms of integrin internalization during remodeling of focal adhesions of migrating IECs are not understood. This study was designed to define the endocytic pathway that mediates internalization of β1-integrin in migrating model IECs. We observed that, in SK-CO15 and T84 colonic epithelial cells, β1-integrin is internalized in a dynamin-dependent manner. Pharmacological inhibition of clathrin-mediated endocytosis or macropinocytosis and small-interfering RNA (siRNA)-mediated knock down of clathrin did not prevent β1-integrin internalization. However, β1-integrin internalization was inhibited following cholesterol extraction and after overexpression of lipid raft protein, caveolin-1. Furthermore, internalized β1-integrin colocalized with the lipid rafts marker cholera toxin, and siRNA-mediated knockdown of caveolin-1 and flotillin-1/2 increased β1-integrin endocytosis. Our data suggest that, in migrating IEC, β1-integrin is internalized via a dynamin-dependent lipid raft-mediated pathway. Such endocytosis is likely to be important for disassembly of integrin-based cell-matrix adhesions and therefore in regulating IEC migration and wound closure.

Ezrin regulates cell-cell and cell-matrix adhesion, a possible role with E-cadherin/beta-catenin

1999 ◽  
Vol 112 (18) ◽  
pp. 3081-3090 ◽  
Author(s):  
S. Hiscox ◽  
W.G. Jiang
Keyword(s):  
Cancer Cells ◽  
Focal Adhesions ◽  
Beta Catenin ◽  
Matrix Adhesion ◽  
Cell Matrix ◽  
Ezrin Expression ◽  
Coated Surfaces ◽  
Cell Matrix Adhesion ◽  
E Cadherin ◽  
Cell Cell

Ezrin, radixin, moesin and merlin form a subfamily of conserved proteins in the band 4.1 superfamily. The function of these proteins is to link the plasma membrane to the actin cytoskeleton. Merlin is defective or absent in schwannomas and meningiomas and has been suggested to function as a tumour suppressor. In this study, we have examined the role of ezrin as a potential regulator of the adhesive and invasive behaviour of tumour cells. We have shown that following inhibition of ezrin expression in colo-rectal cancer cells using antisense oligonucleotides, these cells displayed a reduced cell-cell adhesiveness together with a gain in their motile and invasive behaviour. These cells also displayed increased spreading over matrix-coated surfaces. Immunofluorescence studies revealed that antisense-treated cells also displayed an increased staining of paxillin in areas representing focal adhesions. Furthermore, coprecipitation studies revealed an association of ezrin with E-cadherin and beta-catenin. Induction of the phosphorylation of ezrin by orthovanadate and hepatocyte growth factor/scatter factor resulted in changes similar to those seen with antisense treatment, together with a marked decrease in the association of ezrin with both beta-catenin and E-cadherin. It is concluded that ezrin regulates cell-cell and cell-matrix adhesion, by interacting with cell adhesion molecules E-cadherin and beta-catenin, and may thus play an important role in the control of adhesion and invasiveness of cancer cells.

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