scholarly journals Direct single-molecule quantification reveals unexpectedly high mechanical stability of vinculin—talin/α-catenin linkages

2019 ◽  
Vol 5 (12) ◽  
pp. eaav2720 ◽  
Author(s):  
Shimin Le ◽  
Miao Yu ◽  
Jie Yan
Keyword(s):  
Single Molecule ◽  
Binding Sites ◽  
Mechanical Stability ◽  
Adherens Junctions ◽  
Focal Adhesions ◽  
Adaptor Proteins ◽  
Cell Matrix ◽  
High Mechanical Stability ◽  
Direct Quantification ◽  
Cell Cell

The vinculin-mediated mechanosensing requires establishment of stable mechanical linkages between vinculin to integrin at focal adhesions and to cadherins at adherens junctions through associations with the respective adaptor proteins talin and α-catenin. However, the mechanical stability of these critical vinculin linkages has yet to be determined. Here, we developed a single-molecule detector assay to provide direct quantification of the mechanical lifetime of vinculin association with the vinculin binding sites in both talin and α-catenin, which reveals a surprisingly high mechanical stability of the vinculin—talin and vinculin—α-catenin interfaces that have a lifetime of >1000 s at forces up to 10 pN and can last for seconds to tens of seconds at 15 to 25 pN. Our results suggest that these force-bearing intermolecular interfaces provide sufficient mechanical stability to support the vinculin-mediated mechanotransduction at cell-matrix and cell-cell adhesions.

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 Transition of responsive mechanosensitive elements from focal adhesions to adherens junctions on epithelial differentiation

2018 ◽  
Vol 29 (19) ◽  
pp. 2317-2325 ◽  
Author(s):  
Barbara Noethel ◽  
Lena Ramms ◽  
Georg Dreissen ◽  
Marco Hoffmann ◽  
Ronald Springer ◽  
...  
Keyword(s):  
Mechanical Stress ◽  
Adherens Junctions ◽  
Adhesion Formation ◽  
Focal Adhesions ◽  
Intercellular Junctions ◽  
Cell Junctions ◽  
Epithelial Tissue ◽  
Cell Matrix ◽  
Junction Protein ◽  
Cell Cell

The skin’s epidermis is a multilayered epithelial tissue and the first line of defense against mechanical stress. Its barrier function depends on an integrated assembly and reorganization of cell–matrix and cell–cell junctions in the basal layer and on different intercellular junctions in suprabasal layers. However, how mechanical stress is recognized and which adhesive and cytoskeletal components are involved are poorly understood. Here, we subjected keratinocytes to cyclic stress in the presence or absence of intercellular junctions. Both states not only recognized but also responded to strain by reorienting actin filaments perpendicular to the applied force. Using different keratinocyte mutant strains that altered the mechanical link of the actin cytoskeleton to either cell–matrix or cell–cell junctions, we show that not only focal adhesions but also adherens junctions function as mechanosensitive elements in response to cyclic strain. Loss of paxillin or talin impaired focal adhesion formation and only affected mechanosensitivity in the absence but not presence of intercellular junctions. Further analysis revealed the adherens junction protein α-catenin as a main mechanosensor, with greatest sensitivity conferred on binding to vinculin. Our data reveal a mechanosensitive transition from cell–matrix to cell–cell adhesions on formation of keratinocyte monolayers with vinculin and α-catenin as vital players.

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.

Integrin-linked kinase is localized to cell-matrix focal adhesions but not cell-cell adhesion sites and the focal adhesion localization of integrin-linked kinase is regulated by the PINCH-binding ANK repeats

1999 ◽  
Vol 112 (24) ◽  
pp. 4589-4599 ◽  
Author(s):  
F. Li ◽  
Y. Zhang ◽  
C. Wu
Keyword(s):  
Focal Adhesion ◽  
Critical Role ◽  
Focal Adhesions ◽  
Subcellular Compartment ◽  
Cell Matrix ◽  
Integrin Binding Site ◽  
Integrin Linked Kinase ◽  
Cell Cell

Integrin-linked kinase (ILK) is a ubiquitously expressed protein serine/threonine kinase that has been implicated in integrin-, growth factor- and Wnt-signaling pathways. In this study, we show that ILK is a constituent of cell-matrix focal adhesions. ILK was recruited to focal adhesions in all types of cells examined upon adhesion to a variety of extracellular matrix proteins. By contrast, ILK was absent in E-cadherin-mediated cell-cell adherens junctions. In previous studies, we have identified PINCH, a protein consisting of five LIM domains, as an ILK binding protein. We demonstrate in this study that the ILK-PINCH interaction requires the N-terminal-most ANK repeat (ANK1) of ILK and one (the C-terminal) of the two zinc-binding modules within the LIM1 domain of PINCH. The ILK ANK repeats domain, which is capable of interacting with PINCH in vitro, could also form a complex with PINCH in vivo. However, the efficiency of the complex formation or the stability of the complex was markedly reduced in the absence of the C-terminal domain of ILK. The PINCH binding defective ANK1 deletion ILK mutant, unlike the wild-type ILK, was unable to localize and cluster in focal adhesions, suggesting that the interaction with PINCH is necessary for focal adhesion localization and clustering of ILK. The N-terminal ANK repeats domain, however, is not sufficient for mediating focal adhesion localization of ILK, as an ILK mutant containing the ANK repeats domain but lacking the C-terminal integrin binding site failed to localize in focal adhesions. These results suggest that focal adhesions are a major subcellular compartment where ILK functions in intracellular signal transduction, and provide important evidence for a critical role of PINCH and integrins in regulating ILK cellular function.

scholarly journals Coordination of mitochondrial and cellular dynamics by the actin-based motor Myo19

2021 ◽  
Vol 134 (10) ◽  
Author(s):  
Katarzyna Majstrowicz ◽  
Ulrike Honnert ◽  
Petra Nikolaus ◽  
Vera Schwarz ◽  
Stefanie J. Oeding ◽  
...  
Keyword(s):  
Molecular Level ◽  
Focal Adhesions ◽  
Adaptor Proteins ◽  
Cellular Level ◽  
Ros Generation ◽  
Cellular Processes ◽  
Daughter Cells ◽  
Cell Matrix ◽  
Motor Activities ◽  
Cell Matrix Adhesion

ABSTRACT Myosin XIX (Myo19) is an actin-based motor that competes with adaptors of microtubule-based motors for binding to the outer mitochondrial transmembrane proteins Miro1 and Miro2 (collectively Miro, also known as RhoT1 and RhoT2, respectively). Here, we investigate which mitochondrial and cellular processes depend on the coordination of Myo19 and microtubule-based motor activities. To this end, we created Myo19-deficient HEK293T cells. Mitochondria in these cells were not properly fragmented at mitosis and were partitioned asymmetrically to daughter cells. Respiratory functions of mitochondria were impaired and ROS generation was enhanced. On a cellular level, cell proliferation, cytokinesis and cell–matrix adhesion were negatively affected. On a molecular level, Myo19 regulates focal adhesions in interphase, and mitochondrial fusion and mitochondrially associated levels of fission protein Drp1 and adaptor proteins dynactin and TRAK1 at prometaphase. These alterations were due to a disturbed coordination of Myo19 and microtubule-based motor activities by Miro.

scholarly journals Intermediate filaments control collective migration by restricting traction forces and sustaining cell-cell contacts

2018 ◽  
Author(s):  
Chiara De Pascalis ◽  
Carlos Pérez-González ◽  
Shailaja Seetharaman ◽  
Batiste Boëda ◽  
Benoit Vianay ◽  
...  
Keyword(s):  
Adherens Junctions ◽  
Cell Monolayer ◽  
Focal Adhesions ◽  
Collective Migration ◽  
Traction Forces ◽  
Mechanical Coupling ◽  
Directed Migration ◽  
Cell Protrusion ◽  
Cell Cell

AbstractMesenchymal cell migration relies on the coordinated regulation of the actin and microtubule networks which participate in polarised cell protrusion, adhesion and contraction. During collective migration, most of the traction forces are generated by the acto-myosin network linked to focal adhesions at the front of leader cells, which transmit these pulling forces to the followers. Here, using an in vitro wound healing assay to induce polarisation and collective directed migration of primary astrocytes, we show that the intermediate filament (IF) network composed of vimentin, GFAP and nestin contributes to directed collective movement by controlling the distribution of forces in the migrating cell monolayer. Together with the cytoskeletal linker plectin, these IFs control the organisation and dynamics of the acto-myosin network, promoting the actin-driven treadmilling of adherens junctions, thereby facilitating the polarisation of leader cells. Independently of their effect on adherens junctions, IFs influence the dynamics and localisation of focal adhesions and limit their mechanical coupling to the acto-myosin network. We thus conclude that IFs promote collective directed migration by restricting the generation of traction forces to the front of leader cells, preventing aberrant tractions in the followers and by contributing to the maintenance of lateral cell-cell interactions.

scholarly journals Computational and single-molecule force studies of a macro domain protein reveal a key molecular determinant for mechanical stability

2010 ◽  
Vol 107 (5) ◽  
pp. 1989-1994 ◽  
Author(s):  
Dora L. Guzmán ◽  
Arlo Randall ◽  
Pierre Baldi ◽  
Zhibin Guan
Keyword(s):  
Molecular Dynamics ◽  
Single Molecule ◽  
Mechanical Stability ◽  
Single Molecule Force Spectroscopy ◽  
Load Bearing ◽  
Molecular Determinant ◽  
Macro Domain ◽  
Domain Protein ◽  
Dynamics Simulations ◽  
High Mechanical Stability

Resolving molecular determinants of mechanical stability of proteins is crucial in the rational design of advanced biomaterials for use in biomedical and nanotechnological applications. Here we present an interdisciplinary study combining bioinformatics screening, steered molecular dynamics simulations, protein engineering, and single-molecule force spectroscopy that explores the mechanical properties of a macro domain protein with mixed α + β topology. The unique architecture is defined by a single seven-stranded β-sheet in the core of the protein flanked by five α-helices. Unlike mechanically stable proteins studied thus far, the macro domain provides the distinct advantage of having the key load-bearing hydrogen bonds (H bonds) buried in the hydrophobic core protected from water attacks. This feature allows direct measurement of the force required to break apart the load-bearing H bonds under locally hydrophobic conditions. Steered molecular dynamics simulations predicted extremely high mechanical stability of the macro domain by using constant velocity and constant force methods. Single-molecule force spectroscopy experiments confirm the exceptional mechanical strength of the macro domain, measuring a rupture force as high as 570 pN. Furthermore, through selective deletion of shielding peptide segments, we examined the same key H bonds under hydrophilic environments in which the β-strands are exposed to solvent and verify that the high mechanical stability of the macro domain results from excellent shielding of the load-bearing H bonds from competing water. Our study reveals that shielding water accessibility to the load-bearing strands is a critical molecular determinant for enhancing the mechanical stability of proteins.

scholarly journals Induction of Cell Scattering by Expression of β1 Integrins in β1-Deficient Epithelial Cells Requires Activation of Members of the Rho Family of Gtpases and Downregulation of Cadherin and Catenin Function

1999 ◽  
Vol 147 (6) ◽  
pp. 1325-1340 ◽  
Author(s):  
Clotilde Gimond ◽  
Arjan van der Flier ◽  
Sanne van Delft ◽  
Cord Brakebusch ◽  
Ingrid Kuikman ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
Adherens Junctions ◽  
Interleukin 2 ◽  
Focal Adhesions ◽  
Morphological Transition ◽  
Protein Levels ◽  
Cell Scattering ◽  
Β1 Integrins ◽  
Cell Cell

Adhesion receptors, which connect cells to each other and to the surrounding extracellular matrix (ECM), play a crucial role in the control of tissue structure and of morphogenesis. In this work, we have studied how intercellular adhesion molecules and β1 integrins influence each other using two different β1-null cell lines, epithelial GE11 and fibroblast-like GD25 cells. Expression of β1A or the cytoplasmic splice variant β1D, induced the disruption of intercellular adherens junctions and cell scattering in both GE11 and GD25 cells. In GE11 cells, the morphological change correlated with the redistribution of zonula occluden (ZO)-1 from tight junctions to adherens junctions at high cell confluency. In addition, the expression of β1 integrins caused a dramatic reorganization of the actin cytoskeleton and of focal contacts. Interaction of β1 integrins with their respective ligands was required for a complete morphological transition towards the spindle-shaped fibroblast-like phenotype. The expression of an interleukin-2 receptor (IL2R)-β1A chimera and its incorporation into focal adhesions also induced the disruption of cadherin-based adhesions and the reorganization of ECM–cell contacts, but failed to promote cell migration on fibronectin, in contrast to full-length β1A. This indicates that the disruption of cell–cell adhesion is not simply the consequence of the stimulated cell migration. Expression of β1 integrins in GE11 cells resulted in a decrease in cadherin and α-catenin protein levels accompanied by their redistribution from the cytoskeleton-associated fraction to the detergent-soluble fraction. Regulation of α-catenin protein levels by β1 integrins is likely to play a role in the morphological transition, since overexpression of α-catenin in GE11 cells before β1 prevented the disruption of intercellular adhesions and cell scattering. In addition, using biochemical activity assays for Rho-like GTPases, we show that the expression of β1A, β1D, or IL2R-β1A in GE11 or GD25 cells triggers activation of both RhoA and Rac1, but not of Cdc42. Moreover, dominant negative Rac1 (N17Rac1) inhibited the disruption of cell–cell adhesions when expressed before β1. However, all three GTPases might be involved in the morphological transition, since expression of either N19RhoA, N17Rac1, or N17Cdc42 reversed cell scattering and partially restored cadherin-based adhesions in GE11-β1A cells. Our results indicate that β1 integrins regulate the polarity and motility of epithelial cells by the induction of intracellular molecular events involving a downregulation of α-catenin function and the activation of the Rho-like G proteins Rac1 and RhoA.

scholarly journals Vascular Endothelial-Cadherin Regulates Cytoskeletal Tension, Cell Spreading, and Focal Adhesions by Stimulating RhoA

2004 ◽  
Vol 15 (6) ◽  
pp. 2943-2953 ◽  
Author(s):  
Celeste M. Nelson ◽  
Dana M. Pirone ◽  
John L. Tan ◽  
Christopher S. Chen
Keyword(s):  
Adhesion Formation ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cell Contact ◽  
Matrix Adhesion ◽  
Cell Matrix ◽  
Focal Adhesion Formation ◽  
Cytoskeletal Tension ◽  
Cell Matrix Adhesion ◽  
Cell Cell

Changes in vascular endothelial (VE)-cadherin–mediated cell-cell adhesion and integrin-mediated cell-matrix adhesion coordinate to affect the physical and mechanical rearrangements of the endothelium, although the mechanisms for such cross talk remain undefined. Herein, we describe the regulation of focal adhesion formation and cytoskeletal tension by intercellular VE-cadherin engagement, and the molecular mechanism by which this occurs. Increasing the density of endothelial cells to increase cell-cell contact decreased focal adhesions by decreasing cell spreading. This contact inhibition of cell spreading was blocked by disrupting VE-cadherin engagement with an adenovirus encoding dominant negative VE-cadherin. When changes in cell spreading were prevented by culturing cells on a micropatterned substrate, VE-cadherin–mediated cell-cell contact paradoxically increased focal adhesion formation. We show that VE-cadherin engagement mediates each of these effects by inducing both a transient and sustained activation of RhoA. Both the increase and decrease in cell-matrix adhesion were blocked by disrupting intracellular tension and signaling through the Rho-ROCK pathway. In all, these findings demonstrate that VE-cadherin signals through RhoA and the actin cytoskeleton to cross talk with cell-matrix adhesion and thereby define a novel pathway by which cell-cell contact alters the global mechanical and functional state of cells.

scholarly journals Integrin-linked kinase tunes cell-cell and cell-matrix adhesions to regulate the switch between apoptosis and EMT downstream of TGFβ1

2021 ◽  
pp. mbc.E20-02-0092
Author(s):  
Ayse Nihan Kilinc ◽  
Siyang Han ◽  
Lena Barrett ◽  
Niroshan Anandasivam ◽  
Celeste M. Nelson
Keyword(s):  
Cell Adhesion ◽  
Epithelial Cells ◽  
Focal Adhesions ◽  
Cell Phenotype ◽  
High Cell ◽  
Potent Inducer ◽  
Mesenchymal Transition ◽  
Cell Matrix ◽  
Integrin Linked Kinase ◽  
Cell Cell

Epithelial-mesenchymal transition (EMT) is a morphogenetic process that endows epithelial cells with migratory and invasive potential. Mechanical and chemical signals from the tumor microenvironment can activate the EMT program, thereby permitting cancer cells to invade the surrounding stroma and disseminate to distant organs. Transforming growth factor β1 (TGFβ1) is a potent inducer of EMT that can also induce apoptosis depending on the microenvironmental context. In particular, stiff microenvironments promote EMT while softer ones promote apoptosis. Here, we investigated the molecular signaling downstream of matrix stiffness that regulates the phenotypic switch in response to TGFβ1, and uncovered a critical role for integrin-linked kinase (ILK). Specifically, depleting ILK from mammary epithelial cells precludes their ability to sense the stiffness of their microenvironment. In response to treatment with TGFβ1, ILK-depleted cells undergo apoptosis on both soft and stiff substrata. We found that knockdown of ILK decreases focal adhesions and increases cell-cell adhesions, thus shifting the balance from cell-matrix to cell-cell adhesion. High cell-matrix adhesion promotes EMT whereas high cell-cell adhesion promotes apoptosis downstream of TGFβ1. These results highlight an important role for ILK in controlling cell phenotype by regulating adhesive connections to the local microenvironment.

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