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

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.

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

AJP Renal Physiology ◽

10.1152/ajprenal.00419.2011 ◽

2012 ◽

Vol 302 (1) ◽

pp. F103-F115 ◽

Cited By ~ 16

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.

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Assembly of adherens junctions is required for sphingosine 1-phosphate-induced matriptase accumulation and activation at mammary epithelial cell-cell contacts

AJP Cell Physiology ◽

10.1152/ajpcell.00400.2003 ◽

2004 ◽

Vol 286 (5) ◽

pp. C1159-C1169 ◽

Cited By ~ 32

Author(s):

Ruei-Jiun Hung ◽

Ia-Wen J. Hsu ◽

Jennifer L. Dreiling ◽

Mon-Juan Lee ◽

Cicely A. Williams ◽

...

Keyword(s):

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Adherens Junctions ◽

Adherens Junction ◽

Mammary Epithelial ◽

Cell Junctions ◽

Cell Contacts ◽

Cytoskeletal Rearrangement ◽

Sphingosine 1 Phosphate ◽

Cell Cell

Sphingosine 1-phosphate (S1P), a bioactive phospholipid, simultaneously induces actin cytoskeletal rearrangements and activation of matriptase, a membrane-associated serine protease in human mammary epithelial cells. In this study, we used a monoclonal antibody selective for activated, two-chain matriptase to examine the functional relationship between these two S1P-induced events. Ten minutes after exposure of 184 A1N4 mammary epithelial cells to S1P, matriptase was observed to accumulate at cell-cell contacts. Activated matriptase first began to appear as small spots at cell-cell contacts, and then its deposits elongated along cell-cell contacts. Concomitantly, S1P induced assembly of adherens junctions and subcortical actin belts. Matriptase localization was observed to be coincident with markers of adherens junctions at cell-cell contacts but likely not to be incorporated into the tightly bound adhesion plaque. Disruption of subcortical actin belt formation and prevention of adherens junction assembly led to prevention of accumulation and activation of the protease at cell-cell contacts. These data suggest that S1P-induced accumulation and activation of matriptase depend on the S1P-induced adherens junction assembly. Although MAb M32, directed against one of the low-density lipoprotein receptor class A domains of matriptase, blocked S1P-induced activation of the enzyme, the antibody had no effect on S1P-induced actin cytoskeletal rearrangement. Together, these data indicate that actin cytoskeletal rearrangement is necessary but not sufficient for S1P-induced activation of matriptase at cell-cell contacts. The coupling of matriptase activation to adherens junction assembly and actin cytoskeletal rearrangement may serve to ensure tight control of matriptase activity, restricted to cell-cell junctions of mammary epithelial cells.

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Functional Hierarchy of Simultaneously Expressed Adhesion Receptors: Integrin α2β1 but Not CD44 Mediates MV3 Melanoma Cell Migration and Matrix Reorganization within Three-dimensional Hyaluronan-containing Collagen Matrices

Molecular Biology of the Cell ◽

10.1091/mbc.10.10.3067 ◽

1999 ◽

Vol 10 (10) ◽

pp. 3067-3079 ◽

Cited By ~ 89

Author(s):

Kerstin Maaser ◽

Katarina Wolf ◽

C. Eberhard Klein ◽

Bernd Niggemann ◽

Kurt S. Zänker ◽

...

Keyword(s):

Cell Migration ◽

Cell Tracking ◽

Three Dimensional ◽

Focal Adhesions ◽

Time Lapse ◽

Computer Assisted ◽

Adhesion Receptors ◽

Collagen Matrices ◽

Β1 Integrins ◽

Associated Functions

Haptokinetic cell migration across surfaces is mediated by adhesion receptors including β1 integrins and CD44 providing adhesion to extracellular matrix (ECM) ligands such as collagen and hyaluronan (HA), respectively. Little is known, however, about how such different receptor systems synergize for cell migration through three-dimensionally (3-D) interconnected ECM ligands. In highly motile human MV3 melanoma cells, both β1 integrins and CD44 are abundantly expressed, support migration across collagen and HA, respectively, and are deposited upon migration, whereas only β1 integrins but not CD44 redistribute to focal adhesions. In 3-D collagen lattices in the presence or absence of HA and cross-linking chondroitin sulfate, MV3 cell migration and associated functions such as polarization and matrix reorganization were blocked by anti-β1 and anti-α2 integrin mAbs, whereas mAbs blocking CD44, α3, α5, α6, or αv integrins showed no effect. With use of highly sensitive time-lapse videomicroscopy and computer-assisted cell tracking techniques, promigratory functions of CD44 were excluded. 1) Addition of HA did not increase the migratory cell population or its migration velocity, 2) blocking of the HA-binding Hermes-1 epitope did not affect migration, and 3) impaired migration after blocking or activation of β1 integrins was not restored via CD44. Because α2β1-mediated migration was neither synergized nor replaced by CD44–HA interactions, we conclude that the biophysical properties of 3-D multicomponent ECM impose more restricted molecular functions of adhesion receptors, thereby differing from haptokinetic migration across surfaces.

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Tumorigenic epithelial clusters are less sensitive to moderate osmotic stresses due to low amounts of junctional E-cadherin

10.1101/2020.10.26.355891 ◽

2020 ◽

Author(s):

Danahe Mohammed ◽

Park Young Chan ◽

Jeffrey J. Fredberg ◽

David A. Weitz

Keyword(s):

Breast Cancer ◽

Cell Migration ◽

Epithelial Cells ◽

Osmotic Stress ◽

Metastatic Breast ◽

Tumorigenic Cells ◽

Breast Epithelial ◽

The Impact ◽

E Cadherin ◽

Cell Cell

AbstractThe migration of tumorigenic epithelial cells is a critical step for metastatic breast cancer progression. Although the role of the extracellular matrix in breast cancer cell migration has been extensively described, the effect of osmotic stress on the migration of tumor breast epithelial cohorts remains unclear. Most of our understanding on the effect of osmotic stresses on cell migration comes from studies at the level of the single cell in isolation and does not take into account cell-cell interactions. Here, we study the impact of moderate osmotic stress on the migration of epithelial clusters composed of either non-tumorigenic or tumorigenic epithelial cells. We observe a decrease in migration distance and speed for non-tumorigenic epithelial cells but not for tumorigenic ones. To explain these differences, we investigate how osmotic stress impacts the mechanical properties of cell clusters and affects cell volumes. After application of osmotic stress renal epithelial cells become stiffer whereas non-tumorigenic and tumorigenic breast epithelial cells do not. In addition, tumorigenic cells are shown to be less sensitive to osmotic stress than non-tumorigenic cells, and this difference is associated with lower levels of E-cadherin expression. Using EGTA treatments, we confirm that the establishment of cell-cell adhesive interactions is a key component of the behavior of epithelial clusters in response to osmotic stress. This study provides evidence on the low sensitivity of tumorigenic epithelial clusters to moderate osmotic stress and highlights the importance of cadherin-based junctions in response to osmotic stress.

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

Science Advances ◽

10.1126/sciadv.aav2720 ◽

2019 ◽

Vol 5 (12) ◽

pp. eaav2720 ◽

Cited By ~ 5

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.

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Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Molecular Biology of the Cell ◽

10.1091/mbc.8.11.2329 ◽

1997 ◽

Vol 8 (11) ◽

pp. 2329-2344 ◽

Cited By ~ 110

Author(s):

Cuiling Zhong ◽

Michael S. Kinch ◽

Keith Burridge

Keyword(s):

Epithelial Cells ◽

Adherens Junctions ◽

Focal Adhesions ◽

Dominant Negative ◽

Cell Junctions ◽

Epithelial Cell Line ◽

Cytoskeletal Organization ◽

Epithelial Phenotype ◽

Adhesive Interactions ◽

Mcf10a Cells

Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell–cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.

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

10.1101/328609 ◽

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.

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Adherens junction serves to generate cryptic lamellipodia required for collective migration of epithelial cells

10.1101/2020.04.20.052118 ◽

2020 ◽

Cited By ~ 1

Author(s):

Masayuki Ozawa ◽

Sylvain Hiver ◽

Takaki Yamamoto ◽

Tatsuo Shibata ◽

Srigokul Upadhyayula ◽

...

Keyword(s):

Epithelial Cells ◽

Adherens Junctions ◽

Myosin Ii ◽

Adherens Junction ◽

Cancer Invasion ◽

Biological Processes ◽

Collective Migration ◽

Epithelial Sheets ◽

Lamellipodia Formation ◽

Cell Cell

AbstractCollective migration of epithelial cells plays crucial roles in various biological processes such as cancer invasion. In migrating epithelial sheets, leader cells form lamellipodia to advance, and follower cells also form similar motile apparatus at cell-cell boundaries, which are called cryptic lamellipodia (c-lamellipodia). Using adenocarcinoma-derived epithelial cells, we investigated how c-lamellipodia are generated, and found that they sporadically grew from Ecadherin-based adherens junctions (AJs). WAVE and Arp2/3 complexes were localized along the AJs, and silencing them not only interfered with c-lamellipodia formation but also prevented follower cells from trailing the leaders. Disruption of AJs by removing αE-catenin resulted in uncontrolled c-lamellipodia growth, and this was brought about by myosin II activation and the resultant contraction of AJ-associated actomyosin cables. Additional observations indicated that c-lamellipodia tended to grow at mechanically weak sites of the junction. We conclude that AJs not only tie cells together but also generate c-lamellipodia by recruiting actin regulators, enabling epithelial cells to undergo ordered collective migration.

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Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia.

The Journal of Cell Biology ◽

10.1083/jcb.130.2.461 ◽

1995 ◽

Vol 130 (2) ◽

pp. 461-471 ◽

Cited By ~ 209

Author(s):

M S Kinch ◽

G J Clark ◽

C J Der ◽

K Burridge

Keyword(s):

Epithelial Cells ◽

Actin Cytoskeleton ◽

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Adherens Junctions ◽

Focal Adhesions ◽

Transformed Cells ◽

Beta Catenin ◽

Altered Cell ◽

E Cadherin

Transformed epithelial cells often are characterized by a fibroblastic or mesenchymal morphology. These cells exhibit altered cell-cell and cell-substrate interactions. Here we have identified changes in the adhesions and cytoskeletal interactions of transformed epithelial cells that contribute to their altered morphology. Using MCF-10A human breast epithelial cells as a model system, we have found that transformation by an activated form of ras is characterized by less developed adherens-type junctions between cells but increased focal adhesions. Contributing to the modified adherens junctions of the transformed cells are decreased interactions among beta-catenin, E-cadherin, and the actin cytoskeleton. The ras-transformed cells reveal elevated phosphotyrosine in many proteins, including beta-catenin and p120 Cas. Whereas in the normal cells beta-catenin is found in association with E-cadherin, p120 Cas is not. In the ras-transformed cells, the situation is reversed; tyrosine-phosphorylated p120 Cas, but not tyrosine-phosphorylated beta-catenin, now is detected in E-cadherin complexes. The tyrosine-phosphorylated beta-catenin also shows increased detergent solubility, suggesting a decreased association with the actin cytoskeleton. p120 Cas, whether tyrosine phosphorylated or not, partitions into the detergent soluble fraction, suggesting that it is not tightly bound to the actin cytoskeleton in either the normal or ras-transformed cells. Inhibitors of tyrosine kinases decrease the level of tyrosine phosphorylation and restore a normal epithelial morphology to the ras-transformed cells. In particular, decreased tyrosine phosphorylation of beta-catenin is accompanied by increased interaction with both E-cadherin and the detergent insoluble cytoskeletal fraction. These results suggest that elevated tyrosine phosphorylation of proteins such as beta-catenin and p120 Cas contribute to the altered adherens junctions of ras-transformed epithelia.

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Rab5 Mediates Caspase-8–promoted Cell Motility and Metastasis

Molecular Biology of the Cell ◽

10.1091/mbc.e09-09-0769 ◽

2010 ◽

Vol 21 (2) ◽

pp. 369-376 ◽

Cited By ~ 52

Author(s):

Vicente A. Torres ◽

Ainhoa Mielgo ◽

Simone Barbero ◽

Ruth Hsiao ◽

John A. Wilkins ◽

...

Keyword(s):

Cell Migration ◽

Focal Adhesions ◽

Caspase 8 ◽

Environmental Cues ◽

Dependent Manner ◽

Cell Responses ◽

Β1 Integrins ◽

And Migration

Caspase-8 is a key apical sensory protein that governs cell responses to environmental cues, alternatively promoting apoptosis, proliferation, and cell migration. The proteins responsible for integration of these pathways, however, have remained elusive. Here, we reveal that Rab5 regulates caspase-8–dependent signaling from integrins. Integrin ligation leads to Rab5 activation, association with integrins, and activation of Rac, in a caspase-8–dependent manner. Rab5 activation promotes colocalization and coprecipitation of integrins with caspase-8, concomitant with Rab5 recruitment to integrin-rich regions such as focal adhesions and membrane ruffles. Moreover, caspase-8 expression promotes Rab5-mediated internalization and the recycling of β1 integrins, increasing cell migration independently of caspase catalytic activity. Conversely, Rab5 knockdown prevented caspase-8–mediated integrin signaling for Rac activation, cell migration, and apoptotic signaling, respectively. Similarly, Rab5 was critical for caspase-8–driven cell migration in vivo, because knockdown of Rab5 compromised the ability of caspase-8 to promote metastasis under nonapoptotic conditions. These studies identify Rab5 as a key integrator of caspase-8–mediated signal transduction downstream of integrins, regulating cell survival and migration in vivo and in vitro.

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