αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion

αE-catenin binds the cell–cell adhesion complex of E-cadherin and β-catenin (β-cat) and regulates filamentous actin (F-actin) dynamics. In vitro, binding of αE-catenin to the E-cadherin–β-cat complex lowers αE-catenin affinity for F-actin, and αE-catenin alone can bind F-actin and inhibit Arp2/3 complex–mediated actin polymerization. In cells, to test whether αE-catenin regulates actin dynamics independently of the cadherin complex, the cytosolic αE-catenin pool was sequestered to mitochondria without affecting overall levels of αE-catenin or the cadherin–catenin complex. Sequestering cytosolic αE-catenin to mitochondria alters lamellipodia architecture and increases membrane dynamics and cell migration without affecting cell–cell adhesion. In contrast, sequestration of cytosolic αE-catenin to the plasma membrane reduces membrane dynamics. These results demonstrate that the cytosolic pool of αE-catenin regulates actin dynamics independently of cell–cell adhesion.

Download Full-text

Mammalian Adenylyl Cyclase-associated Protein 1 (CAP1) Regulates Cofilin Function, the Actin Cytoskeleton, and Cell Adhesion

Journal of Biological Chemistry ◽

10.1074/jbc.m113.484535 ◽

2013 ◽

Vol 288 (29) ◽

pp. 20966-20977 ◽

Cited By ~ 51

Author(s):

Haitao Zhang ◽

Pooja Ghai ◽

Huhehasi Wu ◽

Changhui Wang ◽

Jeffrey Field ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Cytoskeleton ◽

Hela Cells ◽

Actin Polymerization ◽

Actin Dynamics ◽

Ras Signaling ◽

Filamentous Actin ◽

Camp Pathway

CAP (adenylyl cyclase-associated protein) was first identified in yeast as a protein that regulates both the actin cytoskeleton and the Ras/cAMP pathway. Although the role in Ras signaling does not extend beyond yeast, evidence supports that CAP regulates the actin cytoskeleton in all eukaryotes including mammals. In vitro actin polymerization assays show that both mammalian and yeast CAP homologues facilitate cofilin-driven actin filament turnover. We generated HeLa cells with stable CAP1 knockdown using RNA interference. Depletion of CAP1 led to larger cell size and remarkably developed lamellipodia as well as accumulation of filamentous actin (F-actin). Moreover, we found that CAP1 depletion also led to changes in cofilin phosphorylation and localization as well as activation of focal adhesion kinase (FAK) and enhanced cell spreading. CAP1 forms complexes with the adhesion molecules FAK and Talin, which likely underlie the cell adhesion phenotypes through inside-out activation of integrin signaling. CAP1-depleted HeLa cells also had substantially elevated cell motility as well as invasion through Matrigel. In summary, in addition to generating in vitro and in vivo evidence further establishing the role of mammalian CAP1 in actin dynamics, we identified a novel cellular function for CAP1 in regulating cell adhesion.

Download Full-text

Changes in E-cadherin rigidity sensing regulate cell adhesion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1618676114 ◽

2017 ◽

Vol 114 (29) ◽

pp. E5835-E5844 ◽

Cited By ~ 34

Author(s):

Caitlin Collins ◽

Aleksandra K. Denisin ◽

Beth L. Pruitt ◽

W. James Nelson

Keyword(s):

Cell Adhesion ◽

Signaling Molecules ◽

Membrane Dynamics ◽

Cell Contact ◽

Adhesion Assay ◽

Cell Periphery ◽

Actin Organization ◽

Rigidity Sensing ◽

E Cadherin ◽

Cell Cell

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion.

Download Full-text

E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton

Journal of Cell Science ◽

10.1242/jcs.111.8.1071 ◽

1998 ◽

Vol 111 (8) ◽

pp. 1071-1080 ◽

Cited By ~ 6

Author(s):

S.M. Reuver ◽

C.C. Garner

Keyword(s):

Cell Adhesion ◽

Model Systems ◽

Molecular Organization ◽

Cell Contact ◽

Adhesion Sites ◽

Associated Proteins ◽

Adhesion Complex ◽

Cortical Cytoskeleton ◽

E Cadherin ◽

Cell Cell

Members of the SAP family of synapse-associated proteins have recently emerged as central players in the molecular organization of synapses. In this study, we have examined the mechanism that localizes one member, SAP97, to sites of cell-cell contact. Utilizing epithelial CACO-2 cells and fibroblast L-cells as model systems, we demonstrate that SAP97 is associated with the submembranous cortical cytoskeleton at cell-cell adhesion sites. Furthermore, we show that its localization into this structure is triggered by E-cadherin. Although SAP97 can be found in an E-cadherin/catenin adhesion complex, this interaction seems to be mediated by the attachment of SAP97 to the cortical cytoskeleton. Our results are consistent with a model in which SAP97 is recruited to sites of cell-cell contact via an E-cadherin induced assembly of the cortical cytoskeleton.

Download Full-text

Biogenesis of Polarized Epithelial Cells During Kidney Development In Situ: Roles of E-Cadherin–mediated Cell–Cell Adhesion and Membrane Cytoskeleton Organization

Molecular Biology of the Cell ◽

10.1091/mbc.9.11.3161 ◽

1998 ◽

Vol 9 (11) ◽

pp. 3161-3177 ◽

Cited By ~ 46

Author(s):

Peter A. Piepenhagen ◽

W. James Nelson

Keyword(s):

Cell Adhesion ◽

Epithelial Cells ◽

Kidney Development ◽

Lateral Membrane ◽

Membrane Cytoskeleton ◽

Triton X ◽

E Cadherin ◽

Cell Cell

Organization of proteins into structurally and functionally distinct plasma membrane domains is an essential characteristic of polarized epithelial cells. Based on studies with cultured kidney cells, we have hypothesized that a mechanism for restricting Na/K-ATPase to the basal-lateral membrane involves E-cadherin–mediated cell–cell adhesion and integration of Na/K-ATPase into the Triton X-100–insoluble ankyrin- and spectrin-based membrane cytoskeleton. In this study, we examined the relevance of these in vitro observations to the generation of epithelial cell polarity in vivo during mouse kidney development. Using differential detergent extraction, immunoblotting, and immunofluorescence histochemistry, we demonstrate the following. First, expression of the 220-kDa splice variant of ankyrin-3 correlates with the development of resistance to Triton X-100 extraction for Na/K-ATPase, E-cadherin, and catenins and precedes maximal accumulation of Na/K-ATPase. Second, expression of the 190-kDa slice variant of ankyrin-3 correlates with maximal accumulation of Na/K-ATPase. Third, Na/K-ATPase, ankyrin-3, and fodrin specifically colocalize at the basal-lateral plasma membrane of all epithelial cells in which they are expressed and during all stages of nephrogenesis. Fourth, the relative immunofluorescence staining intensities of Na/K-ATPase, ankyrin-3, and fodrin become more similar during development until they are essentially identical in adult kidney. Thus, renal epithelial cells in vivo regulate the accumulation of E-cadherin–mediated adherens junctions, the membrane cytoskeleton, and Na/K-ATPase through sequential protein expression and assembly on the basal-lateral membrane. These results are consistent with a mechanism in which generation and maintenance of polarized distributions of these proteins in vivo and in vitro involve cell–cell adhesion, assembly of the membrane cytoskeleton complex, and concomitant integration and retention of Na/K-ATPase in this complex.

Download Full-text

Actin protrusions push on E-cadherin to maintain epithelial cell-cell adhesion

10.1101/644302 ◽

2019 ◽

Author(s):

John Xiao He Li ◽

Vivian W. Tang ◽

William M. Brieher

Keyword(s):

Cell Adhesion ◽

Actin Polymerization ◽

Mdck Cells ◽

Myosin Ii ◽

Cell Junctions ◽

Apical Junctions ◽

Actin Protrusions ◽

Adhesive Contacts ◽

E Cadherin ◽

Cell Cell

AbstractCadherin mediated cell-cell adhesion is actin dependent, but the precise role of actin in maintaining cell-cell adhesion is not fully understood. Actin polymerization-dependent protrusive activity is required to push distally separated cells close enough together to initiate contact. Whether protrusive activity is required to maintain adhesion in confluent sheets of epithelial cells is not known. By electron microscopy as well as live cell imaging, we have identified a population of protruding actin microspikes that operate continuously near apical junctions of polarized MDCK cells. Live imaging shows that microspikes containing E-cadherin extend into gaps between E-cadherin clusters on neighboring cells while reformation of cadherin clusters across the cell-cell boundary triggers microspike withdrawal. We identify Arp2/3, EVL, and CRMP-1 as three actin assembly factors necessary for microspike formation. Depleting these factors from cells using RNAi results in myosin II-dependent unzipping of cadherin adhesive bonds. Therefore, actin polymerization-dependent protrusive activity operates continuously at cadherin cell-cell junctions to keep them shut and to prevent myosin II-dependent contractility from tearing cadherin adhesive contacts apart.

Download Full-text

Inactivation of E-cadherin by Trop-2 drives colon cancer metastasis.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.3_suppl.105 ◽

2021 ◽

Vol 39 (3_suppl) ◽

pp. 105-105

Author(s):

Saverio Alberti ◽

Emanuela Guerra ◽

Donato F. Altomare ◽

Raffaella Depalo ◽

Marco Trerotola

Keyword(s):

Colon Cancer ◽

Cell Adhesion ◽

Cancer Patients ◽

Cancer Metastasis ◽

Colon Cancer Metastasis ◽

Metastatic Relapse ◽

Metastatic Spreading ◽

E Cadherin ◽

Cell Cell

105 Background: Tumor metastasis is the main cause of death of colon cancer patients and the biggest hurdle for cancer cure. We set to identify decisive drivers and of pivotal therapy targets for colon cancer metastasis. Methods: IHC analysis quantified the expression of target molecules in primary tumors and metastases. Cell-cell adhesion capacity was assessed in vitro and in HCT116 colon cancer cell spheroids. Pre-clinical models of orthotopic growth of KM12SM colon cancer cells and metastatic diffusion to the liver were utilized to assess metastatic spreading force of wtTrop-2 and of the constitutively-active, tail-less form of Trop-2 (Δcyto). Xenotransplant and metastasis transcriptomes were analyzed for differential induction of EMT determinants. Kaplan–Meier plots were used to illustrate survival and metastatic relapse in independent case series of colon cancer patients. Results: wtTrop-2 was shown to induce wound-healing. ΔcytoTrop-2 further increased cell migration ability. Both wtTrop-2 and ΔcytoTrop-2 induced resistance to apoptosis in vitro and in vivo. wtTrop-2 strikingly increased the metastatic capacity of KM12SM cells, raising metastasis rates from 45% for control cells to 90% for wtTrop-2 transfectants. The constitutively-active ΔcytoTrop-2 further boosted metastatic spreading, with metastatic livers reaching up to four times their normal size. Cancer metastases revealed high levels of E-cadherin, in the absence of transcriptional down-regulation. EMT transcription factors were largely missing from Trop-2-activated cells. Rather, binding to Trop-2 was shown to cause the release of E-cadherin from the cytoskeleton, loss of cell-cell adhesion and activation of β-catenin. This global, Trop-2/E-cadherin/β-catenin-driven pro-metastatic program was recapitulated in colon cancer patients and was shown to impact on colon cancer metastatic relapse and overall patient survival. Conclusions: We identify Trop-2-driven functional inactivation of E-cadherin as a widespread driver of metastatic diffusion in colon cancer, opening novel avenues for personalized diagnostic procedures and anti-cancer therapies. [Table: see text]

Download Full-text

Formin-mediated actin polymerization at cell–cell junctions stabilizes E-cadherin and maintains monolayer integrity during wound repair

Molecular Biology of the Cell ◽

10.1091/mbc.e16-06-0429 ◽

2016 ◽

Vol 27 (18) ◽

pp. 2844-2856 ◽

Cited By ~ 31

Author(s):

Megha Vaman Rao ◽

Ronen Zaidel-Bar

Keyword(s):

Cell Adhesion ◽

Wound Repair ◽

Actin Polymerization ◽

Cell Junctions ◽

Epithelial Tissue ◽

Collective Cell Migration ◽

Epithelial Junctions ◽

Major Factors ◽

E Cadherin ◽

Cell Cell

Cadherin-mediated cell–cell adhesion is required for epithelial tissue integrity in homeostasis, during development, and in tissue repair. E-cadherin stability depends on F-actin, but the mechanisms regulating actin polymerization at cell–cell junctions remain poorly understood. Here we investigated a role for formin-mediated actin polymerization at cell–cell junctions. We identify mDia1 and Fmnl3 as major factors enhancing actin polymerization and stabilizing E-cadherin at epithelial junctions. Fmnl3 localizes to adherens junctions downstream of Src and Cdc42 and its depletion leads to a reduction in F-actin and E-cadherin at junctions and a weakening of cell–cell adhesion. Of importance, Fmnl3 expression is up-regulated and junctional localization increases during collective cell migration. Depletion of Fmnl3 or mDia1 in migrating monolayers results in dissociation of leader cells and impaired wound repair. In summary, our results show that formin activity at epithelial cell–cell junctions is important for adhesion and the maintenance of epithelial cohesion during dynamic processes, such as wound repair.

Download Full-text

Regulation of the cell-cell adhesion protein, E-cadherin, in dog and human thyrocytes in vitro.

Endocrinology ◽

10.1210/endo.136.7.7789339 ◽

1995 ◽

Vol 136 (7) ◽

pp. 3113-3119 ◽

Cited By ~ 15

Author(s):

G Brabant ◽

C Hoang-Vu ◽

J Behrends ◽

Y Cetin ◽

E Pötter ◽

...

Keyword(s):

Cell Adhesion ◽

Adhesion Protein ◽

Cell Adhesion Protein ◽

E Cadherin ◽

Cell Cell

Download Full-text

TetraThymosinβ Is Required for Actin Dynamics in Caenorhabditis elegans and Acts via Functionally Different Actin-binding Repeats

Molecular Biology of the Cell ◽

10.1091/mbc.e04-03-0225 ◽

2004 ◽

Vol 15 (10) ◽

pp. 4735-4748 ◽

Cited By ~ 28

Author(s):

Marleen Van Troys ◽

Kanako Ono ◽

Daisy Dewitte ◽

Veronique Jonckheere ◽

Natalie De Ruyck ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Actin Filament ◽

Actin Polymerization ◽

Actin Dynamics ◽

Actin Binding ◽

In Vitro Assays ◽

Filamentous Actin ◽

Lethal Mutant ◽

Interaction Sites

Generating specific actin structures via controlled actin polymerization is a prerequisite for eukaryote development and reproduction. We here report on an essential Caenorhabditis elegans protein tetraThymosinβ expressed in developing neurons and crucial during oocyte maturation in adults. TetraThymosinβ has four repeats, each related to the actin monomer-sequestering protein thymosinβ 4 and assists in actin filament elongation. For homologues with similar multirepeat structures, a profilin-like mechanism of ushering actin onto filament barbed ends, based on the formation of a 1:1 complex, is proposed to underlie this activity. We, however, demonstrate that tetraThymosinβ binds multiple actin monomers via different repeats and in addition also interacts with filamentous actin. All repeats need to be functional for attaining full activity in various in vitro assays. The activities on actin are thus a direct consequence of the repeated structure. In containing both G- and F-actin interaction sites, tetraThymosinβ may be reminiscent of nonhomologous multimodular actin regulatory proteins implicated in actin filament dynamics. A mutation that suppresses expression of tetraThymosinβ is homozygous lethal. Mutant organisms develop into adults but display a dumpy phenotype and fail to reproduce as their oocytes lack essential actin structures. This strongly suggests that the activity of tetraThymosinβ is of crucial importance at specific developmental stages requiring actin polymerization.

Download Full-text