Evolutionary rate covariation analysis of E-cadherin identifies Raskol as regulator of cell adhesion and actin dynamics in Drosophila

AbstractThe adherens junction couples the actin cytoskeletons of neighboring cells to provide the foundation for multicellular organization. The core of the adherens junction is the cadherin-catenin complex that arose early in the evolution of multicellularity to link cortical actin to intercellular adhesions. Over time, evolutionary pressures have shaped the signaling and mechanical functions of the adherens junction to meet specific developmental and physiological demands. Evolutionary rate covariation (ERC) identifies genes with correlated fluctuations in evolutionary rate that can reflect shared selective pressures and functions. Here we use ERC to identify genes with evolutionary histories similar to shotgun (shg), which encodes the Drosophila E-cadherin (DE-Cad) ortholog. Core adherens junction components α-catenin and p120-catenin displayed strong ERC correlations with shg, indicating that they evolved under similar selective pressures during evolution between Drosophila species. Further analysis of the shg ERC profile revealed a collection of genes not previously associated with shg function or cadherin-mediated adhesion. We then analyzed the function of a subset of ERC-identified candidate genes by RNAi during border cell (BC) migration and identified novel genes that function to regulate DE-Cad. Among these, we found that the gene CG42684, which encodes a putative GTPase activating protein (GAP), regulates BC migration and adhesion. We named CG42684 raskol (“to split” in Russian) and show that it regulates DE-Cad levels and actin protrusions in BCs. We propose that Raskol functions with DE-Cad to restrict Ras/Rho signaling and help guide BC migration. Our results demonstrate that a coordinated selective pressure has shaped the adherens junction and this can be leveraged to identify novel components of the complexes and signaling pathways that regulate cadherin-mediated adhesion.Author SummaryThe establishment of intercellular adhesions facilitated the genesis of multicellular organisms. The adherens junction, which links the actin cytoskeletons of neighboring cells, arose early in the evolution of multicellularity and selective pressures have shaped its function and molecular composition over time. In this study, we used evolutionary rate covariation (ERC) analysis to examine the evolutionary history of the adherens junction and to identify genes that coevolved with the adherens junction gene shotgun, which encodes the Drosophila E-cadherin (DE-Cad). ERC analysis of shotgun revealed a collection of genes with similar evolutionary histories. We then tested the role of these genes in border cell migration in the fly egg chamber, a process that requires the coordinated regulation of cell-cell adhesion and cell motility. Among these, we found that a previously uncharacterized gene CG42684, which encodes a putative GTPase activating protein (GAP), regulates the collective cell migration of border cells, stabilizes cell-cell adhesions and regulates the actin dynamics. Our results demonstrate that components of the adherens junction share an evolutionary history and that ERC analysis is a powerful method to identify novel components of cell adhesion complexes in Drosophila.

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Evolutionary rate covariation analysis of E-cadherin identifies Raskol as a regulator of cell adhesion and actin dynamics in Drosophila

PLoS Genetics ◽

10.1371/journal.pgen.1007720 ◽

2019 ◽

Vol 15 (2) ◽

pp. e1007720 ◽

Cited By ~ 5

Author(s):

Qanber Raza ◽

Jae Young Choi ◽

Yang Li ◽

Roisin M. O’Dowd ◽

Simon C. Watkins ◽

...

Keyword(s):

Cell Adhesion ◽

Evolutionary Rate ◽

Actin Dynamics ◽

Covariation Analysis ◽

E Cadherin

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Minus end–directed motor KIFC3 suppresses E-cadherin degradation by recruiting USP47 to adherens junctions

Molecular Biology of the Cell ◽

10.1091/mbc.e14-07-1245 ◽

2014 ◽

Vol 25 (24) ◽

pp. 3851-3860 ◽

Cited By ~ 15

Author(s):

Kyoko Sako-Kubota ◽

Nobutoshi Tanaka ◽

Shigenori Nagae ◽

Wenxiang Meng ◽

Masatoshi Takeichi

Keyword(s):

Cell Adhesion ◽

Proteasome Inhibitors ◽

Adherens Junction ◽

Ubiquitin Protein Ligase ◽

Juxtamembrane Region ◽

Specific Protease ◽

Ubiquitin Specific Protease ◽

Epithelial Sheets ◽

E Cadherin ◽

Cell Cell

The adherens junction (AJ) plays a crucial role in maintaining cell–cell adhesion in epithelial tissues. Previous studies show that KIFC3, a minus end–directed kinesin motor, moves into AJs via microtubules that grow from clusters of CAMSAP3 (also known as Nezha), a protein that binds microtubule minus ends. The function of junction-associated KIFC3, however, remains to be elucidated. Here we find that KIFC3 binds the ubiquitin-specific protease USP47, a protease that removes ubiquitin chains from substrates and hence inhibits proteasome-mediated proteolysis, and recruits it to AJs. Depletion of KIFC3 or USP47 promotes cleavage of E-cadherin at a juxtamembrane region of the cytoplasmic domain, resulting in the production of a 90-kDa fragment and the internalization of E-cadherin. This cleavage depends on the E3 ubiquitin protein ligase Hakai and is inhibited by proteasome inhibitors. E-cadherin ubiquitination consistently increases after depletion of KIFC3 or USP47. These findings suggest that KIFC3 suppresses the ubiquitination and resultant degradation of E-cadherin by recruiting USP47 to AJs, a process that may be involved in maintaining stable cell–cell adhesion in epithelial sheets.

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Structure-Based Virtual Screening Allows the Identification of Efficient Modulators of E-Cadherin-Mediated Cell–Cell Adhesion

International Journal of Molecular Sciences ◽

10.3390/ijms20143404 ◽

2019 ◽

Vol 20 (14) ◽

pp. 3404 ◽

Cited By ~ 9

Author(s):

Andrea Dalle Vedove ◽

Federico Falchi ◽

Stefano Donini ◽

Aurelie Dobric ◽

Sebastien Germain ◽

...

Keyword(s):

Cell Adhesion ◽

Virtual Screening ◽

Adherens Junction ◽

Large Family ◽

Calcium Dependent ◽

Molecule Inhibitor ◽

Dependent Cell ◽

Cell Adhesion Proteins ◽

E Cadherin ◽

Cell Cell

Cadherins are a large family of transmembrane calcium-dependent cell adhesion proteins that orchestrate adherens junction formation and are crucially involved in tissue morphogenesis. Due to their important role in cancer development and metastasis, cadherins can be considered attractive targets for drug discovery. A recent crystal structure of the complex of a cadherin extracellular portion and a small molecule inhibitor allowed the identification of a druggable interface, thus providing a viable strategy for the design of cadherin dimerization modulators. Here, we report on a structure-based virtual screening approach that led to the identification of efficient and selective modulators of E-cadherin-mediated cell–cell adhesion. Of all the putative inhibitors that were identified and experimentally tested by cell adhesion assays using human pancreatic tumor BxPC-3 cells expressing both E-cadherin and P-cadherin, two compounds turned out to be effective in inhibiting stable cell–cell adhesion at micromolar concentrations. Moreover, at the same concentrations, one of them also showed anti-invasive properties in cell invasion assays. These results will allow further development of novel and selective cadherin-mediated cell–cell adhesion modulators for the treatment of a variety of cadherin-expressing solid tumors and for improving the efficiency of drug delivery across biological barriers.

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Remodeling the intercalated disc leads to cardiomyopathy in mice misexpressing cadherins in the heart

Journal of Cell Science ◽

10.1242/jcs.115.8.1623 ◽

2002 ◽

Vol 115 (8) ◽

pp. 1623-1634 ◽

Cited By ~ 2

Author(s):

M. Celeste Ferreira-Cornwell ◽

Yang Luo ◽

Navneet Narula ◽

Jennifer M. Lenox ◽

Melanie Lieberman ◽

...

Keyword(s):

Cell Adhesion ◽

Transgenic Mice ◽

Dilated Cardiomyopathy ◽

Cyclin D1 ◽

Connexin 43 ◽

Ectopic Expression ◽

Adherens Junction ◽

Transgenic Animals ◽

Intercalated Disc ◽

E Cadherin

The contractile force of the cardiomyocyte is transmitted through the adherens junction, a component of the intercalated disc, enabling the myocardium to function as a syncytium. The cadherin family of cell adhesion receptors, located in the adherens junction, interact homophilically to mediate strong cell-cell adhesion. Ectopic expression of cadherins is associated with changes in tumor cell behavior and pathology. To examine the effect of cadherin specificity on cardiac structure and function, we expressed either the epithelial cadherin, E-cadherin, or N-cadherin in the heart of transgenic mice. E-cadherin was localized to the intercalated disc structure in these animals similar to endogenous N-cadherin. Both N- and E-cadherin transgenic animals developed dilated cardiomyopathy. However, misexpression of E-cadherin led to earlier onset and increased mortality compared with N-cadherin mice. A dramatic decrease in connexin 43 was associated with the hypertrophic response in E-cadherin transgenic mice. Myofibril organization appeared normal although, vinculin, which normally localizes to the intercalated disc, was redistributed to the cytoplasm in the E-cadherin transgenic mice. Furthermore, E-cadherin induced cyclin D1, nuclear reduplication, and karyokinesis in the absence of cytokinesis, resulting in myocytes with two closely opposed nuclei. By contrast, N-cadherin overexpressing transgenic mice did not exhibit an increase in cyclin D1,suggesting that E-cadherin may provide a specific growth signal to the myocyte. This study demonstrates that modulation of cadherin-mediated adhesion can lead to dilated cardiomyopathy and that E-cadherin can stimulate DNA replication in myocytes normally withdrawn from the cell cycle.

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HER2 activation results in β-catenin-dependent changes in pulmonary epithelial permeability

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00237.2014 ◽

2015 ◽

Vol 308 (2) ◽

pp. L199-L207 ◽

Cited By ~ 14

Author(s):

James H. Finigan ◽

Vihas T. Vasu ◽

Jyoti V. Thaikoottathil ◽

Rangnath Mishra ◽

Mohammad A. Shatat ◽

...

Keyword(s):

Cell Adhesion ◽

Growth Factor Receptor ◽

Adherens Junction ◽

Epithelial Barrier ◽

Epithelial Barrier Function ◽

Neuregulin 1 ◽

Pulmonary Epithelial Permeability ◽

Her2 Signaling ◽

E Cadherin

The receptor tyrosine kinase human epidermal growth factor receptor-2 (HER2) is known to regulate pulmonary epithelial barrier function; however, the mechanisms behind this effect remain unidentified. We hypothesized that HER2 signaling alters the epithelial barrier through an interaction with the adherens junction (AJ) protein β-catenin, leading to dissolution of the AJ. In quiescent pulmonary epithelial cells, HER2 and β-catenin colocalized along the lateral intercellular junction. HER2 activation by the ligand neuregulin-1 was associated with tyrosine phosphorylation of β-catenin, dissociation of β-catenin from E-cadherin, and decreased E-cadherin-mediated cell adhesion. All effects were blocked with the HER2 inhibitor lapatinib. β-Catenin knockdown using shRNA significantly attenuated neuregulin-1-induced decreases in pulmonary epithelial resistance in vitro. Our data indicate that HER2 interacts with β-catenin, leading to dissolution of the AJ, decreased cell-cell adhesion, and disruption of the pulmonary epithelial barrier.

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Involvement of the Tyrosine Kinase Fer in Cell Adhesion

Molecular and Cellular Biology ◽

10.1128/mcb.18.10.5762 ◽

1998 ◽

Vol 18 (10) ◽

pp. 5762-5770 ◽

Cited By ~ 78

Author(s):

Roberto Rosato ◽

Jacqueline M. Veltmaat ◽

John Groffen ◽

Nora Heisterkamp

Keyword(s):

Cell Adhesion ◽

Tyrosine Kinase ◽

Tyrosine Kinases ◽

Negative Selection ◽

Expression System ◽

Adherens Junction ◽

Embryonic Fibroblasts ◽

Strong Negative Selection ◽

Permanent Cell ◽

E Cadherin

ABSTRACT The Fer protein belongs to the fes/fps family of nontransmembrane receptor tyrosine kinases. Lack of success in attempts to establish a permanent cell line overexpressing it at significant levels suggested a strong negative selection against too much Fer protein and pointed to a critical cellular function for Fer. Using a tetracycline-regulatable expression system, overexpression of Fer in embryonic fibroblasts was shown to evoke a massive rounding up, and the subsequent detachment of the cells from the substratum, which eventually led to cell death. Induction of Fer expression coincided with increased complex formation between Fer and the cadherin/src-associated substrate p120 cas and elevated tyrosine phosphorylation of p120 cas . β-Catenin also exhibited clearly increased phosphotyrosine levels, and Fer and β-catenin were found to be in complex. Significantly, although the levels of α-catenin, β-catenin, and E-cadherin were unaffected by Fer overexpression, decreased amounts of α-catenin and β-catenin were coimmunoprecipitated with E-cadherin, demonstrating a dissolution of adherens junction complexes. A concomitant decrease in levels of phosphotyrosine in the focal adhesion-associated protein p130 was also observed. Together, these results provide a mechanism for explaining the phenotype of cells overexpressing Fer and indicate that the Fer tyrosine kinase has a function in the regulation of cell-cell adhesion.

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E-cadherin endocytosis is modulated by p120-catenin through the opposing actions of RhoA and Arf1

10.1101/477760 ◽

2018 ◽

Cited By ~ 1

Author(s):

Joshua Greig ◽

Natalia A. Bulgakova

Keyword(s):

Plasma Membrane ◽

Cell Adhesion ◽

Actin Dynamics ◽

P120 Catenin ◽

Vital Importance ◽

C Terminus ◽

Opposing Effects ◽

Actin Remodelling ◽

Tissue Tension ◽

E Cadherin

AbstractThe regulation of E-cadherin at the plasma membrane by endocytosis is of vital importance for developmental and disease. p120-catenin, which binds to the E-cadherin C-terminus, can both promote and inhibit E-cadherin endocytosis. However, little is known about what determines the directionality of p120-catenin activity, and the molecules downstream. Here, we have discovered that p120-catenin fine-tunes the clathrin-mediated endocytosis of E-cadherin in Drosophila embryonic epidermal cells. It simultaneously activated two actin-remodelling pathways with opposing effects: RhoA, which stabilized E-cadherin at the membrane, and Arf1, which promoted internalization. Epistasis experiments revealed that RhoA additionally inhibited Arf1. E-cadherin was efficiently endocytosed only in the presence of intermediate p120-catenin amounts with too little and too much p120-catenin inhibiting E-cadherin endocytosis. Finally, we found that p120-catenin levels altered the tension of the plasma membrane. Altogether, this shows that p120-catenin is a central hub which co-ordinates cell adhesion, endocytosis, and actin dynamics with tissue tension.

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Downregulation of Rap1GAP in Human Tumor Cells Alters Cell/Matrix and Cell/Cell Adhesion

Molecular and Cellular Biology ◽

10.1128/mcb.01345-09 ◽

2010 ◽

Vol 30 (13) ◽

pp. 3262-3274 ◽

Cited By ~ 29

Author(s):

Oxana M. Tsygankova ◽

Changqing Ma ◽

Waixing Tang ◽

Christopher Korch ◽

Michael D. Feldman ◽

...

Keyword(s):

Cell Adhesion ◽

Tumor Cells ◽

Adherens Junction ◽

Protein Distribution ◽

P120 Catenin ◽

Cell Contacts ◽

Cell Matrix ◽

Junction Protein ◽

E Cadherin ◽

Cell Cell

ABSTRACT Rap1GAP expression is decreased in human tumors. The significance of its downregulation is unknown. We show that Rap1GAP expression is decreased in primary colorectal carcinomas. To elucidate the advantages conferred on tumor cells by loss of Rap1GAP, Rap1GAP expression was silenced in human colon carcinoma cells. Suppressing Rap1GAP induced profound alterations in cell adhesion. Rap1GAP-depleted cells exhibited defects in cell/cell adhesion that included an aberrant distribution of adherens junction proteins. Depletion of Rap1GAP enhanced adhesion and spreading on collagen. Silencing of Rap expression normalized spreading and restored E-cadherin, β-catenin, and p120-catenin to cell/cell contacts, indicating that unrestrained Rap activity underlies the alterations in cell adhesion. The defects in adherens junction protein distribution required integrin signaling as E-cadherin and p120-catenin were restored at cell/cell contacts when cells were plated on poly-l-lysine. Unexpectedly, Src activity was increased in Rap1GAP-depleted cells. Inhibition of Src impaired spreading and restored E-cadherin at cell/cell contacts. These findings provide the first evidence that Rap1GAP contributes to cell/cell adhesion and highlight a role for Rap1GAP in regulating cell/matrix and cell/cell adhesion. The frequent downregulation of Rap1GAP in epithelial tumors where alterations in cell/cell and cell/matrix adhesion are early steps in tumor dissemination supports a role for Rap1GAP depletion in tumor progression.

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

The Journal of Cell Biology ◽

10.1083/jcb.200910041 ◽

2010 ◽

Vol 189 (2) ◽

pp. 339-352 ◽

Cited By ~ 111

Author(s):

Jacqueline M. Benjamin ◽

Adam V. Kwiatkowski ◽

Changsong Yang ◽

Farida Korobova ◽

Sabine Pokutta ◽

...

Keyword(s):

Cell Adhesion ◽

Actin Polymerization ◽

Membrane Dynamics ◽

Actin Dynamics ◽

Filamentous Actin ◽

Vitro Binding ◽

Adhesion Complex ◽

E Cadherin ◽

Cell Cell

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.

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