Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction

Cohen, Daniel; Gloerich, Martijn; Nelson, W. James

doi:10.1073/pnas.1612208113

Cited by 40 publications

(38 citation statements)

References 46 publications

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“…Substrates immobilized with Ecad-Fc mimic cell-cell interactions, and cellular E-cadherin protein dynamics at the Ecad-Fc interface are similar to those found at endogenous cellcell junctions (27,28). Saturating amounts of Ecad-Fc were bound to substrates to ensure maximum engagement of E-cadherin in adhering cells (23).…”

Section: Resultsmentioning

confidence: 93%

Changes in E-cadherin rigidity sensing regulate cell adhesion

Collins

Denisin

Pruitt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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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 hightraction-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 cellcell adhesion.C ell adhesion is essential for tissue structure and function. Cells use specialized types of adhesions to interact with the surrounding environment, including integrin-based focal adhesions at cell-extracellular matrix (cell-ECM) contacts and cadherin-based adhesions at cell-cell contacts (1). Integrins bind to the ECM and intracellular proteins that link to the actin cytoskeleton and important signaling pathways (2). Similarly, cadherins regulate cellcell recognition and adhesion (3) and, through cytoplasmic adaptor proteins (catenins, vinculin) (4, 5), also link to the actin cytoskeleton and other proteins with signaling and scaffolding functions (6).Initiation of cell-cell adhesion requires significant reorganization of the actin cytoskeleton and is tightly controlled by the activities of actin nucleating proteins and Rho GTPases. Adhesion is initiated when filopodia from opposing cells come into contact with one another (7,8), and this process is regulated by Cdc42 activity (9, 10) and formin-dependent actin polymerization (11)(12)(13)(14). Intermediate stages of cell-cell contact formation involve lateral expansion of the contact by Rac1-induced and Arp2/3-dependent lamellipodial activity (15, 16). Finally, com...

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Section: Resultsmentioning

confidence: 93%

Changes in E-cadherin rigidity sensing regulate cell adhesion

Collins

Denisin

Pruitt

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…4 A and C). To test whether this tension-sensitive cortical recruitment of LGN was due to its recruitment to E-cadherin adhesions specifically, we artificially formed E-cadherin-dependent cell adhesions on microfabricated silicone sidewalls functionalized with the extracellular domain of E-cadherin (41). Single MDCK cells bound to these functionalized sidewalls and LGN was recruited to the E-cadherin complex that formed at the cell-sidewall interface (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…E-cadherin:Fc functionalized silicone sidewalls were fabricated as described (8,41). In brief, scaffolds were constructed from 250-mm thick silicone sheeting (Bisco HT-6240, Stockwell Elastomers) cut into microwell inserts measuring 14 × 11 mm using a computer-controlled razor writer (Cameo, Silhouette), plasma activated using atmospheric plasma (50 W, 45 s, 500 mTorr, PDC-001, Harrick Plasma), silanized by immersion in a solution of 2% triethoxysilylundecanal (Gelest) and 2% triethylamine (Sigma) in pure ethanol (Goldshield), followed by baking at 85°C for 3 h. A total of 250 mg/mL Protein A/G (Thermo-Pierce) in sodium cyanoborohydride coupling buffer (Sigma) was added to each well and allowed to react with the silanized stencils overnight at 4°C.…”

Section: Analysis Of Cortical Myosin Recruitmentmentioning

confidence: 99%

E-cadherin and LGN align epithelial cell divisions with tissue tension independently of cell shape

Hart

Tan

Siemers

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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104

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Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of Ecadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of trans engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.cell-cell adhesion | cell division orientation | mitotic spindle | mechanotransduction

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“…we placed silicone microwells into the dishes as described in [ 36 ] at densities from [1-2x10 6 430 cells/mL] which ultimately allowed for single cells, low density confluent monolayers, and high 431 density confluent monolayers to be captured. Silicone microwells consisted of 3x3 arrays of 9 432 mm 2 microwells into which we added 4 µL of suspended cells in media, allowed them to adhere 433 for 30 min in the incubator (6 hrs for keratinocytes), added media and returned them to the 434 incubator overnight prior to imaging.…”

Section: Preparation Of Training Samples 424mentioning

confidence: 99%

“…This is why context is extremely important for FRM 30 and why the work we present here focuses on evaluating practical uses of FRM with respect to 31 given P values. 32 33 Our goal here is not to improve FRM performance, but rather to provide a standardized 34 implementation of it and demonstrate its practical performance for every-day tasks such as 35 nuclear localization and tracking, characterizing cell morphology, cell-cell junction detection and 36 analysis, and re-analyzing legacy data and data collected on different systems ( Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

Practical Fluorescence Reconstruction Microscopy for Large Samples and Low-Magnification Imaging

LaChance

Cohen

2020

Preprint

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Fluorescence reconstruction microscopy (FRM) is an approach where transmitted light images are passed into a convolutional neural network which then outputs predicted epifluorescence images. This approach enables many benefits including reduced phototoxicity, freeing up of fluorescence channels, simplified sample preparation, and the ability to re-process legacy data for new insights. However, current FRM benchmarks are single scores that are difficult to relate to how useful for trustworthy and FRM predicition is. Here, we relate the conventional benchmark to practical and familiar cell biology analyses to demonstrate that FRM should be judged in context. We further demonstrate that it performs remarkably well even with lowermagnification microscopy data, as are often collected in high content imaging. Specifically, we present promising results for nuclei, cell-cell junctions, and fine feature reconstruction; provide experimental design guidelines; and provide the code, sample data, and user manual to enable more widespread adoption of FRM. where S = Scaling B/ Scaling A Ex:S Zeiss->Nikon = 1.4

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Epithelial self-healing is recapitulated by a 3D biomimetic E-cadherin junction

Cited by 40 publications

References 46 publications

Changes in E-cadherin rigidity sensing regulate cell adhesion

Changes in E-cadherin rigidity sensing regulate cell adhesion

E-cadherin and LGN align epithelial cell divisions with tissue tension independently of cell shape

Practical Fluorescence Reconstruction Microscopy for Large Samples and Low-Magnification Imaging

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