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

Rao, Megha Vaman; Zaidel-Bar, Ronen

doi:10.1091/mbc.e16-06-0429

Cited by 58 publications

(56 citation statements)

References 61 publications

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“…In this context, FMNL3 recruitment to junctions is tension dependent and regulated by Cdc42 (14). Our results indicate that the requirement for Cdc42 and formin activity becomes less important as substrate modulus increases, which could be related to the roles of mDia1 and FMNL3 in stabilizing early junctions and decreasing E-cadherin mobility (14).…”

Section: Discussionmentioning

confidence: 78%

“…Small molecule inhibitors were added to inhibit formins (20 μM SMIFH2) and Arp2/3 (100 μM CK666) activity. These concentrations have been shown previously to disrupt formin and Arp2/3 activities in MDCK cells without inducing cytotoxic or off-target effects (14,(43)(44)(45). Importantly, the effects of these inhibitor concentrations are reversible so that recovery of normal function can be monitored.…”

Section: Resultsmentioning

confidence: 97%

“…However, formin1 binds directly to the E-cadherin complex through α-catenin (67), and it is possible that forces at E-cadherin adhesions activate α-catenin binding to formins via force-dependent changes in α-catenin conformation (68), perhaps similar to that required for binding to F-actin (5). In this context, FMNL3 recruitment to junctions is tension dependent and regulated by Cdc42 (14). Our results indicate that the requirement for Cdc42 and formin activity becomes less important as substrate modulus increases, which could be related to the roles of mDia1 and FMNL3 in stabilizing early junctions and decreasing E-cadherin mobility (14).…”

Section: Discussionmentioning

confidence: 99%

“…MDCK cells expressing GFP-LifeAct were imaged 24 h after transfection. All inhibitors were diluted in DMSO and used at concentrations previously shown to inhibit activities without inducing cytotoxic or off-target effects in MDCK cells: 20 μM SMIFH2 (14), 100 μM CK666 (45), 20 μM ML-141 (52), and 100 μM NSC-23766 (54). Threedimensional hanging-drop assays used single-cell suspensions in droplets hanging from an inverted 35-mm Petri dish lid.…”

Section: Methodsmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

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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: Discussionmentioning

confidence: 78%

Section: Resultsmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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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Loss of scinderin decreased expression of epidermal growth factor receptor and promoted apoptosis of castration‐resistant prostate cancer cells

Lai

Zhao³

et al. 2018

FEBS Open Bio

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Most patients with prostate cancer will eventually develop the castration‐resistant form characterised by metastasis. Cytoskeleton constituents, including F‐actin, play important roles in maintaining epithelial integrity and their disruption is a major cause of cancer progression. We previously showed that scinderin (SCIN), an important regulator of F‐actin organisation, is highly expressed in poorly differentiated cancer tissues. This study aimed to explore the mechanism of its regulation of cell proliferation. We discovered that SCIN knockdown significantly downregulated epidermal growth factor receptor (EGFR) protein expression, and inhibited epidermal growth factor (EGF)‐mediated cell proliferation and activation of the downstream mitogen‐activated protein kinase kinase (MEK)/extracellular signal‐regulated kinase (ERK) signalling pathway. Silencing of SCIN promoted apoptosis in two cell lines (PC‐3 and DU145), inhibited B‐cell lymphoma‐extra‐large (Bcl‐xl) expression and activated caspase signalling. Furthermore, in vivo studies showed that SCIN deletion slowed tumour growth and decreased EGFR expression. Thus, we conclude that SCIN promotes prostate cancer cell survival by stabilising EGFR and MEK/ERK signalling.

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Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry

2018

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Preparing a single cell suspension is a critical step in any solid tissue flow cytometry experiment. Tissue dissection, enzymatic digestion, and mechanical dissociation are three significant steps leading to the degradation of the extracellular matrix and the isolation of single cells, allowing the generation of high‐quality flow cytometry data. Cells and the extracellular matrix contain various proteins and other structures which must be considered when designing a tissue digestion protocol to preserve the viability of cells and the presence of relevant antigens while digesting matrix components and cleaving cell–cell junctions. Evaluation of the single cell suspension is essential before proceeding with the labeling of the cells as high viability and absence of cell debris and aggregates are critical for flow cytometry. The information presented should be used as a general guide of steps to consider when preparing a single cell suspension from solid tissues for flow cytometry experiments. © 2018 International Society for Advancement of Cytometry

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

Cited by 58 publications

References 61 publications

Changes in E-cadherin rigidity sensing regulate cell adhesion

Changes in E-cadherin rigidity sensing regulate cell adhesion

Loss of scinderin decreased expression of epidermal growth factor receptor and promoted apoptosis of castration‐resistant prostate cancer cells

Best Practices for Preparing a Single Cell Suspension from Solid Tissues for Flow Cytometry

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