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Integrin-dependent actomyosin contraction regulates epithelial cell scattering.

de Rooij J, Kerstens A, Danuser G, Schwartz MA, Waterman-Storer CM - J. Cell Biol. (2005)

Bottom Line: Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions.Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates.We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
The scattering of Madin-Darby canine kidney cells in vitro mimics key aspects of epithelial-mesenchymal transitions during development, carcinoma cell invasion, and metastasis. Scattering is induced by hepatocyte growth factor (HGF) and is thought to involve disruption of cadherin-dependent cell-cell junctions. Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions. We show that HGF does not trigger any detectable decrease in E-cadherin function, but increases integrin-mediated adhesion. Time-lapse imaging suggests that tension on cell-cell junctions may disrupt cell-cell adhesion. Varying the density and type of extracellular matrix proteins shows that scattering correlates with stronger integrin adhesion and increased phosphorylation of the myosin regulatory light chain. To directly test the role of integrin-dependent traction forces, substrate compliance was varied. Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates. We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.

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Cell–cell adhesions are pulled apart during scattering. (A) Visualizing GFP-cadherin during scattering. MDCK cells stably expressing GFP-E-cadherin on 3 μg/ml Cn were imaged by time-lapse epifluorescence microscopy. HGF was added after 2 h and imaging continued. Images are from time-lapse Video 2. Images show dynamic adherens junctions that reorganized into radial streaks before breakage. (B) Scattering MDCK cells expressing GFP–ZO-1 were observed in the same manner as in A (Video 3). Cells just before the moment of cell–cell disruption are shown, demonstrating similar radial streaks as seen in GFP-E-cadherin. (C) Cells treated with HGF for 5 h were fixed and stained for α-catenin (red), paxillin (blue), and F-actin (green), to simultaneously visualize adherens junction, focal adhesions, and the actin cytoskeleton. In cells with disrupting adherens junctions, F-actin bundles terminate in focal adhesions just adjacent to cell–cell junctions.
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fig2: Cell–cell adhesions are pulled apart during scattering. (A) Visualizing GFP-cadherin during scattering. MDCK cells stably expressing GFP-E-cadherin on 3 μg/ml Cn were imaged by time-lapse epifluorescence microscopy. HGF was added after 2 h and imaging continued. Images are from time-lapse Video 2. Images show dynamic adherens junctions that reorganized into radial streaks before breakage. (B) Scattering MDCK cells expressing GFP–ZO-1 were observed in the same manner as in A (Video 3). Cells just before the moment of cell–cell disruption are shown, demonstrating similar radial streaks as seen in GFP-E-cadherin. (C) Cells treated with HGF for 5 h were fixed and stained for α-catenin (red), paxillin (blue), and F-actin (green), to simultaneously visualize adherens junction, focal adhesions, and the actin cytoskeleton. In cells with disrupting adherens junctions, F-actin bundles terminate in focal adhesions just adjacent to cell–cell junctions.

Mentions: To examine the dynamic behavior of junctional E-cadherin during scattering, MDCK cells stably expressing GFP-E-cadherin were examined by time-lapse fluorescence imaging. GFP-E-cadherin showed localization similar to endogenous E-cadherin at all times during scattering, and expression of GFP-E-cadherin did not inhibit scattering or cell migration (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200506152/DC1), thus confirming the observations by Iino et al. (2001) that GFP-E-cadherin behaves essentially like the endogenous protein. We observed that E-cadherin junctions were dynamic, undergoing continual rearrangement even in the absence of HGF (Fig. 2 A and Video 2). Soon after the addition of HGF, before junctions were disrupted, E-cadherin rearranged into linear structures perpendicular to cell edges. These structures broke down and fluorescence intensity abruptly dropped only as cells pulled apart, with no apparent drop in fluorescence intensity before that point. Time-lapse imaging of a GFP fusion of zona-occludens-1 (ZO-1), to label tight junctions, revealed similar behavior in which linear streaks of fluorescence aligned perpendicular to the cell edge and remained present until the cells pulled apart (Fig. 2 B and Video 3). The linear structures and the mechanism of junction breakdown suggest that increased centripetal tension, perpendicular to the junctions, is pulling the cells apart. Furthermore, as migrating cells contacted one another, new junctions formed at the sites of contact, with GFP-E-cadherin fluorescence immediately accumulating at these sites, as was described for newly forming contacts in the absence of HGF (Adams et al., 1998). This result again indicates that the ability of E-cadherin to form homotypic interactions is not impaired by HGF.


Integrin-dependent actomyosin contraction regulates epithelial cell scattering.

de Rooij J, Kerstens A, Danuser G, Schwartz MA, Waterman-Storer CM - J. Cell Biol. (2005)

Cell–cell adhesions are pulled apart during scattering. (A) Visualizing GFP-cadherin during scattering. MDCK cells stably expressing GFP-E-cadherin on 3 μg/ml Cn were imaged by time-lapse epifluorescence microscopy. HGF was added after 2 h and imaging continued. Images are from time-lapse Video 2. Images show dynamic adherens junctions that reorganized into radial streaks before breakage. (B) Scattering MDCK cells expressing GFP–ZO-1 were observed in the same manner as in A (Video 3). Cells just before the moment of cell–cell disruption are shown, demonstrating similar radial streaks as seen in GFP-E-cadherin. (C) Cells treated with HGF for 5 h were fixed and stained for α-catenin (red), paxillin (blue), and F-actin (green), to simultaneously visualize adherens junction, focal adhesions, and the actin cytoskeleton. In cells with disrupting adherens junctions, F-actin bundles terminate in focal adhesions just adjacent to cell–cell junctions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171213&req=5

fig2: Cell–cell adhesions are pulled apart during scattering. (A) Visualizing GFP-cadherin during scattering. MDCK cells stably expressing GFP-E-cadherin on 3 μg/ml Cn were imaged by time-lapse epifluorescence microscopy. HGF was added after 2 h and imaging continued. Images are from time-lapse Video 2. Images show dynamic adherens junctions that reorganized into radial streaks before breakage. (B) Scattering MDCK cells expressing GFP–ZO-1 were observed in the same manner as in A (Video 3). Cells just before the moment of cell–cell disruption are shown, demonstrating similar radial streaks as seen in GFP-E-cadherin. (C) Cells treated with HGF for 5 h were fixed and stained for α-catenin (red), paxillin (blue), and F-actin (green), to simultaneously visualize adherens junction, focal adhesions, and the actin cytoskeleton. In cells with disrupting adherens junctions, F-actin bundles terminate in focal adhesions just adjacent to cell–cell junctions.
Mentions: To examine the dynamic behavior of junctional E-cadherin during scattering, MDCK cells stably expressing GFP-E-cadherin were examined by time-lapse fluorescence imaging. GFP-E-cadherin showed localization similar to endogenous E-cadherin at all times during scattering, and expression of GFP-E-cadherin did not inhibit scattering or cell migration (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200506152/DC1), thus confirming the observations by Iino et al. (2001) that GFP-E-cadherin behaves essentially like the endogenous protein. We observed that E-cadherin junctions were dynamic, undergoing continual rearrangement even in the absence of HGF (Fig. 2 A and Video 2). Soon after the addition of HGF, before junctions were disrupted, E-cadherin rearranged into linear structures perpendicular to cell edges. These structures broke down and fluorescence intensity abruptly dropped only as cells pulled apart, with no apparent drop in fluorescence intensity before that point. Time-lapse imaging of a GFP fusion of zona-occludens-1 (ZO-1), to label tight junctions, revealed similar behavior in which linear streaks of fluorescence aligned perpendicular to the cell edge and remained present until the cells pulled apart (Fig. 2 B and Video 3). The linear structures and the mechanism of junction breakdown suggest that increased centripetal tension, perpendicular to the junctions, is pulling the cells apart. Furthermore, as migrating cells contacted one another, new junctions formed at the sites of contact, with GFP-E-cadherin fluorescence immediately accumulating at these sites, as was described for newly forming contacts in the absence of HGF (Adams et al., 1998). This result again indicates that the ability of E-cadherin to form homotypic interactions is not impaired by HGF.

Bottom Line: Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions.Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates.We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
The scattering of Madin-Darby canine kidney cells in vitro mimics key aspects of epithelial-mesenchymal transitions during development, carcinoma cell invasion, and metastasis. Scattering is induced by hepatocyte growth factor (HGF) and is thought to involve disruption of cadherin-dependent cell-cell junctions. Scattering is enhanced on collagen and fibronectin, as compared with laminin1, suggesting possible cross talk between integrins and cell-cell junctions. We show that HGF does not trigger any detectable decrease in E-cadherin function, but increases integrin-mediated adhesion. Time-lapse imaging suggests that tension on cell-cell junctions may disrupt cell-cell adhesion. Varying the density and type of extracellular matrix proteins shows that scattering correlates with stronger integrin adhesion and increased phosphorylation of the myosin regulatory light chain. To directly test the role of integrin-dependent traction forces, substrate compliance was varied. Rigid substrates that produce high traction forces promoted scattering, in comparison to more compliant substrates. We conclude that integrin-dependent actomyosin traction force mediates the disruption of cell-cell adhesion during epithelial cell scattering.

Show MeSH
Related in: MedlinePlus