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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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Related in: MedlinePlus

Scattering is promoted by increasing ECM concentration and is more efficient on Cn and Fn than on Ln 1. MDCK cells plated on the indicated ECM proteins were imaged by time-lapse phase-contrast microscopy. HGF was added after 2 h and imaging continued for 16 h. (A) Representative images from the time-lapse series (Video 4) showing the differences between scattering on the three different ECM proteins at saturating concentrations (3 μg/ml type I Cn, 10 μg/ml Fn, 10 μg/ml Ln1). By 6 h, cells have begun to scatter on Cn and Fn, but have not initiated on Ln1. (B) For all matrices, t1/2 of scattering is reached faster on increasing matrix concentration and is saturable. Quantification of scattering from three time-lapses per condition from one representative experiment is shown. The top graph shows the time at which 50% of the islands initiated scattering (as measured by the disruption of at least three cell–cell junctions per island). The bottom graph depicts the progression of scattering at saturating ECM concentrations. Scattering progresses with similar kinetics on Fn and Cn, and much more slowly on Ln1. (C) Cells on Ln 1 do not scatter as completely as cells on other matrices. The extent of scattering, quantified as the average number of cell–cell contacts per cell at 14 h of HGF on the indicated matrix (when scattering was complete) or in the absence of HGF on 3 μg/ml Cn. Data are means ± SEM, and at least 250 cells were counted per condition. (D) Scattering is not specific to β1 integrins. Cells were induced to scatter as in A on 3 μg/ml Cn or 3 μg/ml Vn in the absence or presence of the β1 integrin–blocking antibody AIIB2 (10 μg/ml) and followed by time-lapse imaging. Three time-lapse series with identical results were obtained (Video 5) and representative images at 12 h after HGF are shown (right). As a control, the effect of AIIB2 on the adhesion to Cn and Vn was measured using an adhesion assay (left), showing that β1 integrins are not involved in the adhesion to Vn. Data are means ± SD; n = 3.
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fig3: Scattering is promoted by increasing ECM concentration and is more efficient on Cn and Fn than on Ln 1. MDCK cells plated on the indicated ECM proteins were imaged by time-lapse phase-contrast microscopy. HGF was added after 2 h and imaging continued for 16 h. (A) Representative images from the time-lapse series (Video 4) showing the differences between scattering on the three different ECM proteins at saturating concentrations (3 μg/ml type I Cn, 10 μg/ml Fn, 10 μg/ml Ln1). By 6 h, cells have begun to scatter on Cn and Fn, but have not initiated on Ln1. (B) For all matrices, t1/2 of scattering is reached faster on increasing matrix concentration and is saturable. Quantification of scattering from three time-lapses per condition from one representative experiment is shown. The top graph shows the time at which 50% of the islands initiated scattering (as measured by the disruption of at least three cell–cell junctions per island). The bottom graph depicts the progression of scattering at saturating ECM concentrations. Scattering progresses with similar kinetics on Fn and Cn, and much more slowly on Ln1. (C) Cells on Ln 1 do not scatter as completely as cells on other matrices. The extent of scattering, quantified as the average number of cell–cell contacts per cell at 14 h of HGF on the indicated matrix (when scattering was complete) or in the absence of HGF on 3 μg/ml Cn. Data are means ± SEM, and at least 250 cells were counted per condition. (D) Scattering is not specific to β1 integrins. Cells were induced to scatter as in A on 3 μg/ml Cn or 3 μg/ml Vn in the absence or presence of the β1 integrin–blocking antibody AIIB2 (10 μg/ml) and followed by time-lapse imaging. Three time-lapse series with identical results were obtained (Video 5) and representative images at 12 h after HGF are shown (right). As a control, the effect of AIIB2 on the adhesion to Cn and Vn was measured using an adhesion assay (left), showing that β1 integrins are not involved in the adhesion to Vn. Data are means ± SD; n = 3.

Mentions: Previous studies have suggested that scattering is dependent on the type of ECM provided or the specific integrins engaged (Clark, 1994; Sander et al., 1998; Gimond et al., 1999). To further investigate these effects, we first systematically assayed the influence of ECM on scattering in our model system. For that purpose, we developed a multiwell plate live-cell microscopy assay on an automated digital microscope system with a robotic stage. MDCK cells were plated in 48-well plates in which the wells were coated with a range of concentrations of different ECM proteins (type 1 Cn, Fn, and Ln1). HGF-induced scattering was then followed by time-lapse phase-contrast imaging for all conditions simultaneously (Fig. 3 A and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200506152/DC1). For quantification, scattering initiation was scored as the percentage of cell islands (groups of 5–16 cells that form when epithelial cells are cultured subconfluently) in which three or more cells simultaneously had disrupted contacts with neighboring cells. Sample time courses for the evolution of scattering on different ECM proteins are shown in Fig. 3 B (bottom). To compare the effects of the three different matrix proteins over a range of concentrations, we determined the time at which 50% of the islands had initiated scattering (t1/2 of scattering). Increasing concentration of any type of ECM caused faster scattering (lower t1/2) to a saturation point (Fig. 3 B, top). The minimal t1/2 of scattering depended on the type of ECM, with cells scattering fastest on Cn, followed closely by Fn, and scattering slowest on Ln1 (Fig. 3 B, top). In addition to the delay in achieving half-scattering, the maximal extent of scattering was also decreased on Ln1 (Fig. 3 A and Video 4). When the number of junctions with neighboring cells was scored after 14 h in the presence of HGF (the time of maximal scattering), cells on Ln1 had significantly more cell contacts (i.e., less scattering) than cells on either Fn or Cn (Fig. 3 C). Thus, as previously observed (Clark, 1994; Sander et al., 1998), scattering is induced by different ECM proteins to different extents. Importantly, we find that in all cases scattering is promoted by increasing concentrations of ECM.


Integrin-dependent actomyosin contraction regulates epithelial cell scattering.

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

Scattering is promoted by increasing ECM concentration and is more efficient on Cn and Fn than on Ln 1. MDCK cells plated on the indicated ECM proteins were imaged by time-lapse phase-contrast microscopy. HGF was added after 2 h and imaging continued for 16 h. (A) Representative images from the time-lapse series (Video 4) showing the differences between scattering on the three different ECM proteins at saturating concentrations (3 μg/ml type I Cn, 10 μg/ml Fn, 10 μg/ml Ln1). By 6 h, cells have begun to scatter on Cn and Fn, but have not initiated on Ln1. (B) For all matrices, t1/2 of scattering is reached faster on increasing matrix concentration and is saturable. Quantification of scattering from three time-lapses per condition from one representative experiment is shown. The top graph shows the time at which 50% of the islands initiated scattering (as measured by the disruption of at least three cell–cell junctions per island). The bottom graph depicts the progression of scattering at saturating ECM concentrations. Scattering progresses with similar kinetics on Fn and Cn, and much more slowly on Ln1. (C) Cells on Ln 1 do not scatter as completely as cells on other matrices. The extent of scattering, quantified as the average number of cell–cell contacts per cell at 14 h of HGF on the indicated matrix (when scattering was complete) or in the absence of HGF on 3 μg/ml Cn. Data are means ± SEM, and at least 250 cells were counted per condition. (D) Scattering is not specific to β1 integrins. Cells were induced to scatter as in A on 3 μg/ml Cn or 3 μg/ml Vn in the absence or presence of the β1 integrin–blocking antibody AIIB2 (10 μg/ml) and followed by time-lapse imaging. Three time-lapse series with identical results were obtained (Video 5) and representative images at 12 h after HGF are shown (right). As a control, the effect of AIIB2 on the adhesion to Cn and Vn was measured using an adhesion assay (left), showing that β1 integrins are not involved in the adhesion to Vn. Data are means ± SD; n = 3.
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Related In: Results  -  Collection

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fig3: Scattering is promoted by increasing ECM concentration and is more efficient on Cn and Fn than on Ln 1. MDCK cells plated on the indicated ECM proteins were imaged by time-lapse phase-contrast microscopy. HGF was added after 2 h and imaging continued for 16 h. (A) Representative images from the time-lapse series (Video 4) showing the differences between scattering on the three different ECM proteins at saturating concentrations (3 μg/ml type I Cn, 10 μg/ml Fn, 10 μg/ml Ln1). By 6 h, cells have begun to scatter on Cn and Fn, but have not initiated on Ln1. (B) For all matrices, t1/2 of scattering is reached faster on increasing matrix concentration and is saturable. Quantification of scattering from three time-lapses per condition from one representative experiment is shown. The top graph shows the time at which 50% of the islands initiated scattering (as measured by the disruption of at least three cell–cell junctions per island). The bottom graph depicts the progression of scattering at saturating ECM concentrations. Scattering progresses with similar kinetics on Fn and Cn, and much more slowly on Ln1. (C) Cells on Ln 1 do not scatter as completely as cells on other matrices. The extent of scattering, quantified as the average number of cell–cell contacts per cell at 14 h of HGF on the indicated matrix (when scattering was complete) or in the absence of HGF on 3 μg/ml Cn. Data are means ± SEM, and at least 250 cells were counted per condition. (D) Scattering is not specific to β1 integrins. Cells were induced to scatter as in A on 3 μg/ml Cn or 3 μg/ml Vn in the absence or presence of the β1 integrin–blocking antibody AIIB2 (10 μg/ml) and followed by time-lapse imaging. Three time-lapse series with identical results were obtained (Video 5) and representative images at 12 h after HGF are shown (right). As a control, the effect of AIIB2 on the adhesion to Cn and Vn was measured using an adhesion assay (left), showing that β1 integrins are not involved in the adhesion to Vn. Data are means ± SD; n = 3.
Mentions: Previous studies have suggested that scattering is dependent on the type of ECM provided or the specific integrins engaged (Clark, 1994; Sander et al., 1998; Gimond et al., 1999). To further investigate these effects, we first systematically assayed the influence of ECM on scattering in our model system. For that purpose, we developed a multiwell plate live-cell microscopy assay on an automated digital microscope system with a robotic stage. MDCK cells were plated in 48-well plates in which the wells were coated with a range of concentrations of different ECM proteins (type 1 Cn, Fn, and Ln1). HGF-induced scattering was then followed by time-lapse phase-contrast imaging for all conditions simultaneously (Fig. 3 A and Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200506152/DC1). For quantification, scattering initiation was scored as the percentage of cell islands (groups of 5–16 cells that form when epithelial cells are cultured subconfluently) in which three or more cells simultaneously had disrupted contacts with neighboring cells. Sample time courses for the evolution of scattering on different ECM proteins are shown in Fig. 3 B (bottom). To compare the effects of the three different matrix proteins over a range of concentrations, we determined the time at which 50% of the islands had initiated scattering (t1/2 of scattering). Increasing concentration of any type of ECM caused faster scattering (lower t1/2) to a saturation point (Fig. 3 B, top). The minimal t1/2 of scattering depended on the type of ECM, with cells scattering fastest on Cn, followed closely by Fn, and scattering slowest on Ln1 (Fig. 3 B, top). In addition to the delay in achieving half-scattering, the maximal extent of scattering was also decreased on Ln1 (Fig. 3 A and Video 4). When the number of junctions with neighboring cells was scored after 14 h in the presence of HGF (the time of maximal scattering), cells on Ln1 had significantly more cell contacts (i.e., less scattering) than cells on either Fn or Cn (Fig. 3 C). Thus, as previously observed (Clark, 1994; Sander et al., 1998), scattering is induced by different ECM proteins to different extents. Importantly, we find that in all cases scattering is promoted by increasing concentrations of ECM.

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