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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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Substrate rigidity regulates scattering. (A–C) Cells were plated for 20 h on Fn-coated acrylamide substrates of increasing rigidity (determined by the percentage of bisacrylamide), stimulated with HGF, and observed by phase-contrast timelapse imaging. (A) Representative pictures of key time points after HGF stimulation show increased scattering on more rigid substrates (B; Video 6). (top) Scattering time course, quantified as in Fig. 3 A. (bottom) Extent of scattering at 10 h HGF, quantified as in Fig. 3 C. Data are means ± SEM. (C) Single cell migration exhibits a biphasic response to substrate stiffness. Velocity was determined at 8–10 h after HGF stimulation, when maximal velocity was reached. Data are means ± SEM. The software was unable to track cells grown at the 0.05% bisacrylamide-containing substrate because of cracks in the substrate (as in A, left) interfering in the segmentation algorithm. (D) Substrate stiffness promotes the formation of larger peripheral adhesions associated with thick actin bundles. Cells on flexible substrates for 16 h were fixed and stained as indicated.
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fig7: Substrate rigidity regulates scattering. (A–C) Cells were plated for 20 h on Fn-coated acrylamide substrates of increasing rigidity (determined by the percentage of bisacrylamide), stimulated with HGF, and observed by phase-contrast timelapse imaging. (A) Representative pictures of key time points after HGF stimulation show increased scattering on more rigid substrates (B; Video 6). (top) Scattering time course, quantified as in Fig. 3 A. (bottom) Extent of scattering at 10 h HGF, quantified as in Fig. 3 C. Data are means ± SEM. (C) Single cell migration exhibits a biphasic response to substrate stiffness. Velocity was determined at 8–10 h after HGF stimulation, when maximal velocity was reached. Data are means ± SEM. The software was unable to track cells grown at the 0.05% bisacrylamide-containing substrate because of cracks in the substrate (as in A, left) interfering in the segmentation algorithm. (D) Substrate stiffness promotes the formation of larger peripheral adhesions associated with thick actin bundles. Cells on flexible substrates for 16 h were fixed and stained as indicated.

Mentions: To test the hypothesis that the modulation of scattering by ECM type is due to ECM-specific effects on cytoskeletal contraction, we sought to alter mechanotransduction between the cytoskeleton and the substrate without changing the type or concentration of ECM. We therefore used polyacrylamide substrates cross-linked with different amounts of bisacrylamide to vary their stiffness. We developed a modified gel that binds ECM protein due to the incorporation of a positively charged acrylamide monomer, and thus does not require covalent attachment of the ECM protein. Gels of various degrees of stiffness were coated with the same amount of Fn (see Materials and methods). It is well established that decreasing substrate rigidity leads to a decrease in tractions applied to the substrate by the cells (Lo et al., 2000). Cells on Fn-coated flexible substrates were examined by time-lapse phase-contrast imaging during HGF-induced scattering. Quantification revealed that both the time course and extent of scattering were inhibited by decreasing substrate rigidity (Fig. 7, A and B; and Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200506152/DC1). Quantifying single cell migration velocity showed a peak at intermediate rigidity (0.15% bisacrylamide) and a decline at higher rigidity (Fig. 7 C), which is similar to the observations of Peyton and Putnam (2005). Therefore, effects on migration speed cannot account for the differences in scattering. Finally, fluorescent staining of paxillin and F-actin showed that increasing substrate rigidity correlated with increases in the size and peripheral distribution of focal adhesions. Indeed, cells on very compliant Fn-coated substrates displayed a cytoskeletal phenotype similar to that observed for cells plated on Ln1-coated glass (compare Fig. 7 D with Fig. 5). Thus, the ability of cells to develop strong tractions promotes HGF-induced cell scattering. Together with the effects of ECM on cytoskeletal organization and scattering, we conclude that ECM modulates disruption of cadherin-dependent cell–cell adhesions and cell scattering through its effects on cytoskeletal contractility.


Integrin-dependent actomyosin contraction regulates epithelial cell scattering.

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

Substrate rigidity regulates scattering. (A–C) Cells were plated for 20 h on Fn-coated acrylamide substrates of increasing rigidity (determined by the percentage of bisacrylamide), stimulated with HGF, and observed by phase-contrast timelapse imaging. (A) Representative pictures of key time points after HGF stimulation show increased scattering on more rigid substrates (B; Video 6). (top) Scattering time course, quantified as in Fig. 3 A. (bottom) Extent of scattering at 10 h HGF, quantified as in Fig. 3 C. Data are means ± SEM. (C) Single cell migration exhibits a biphasic response to substrate stiffness. Velocity was determined at 8–10 h after HGF stimulation, when maximal velocity was reached. Data are means ± SEM. The software was unable to track cells grown at the 0.05% bisacrylamide-containing substrate because of cracks in the substrate (as in A, left) interfering in the segmentation algorithm. (D) Substrate stiffness promotes the formation of larger peripheral adhesions associated with thick actin bundles. Cells on flexible substrates for 16 h were fixed and stained as indicated.
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fig7: Substrate rigidity regulates scattering. (A–C) Cells were plated for 20 h on Fn-coated acrylamide substrates of increasing rigidity (determined by the percentage of bisacrylamide), stimulated with HGF, and observed by phase-contrast timelapse imaging. (A) Representative pictures of key time points after HGF stimulation show increased scattering on more rigid substrates (B; Video 6). (top) Scattering time course, quantified as in Fig. 3 A. (bottom) Extent of scattering at 10 h HGF, quantified as in Fig. 3 C. Data are means ± SEM. (C) Single cell migration exhibits a biphasic response to substrate stiffness. Velocity was determined at 8–10 h after HGF stimulation, when maximal velocity was reached. Data are means ± SEM. The software was unable to track cells grown at the 0.05% bisacrylamide-containing substrate because of cracks in the substrate (as in A, left) interfering in the segmentation algorithm. (D) Substrate stiffness promotes the formation of larger peripheral adhesions associated with thick actin bundles. Cells on flexible substrates for 16 h were fixed and stained as indicated.
Mentions: To test the hypothesis that the modulation of scattering by ECM type is due to ECM-specific effects on cytoskeletal contraction, we sought to alter mechanotransduction between the cytoskeleton and the substrate without changing the type or concentration of ECM. We therefore used polyacrylamide substrates cross-linked with different amounts of bisacrylamide to vary their stiffness. We developed a modified gel that binds ECM protein due to the incorporation of a positively charged acrylamide monomer, and thus does not require covalent attachment of the ECM protein. Gels of various degrees of stiffness were coated with the same amount of Fn (see Materials and methods). It is well established that decreasing substrate rigidity leads to a decrease in tractions applied to the substrate by the cells (Lo et al., 2000). Cells on Fn-coated flexible substrates were examined by time-lapse phase-contrast imaging during HGF-induced scattering. Quantification revealed that both the time course and extent of scattering were inhibited by decreasing substrate rigidity (Fig. 7, A and B; and Video 6, available at http://www.jcb.org/cgi/content/full/jcb.200506152/DC1). Quantifying single cell migration velocity showed a peak at intermediate rigidity (0.15% bisacrylamide) and a decline at higher rigidity (Fig. 7 C), which is similar to the observations of Peyton and Putnam (2005). Therefore, effects on migration speed cannot account for the differences in scattering. Finally, fluorescent staining of paxillin and F-actin showed that increasing substrate rigidity correlated with increases in the size and peripheral distribution of focal adhesions. Indeed, cells on very compliant Fn-coated substrates displayed a cytoskeletal phenotype similar to that observed for cells plated on Ln1-coated glass (compare Fig. 7 D with Fig. 5). Thus, the ability of cells to develop strong tractions promotes HGF-induced cell scattering. Together with the effects of ECM on cytoskeletal organization and scattering, we conclude that ECM modulates disruption of cadherin-dependent cell–cell adhesions and cell scattering through its effects on cytoskeletal contractility.

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