Limits...
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.

Show MeSH

Related in: MedlinePlus

Myosin II regulatory light chain phosphorylation and distribution are regulated by ECM type and concentration. (A) MLC phosphorylation increases with concentration of matrix. Lysates from cells plated on the indicated ECM were analyzed for pMLC by Western blotting. Total myosin IIA heavy chain was analyzed as a loading control. The induction of pMLC by increasing Cn was quantified relative to the lowest concentration used. Data are means ± SD; n = 3. (B) pMLC is greater in cells plated on Cn or Fn than on Ln 1. pMLC content was compared in cells on saturating concentrations of 3 μg/ml Cn, 10 μg/ml Fn, and 10 μg/ml Ln1. Levels of pMLC on Ln1 relative to Cn in the absence and presence of HGF (30 min) were calculated. Data are means ± SD; n = 5. (C) HGF induces a transient increase in pMLC that peaks at 30 min after application. Cells on Cn (3 μg/ml) or Ln1 (10 μg/ml) were stimulated with HGF for the indicated periods of time. Levels of pMLC relative to unstimulated samples on either Cn or Ln1 are shown. (D) HGF induces localization of pMLC to actin bundles near cell–cell junctions on Cn, but not on Ln1. Cells were plated on either 3 μg/ml Cn- or 10 μg/ml Ln1-coated coverslips and stimulated for the indicated times with HGF. Cells were fixed, and actin and pMLC were localized.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171213&req=5

fig6: Myosin II regulatory light chain phosphorylation and distribution are regulated by ECM type and concentration. (A) MLC phosphorylation increases with concentration of matrix. Lysates from cells plated on the indicated ECM were analyzed for pMLC by Western blotting. Total myosin IIA heavy chain was analyzed as a loading control. The induction of pMLC by increasing Cn was quantified relative to the lowest concentration used. Data are means ± SD; n = 3. (B) pMLC is greater in cells plated on Cn or Fn than on Ln 1. pMLC content was compared in cells on saturating concentrations of 3 μg/ml Cn, 10 μg/ml Fn, and 10 μg/ml Ln1. Levels of pMLC on Ln1 relative to Cn in the absence and presence of HGF (30 min) were calculated. Data are means ± SD; n = 5. (C) HGF induces a transient increase in pMLC that peaks at 30 min after application. Cells on Cn (3 μg/ml) or Ln1 (10 μg/ml) were stimulated with HGF for the indicated periods of time. Levels of pMLC relative to unstimulated samples on either Cn or Ln1 are shown. (D) HGF induces localization of pMLC to actin bundles near cell–cell junctions on Cn, but not on Ln1. Cells were plated on either 3 μg/ml Cn- or 10 μg/ml Ln1-coated coverslips and stimulated for the indicated times with HGF. Cells were fixed, and actin and pMLC were localized.

Mentions: To further investigate whether the differential scattering of MDCK cells on ECM correlates with cytoskeletal contraction, we investigated the phosphorylation of the myosin II regulatory light chain (MLC), which is the main regulatory event leading to actomyosin contractility (Somlyo and Somlyo, 1994; Bresnick, 1999). Western blotting for MLC phosphorylated at serine 19 (pMLC) showed that increasing concentrations of Cn increased pMLC (Fig. 6 A), which correlates with better scattering on high ECM coating concentrations (Fig. 3 A). Similar results were obtained with Fn and Ln1 (unpublished data). Moreover, at ECM concentrations corresponding to maximal scattering efficiency, pMLC was higher on Cn than on Ln1 (Fig. 6 B), again correlating with scattering efficiency on the two substrate types (Fig. 3 C). The addition of HGF induced a transient increase in total levels of pMLC on all ECM substrates (Fig. 6 C; Fn not depicted). These results suggest that myosin II–dependent contractility correlates with the differences in scattering observed for different ECM conditions.


Integrin-dependent actomyosin contraction regulates epithelial cell scattering.

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

Myosin II regulatory light chain phosphorylation and distribution are regulated by ECM type and concentration. (A) MLC phosphorylation increases with concentration of matrix. Lysates from cells plated on the indicated ECM were analyzed for pMLC by Western blotting. Total myosin IIA heavy chain was analyzed as a loading control. The induction of pMLC by increasing Cn was quantified relative to the lowest concentration used. Data are means ± SD; n = 3. (B) pMLC is greater in cells plated on Cn or Fn than on Ln 1. pMLC content was compared in cells on saturating concentrations of 3 μg/ml Cn, 10 μg/ml Fn, and 10 μg/ml Ln1. Levels of pMLC on Ln1 relative to Cn in the absence and presence of HGF (30 min) were calculated. Data are means ± SD; n = 5. (C) HGF induces a transient increase in pMLC that peaks at 30 min after application. Cells on Cn (3 μg/ml) or Ln1 (10 μg/ml) were stimulated with HGF for the indicated periods of time. Levels of pMLC relative to unstimulated samples on either Cn or Ln1 are shown. (D) HGF induces localization of pMLC to actin bundles near cell–cell junctions on Cn, but not on Ln1. Cells were plated on either 3 μg/ml Cn- or 10 μg/ml Ln1-coated coverslips and stimulated for the indicated times with HGF. Cells were fixed, and actin and pMLC were localized.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171213&req=5

fig6: Myosin II regulatory light chain phosphorylation and distribution are regulated by ECM type and concentration. (A) MLC phosphorylation increases with concentration of matrix. Lysates from cells plated on the indicated ECM were analyzed for pMLC by Western blotting. Total myosin IIA heavy chain was analyzed as a loading control. The induction of pMLC by increasing Cn was quantified relative to the lowest concentration used. Data are means ± SD; n = 3. (B) pMLC is greater in cells plated on Cn or Fn than on Ln 1. pMLC content was compared in cells on saturating concentrations of 3 μg/ml Cn, 10 μg/ml Fn, and 10 μg/ml Ln1. Levels of pMLC on Ln1 relative to Cn in the absence and presence of HGF (30 min) were calculated. Data are means ± SD; n = 5. (C) HGF induces a transient increase in pMLC that peaks at 30 min after application. Cells on Cn (3 μg/ml) or Ln1 (10 μg/ml) were stimulated with HGF for the indicated periods of time. Levels of pMLC relative to unstimulated samples on either Cn or Ln1 are shown. (D) HGF induces localization of pMLC to actin bundles near cell–cell junctions on Cn, but not on Ln1. Cells were plated on either 3 μg/ml Cn- or 10 μg/ml Ln1-coated coverslips and stimulated for the indicated times with HGF. Cells were fixed, and actin and pMLC were localized.
Mentions: To further investigate whether the differential scattering of MDCK cells on ECM correlates with cytoskeletal contraction, we investigated the phosphorylation of the myosin II regulatory light chain (MLC), which is the main regulatory event leading to actomyosin contractility (Somlyo and Somlyo, 1994; Bresnick, 1999). Western blotting for MLC phosphorylated at serine 19 (pMLC) showed that increasing concentrations of Cn increased pMLC (Fig. 6 A), which correlates with better scattering on high ECM coating concentrations (Fig. 3 A). Similar results were obtained with Fn and Ln1 (unpublished data). Moreover, at ECM concentrations corresponding to maximal scattering efficiency, pMLC was higher on Cn than on Ln1 (Fig. 6 B), again correlating with scattering efficiency on the two substrate types (Fig. 3 C). The addition of HGF induced a transient increase in total levels of pMLC on all ECM substrates (Fig. 6 C; Fn not depicted). These results suggest that myosin II–dependent contractility correlates with the differences in scattering observed for different ECM conditions.

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