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The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin.

Qin Y, Capaldo C, Gumbiner BM, Macara IG - J. Cell Biol. (2005)

Bottom Line: These effects are independent of Rac activation or Scrib binding to betaPIX.Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion.Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Signaling, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

ABSTRACT
Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to betaPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.

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βPIX is not involved in Scrib function at adherens junctions. (A) MDCK II cells were transiently transfected with either pS-Luciferase control vector (Luc) or three pSUPER constructs targeting different sequences of canine βPIX mRNA (PixKD). Proteins were analyzed by SDS-PAGE and immunoblot. (B) Quantification of Boyden chamber migration assay with Luc, ScrbKD, and ScrbKD plus PixKD cells. Error bars represent mean ± SD. (C) Immunoblot showing Scrib single KD and Scrib and βPIX double KD. (D) Rac pull-down assays with control and ScrbKD cells in both normal medium (top) and low-calcium medium (bottom). (E) Immunoblot analysis of phospho-ERK in control and ScrbKD cells with and without stimulation by 15 ng/ml HGF.
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fig4: βPIX is not involved in Scrib function at adherens junctions. (A) MDCK II cells were transiently transfected with either pS-Luciferase control vector (Luc) or three pSUPER constructs targeting different sequences of canine βPIX mRNA (PixKD). Proteins were analyzed by SDS-PAGE and immunoblot. (B) Quantification of Boyden chamber migration assay with Luc, ScrbKD, and ScrbKD plus PixKD cells. Error bars represent mean ± SD. (C) Immunoblot showing Scrib single KD and Scrib and βPIX double KD. (D) Rac pull-down assays with control and ScrbKD cells in both normal medium (top) and low-calcium medium (bottom). (E) Immunoblot analysis of phospho-ERK in control and ScrbKD cells with and without stimulation by 15 ng/ml HGF.

Mentions: Of four pSUPER constructs tested, three efficiently suppressed βPIX expression (Fig. 4 A). In particular, the PixKD1 shRNA reduced expression of the protein by >90%. However, loss of βPIX had no detectable effect on cell migration as measured using the Boyden chamber assay (Fig. 4 B). Moreover, in our hands, overexpression of βPIX did not increase cell migration in the filter assay (unpublished data). We then performed double KD experiments in which the expression of both Scrib and βPIX was suppressed (Fig. 4 C). We reasoned that one function of Scrib might be to sequester and inactivate βPIX. In this case, loss of Scrib would release the βPIX, leading to inappropriate activation of Rac and increased migration. If this hypothesis were correct, a double KD would reverse the migration phenotype by removing the excess free βPIX from the cell. As depicted in Fig. 3 (A and B), migration through filters was increased by Scrib KD. However, the coordinate loss of βPIX did not significantly perturb this effect (Fig. 4 B). Note that cotransfection of the PixKD shRNA did not interfere with gene silencing of Scrib (Fig. 4 C). We therefore conclude that Scrib function in cell adhesion and migration is independent of βPIX binding.


The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin.

Qin Y, Capaldo C, Gumbiner BM, Macara IG - J. Cell Biol. (2005)

βPIX is not involved in Scrib function at adherens junctions. (A) MDCK II cells were transiently transfected with either pS-Luciferase control vector (Luc) or three pSUPER constructs targeting different sequences of canine βPIX mRNA (PixKD). Proteins were analyzed by SDS-PAGE and immunoblot. (B) Quantification of Boyden chamber migration assay with Luc, ScrbKD, and ScrbKD plus PixKD cells. Error bars represent mean ± SD. (C) Immunoblot showing Scrib single KD and Scrib and βPIX double KD. (D) Rac pull-down assays with control and ScrbKD cells in both normal medium (top) and low-calcium medium (bottom). (E) Immunoblot analysis of phospho-ERK in control and ScrbKD cells with and without stimulation by 15 ng/ml HGF.
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Related In: Results  -  Collection

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fig4: βPIX is not involved in Scrib function at adherens junctions. (A) MDCK II cells were transiently transfected with either pS-Luciferase control vector (Luc) or three pSUPER constructs targeting different sequences of canine βPIX mRNA (PixKD). Proteins were analyzed by SDS-PAGE and immunoblot. (B) Quantification of Boyden chamber migration assay with Luc, ScrbKD, and ScrbKD plus PixKD cells. Error bars represent mean ± SD. (C) Immunoblot showing Scrib single KD and Scrib and βPIX double KD. (D) Rac pull-down assays with control and ScrbKD cells in both normal medium (top) and low-calcium medium (bottom). (E) Immunoblot analysis of phospho-ERK in control and ScrbKD cells with and without stimulation by 15 ng/ml HGF.
Mentions: Of four pSUPER constructs tested, three efficiently suppressed βPIX expression (Fig. 4 A). In particular, the PixKD1 shRNA reduced expression of the protein by >90%. However, loss of βPIX had no detectable effect on cell migration as measured using the Boyden chamber assay (Fig. 4 B). Moreover, in our hands, overexpression of βPIX did not increase cell migration in the filter assay (unpublished data). We then performed double KD experiments in which the expression of both Scrib and βPIX was suppressed (Fig. 4 C). We reasoned that one function of Scrib might be to sequester and inactivate βPIX. In this case, loss of Scrib would release the βPIX, leading to inappropriate activation of Rac and increased migration. If this hypothesis were correct, a double KD would reverse the migration phenotype by removing the excess free βPIX from the cell. As depicted in Fig. 3 (A and B), migration through filters was increased by Scrib KD. However, the coordinate loss of βPIX did not significantly perturb this effect (Fig. 4 B). Note that cotransfection of the PixKD shRNA did not interfere with gene silencing of Scrib (Fig. 4 C). We therefore conclude that Scrib function in cell adhesion and migration is independent of βPIX binding.

Bottom Line: These effects are independent of Rac activation or Scrib binding to betaPIX.Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion.Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein.

View Article: PubMed Central - PubMed

Affiliation: Center for Cell Signaling, Department of Microbiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

ABSTRACT
Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin-Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to betaPIX. Rather, Scrib depletion disrupts E-cadherin-mediated cell-cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin-alpha-catenin fusion protein but not by E-cadherin-green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.

Show MeSH
Related in: MedlinePlus