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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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Suppression of Scrib expression increases cell motility and attenuates orientated migration. (A) Motility of cells transfected with Luc, ScrbKD1 shRNA, or ScrbKD1 plus GFP-tagged human Scrib was measured using a Boyden chamber assay. Images were captured after crystal violet staining of the cells at the bottom side of the filter. (B) Quantification of cell migration by measuring A595 of eluted crystal violet. Error bars represent mean ± SD. (C) Immunoblot analysis of transfected cells with anti-Scrib antibody. Equal protein concentrations were loaded and normalized using anti-tubulin. (D) Rose plot of individual cell tracks from time-lapse movies of the wounding assay.
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fig3: Suppression of Scrib expression increases cell motility and attenuates orientated migration. (A) Motility of cells transfected with Luc, ScrbKD1 shRNA, or ScrbKD1 plus GFP-tagged human Scrib was measured using a Boyden chamber assay. Images were captured after crystal violet staining of the cells at the bottom side of the filter. (B) Quantification of cell migration by measuring A595 of eluted crystal violet. Error bars represent mean ± SD. (C) Immunoblot analysis of transfected cells with anti-Scrib antibody. Equal protein concentrations were loaded and normalized using anti-tubulin. (D) Rose plot of individual cell tracks from time-lapse movies of the wounding assay.

Mentions: To determine whether Scrib regulates MDCK cell motility, transfected cells were plated onto 8-μm filters in Boyden chambers and incubated in normal medium containing 10% serum (both above and below the filter). The same number of cells was plated onto each filter. After 16–20 h, cells that had migrated through the pores to the bottom surface of the filters were stained with crystal violet. Loss of Scrib substantially increased the number of cells that had migrated through the filter (Fig. 3, A and B). Importantly, when we expressed a GFP fusion of a human Scrib in the cells transfected with the pS-ScrbKD1 vector, the number of cells migrating through the filter was reduced to control levels (Fig. 3, A and B). It was conceivable that the reversion to a wild-type phenotype was caused by reexpression of the endogenous protein. To test for this possibility, we blotted cell lysates for Scrib (Fig. 3 C). The endogenous Scrib protein level was reduced upon expression of the ScrbKD1 shRNA and did not respond to coexpression of the human GFP–Scrib fusion. The GFP–Scrib fusion can be distinguished by its lower mobility on SDS-PAGE and was expressed at approximately two to three times the level of the endogenous protein in control cells (Fig. 3 C). These data prove that the effects of Scrib RNAi on motility are indeed caused by loss of the Scrib protein rather than by off-target effects of the shRNA.


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)

Suppression of Scrib expression increases cell motility and attenuates orientated migration. (A) Motility of cells transfected with Luc, ScrbKD1 shRNA, or ScrbKD1 plus GFP-tagged human Scrib was measured using a Boyden chamber assay. Images were captured after crystal violet staining of the cells at the bottom side of the filter. (B) Quantification of cell migration by measuring A595 of eluted crystal violet. Error bars represent mean ± SD. (C) Immunoblot analysis of transfected cells with anti-Scrib antibody. Equal protein concentrations were loaded and normalized using anti-tubulin. (D) Rose plot of individual cell tracks from time-lapse movies of the wounding assay.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171311&req=5

fig3: Suppression of Scrib expression increases cell motility and attenuates orientated migration. (A) Motility of cells transfected with Luc, ScrbKD1 shRNA, or ScrbKD1 plus GFP-tagged human Scrib was measured using a Boyden chamber assay. Images were captured after crystal violet staining of the cells at the bottom side of the filter. (B) Quantification of cell migration by measuring A595 of eluted crystal violet. Error bars represent mean ± SD. (C) Immunoblot analysis of transfected cells with anti-Scrib antibody. Equal protein concentrations were loaded and normalized using anti-tubulin. (D) Rose plot of individual cell tracks from time-lapse movies of the wounding assay.
Mentions: To determine whether Scrib regulates MDCK cell motility, transfected cells were plated onto 8-μm filters in Boyden chambers and incubated in normal medium containing 10% serum (both above and below the filter). The same number of cells was plated onto each filter. After 16–20 h, cells that had migrated through the pores to the bottom surface of the filters were stained with crystal violet. Loss of Scrib substantially increased the number of cells that had migrated through the filter (Fig. 3, A and B). Importantly, when we expressed a GFP fusion of a human Scrib in the cells transfected with the pS-ScrbKD1 vector, the number of cells migrating through the filter was reduced to control levels (Fig. 3, A and B). It was conceivable that the reversion to a wild-type phenotype was caused by reexpression of the endogenous protein. To test for this possibility, we blotted cell lysates for Scrib (Fig. 3 C). The endogenous Scrib protein level was reduced upon expression of the ScrbKD1 shRNA and did not respond to coexpression of the human GFP–Scrib fusion. The GFP–Scrib fusion can be distinguished by its lower mobility on SDS-PAGE and was expressed at approximately two to three times the level of the endogenous protein in control cells (Fig. 3 C). These data prove that the effects of Scrib RNAi on motility are indeed caused by loss of the Scrib protein rather than by off-target effects of the shRNA.

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