Limits...
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.

Show MeSH

Related in: MedlinePlus

Decreased adhesiveness of cells lacking Scrib. (A) 3 × 104 control and ScrbKD cells were seeded into hanging drop cultures and allowed to aggregate overnight. After trituration by passing the cell cluster 10 times through a 200-μl pipette tip, images were captured by phase-contrast microscopy using a 10× objective. (B) E-cadherin is not preferentially lost from the ScrbKD cells in suspension culture. (C) Quantification of the degree of aggregation shown in A. Data are presented as the area of the aggregated cells/number of individual nonaggregated cells and represent means of 10–12 images from triplicates of each sample ± SD. (D) Effect of βPIX silencing on cell aggregation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171311&req=5

fig5: Decreased adhesiveness of cells lacking Scrib. (A) 3 × 104 control and ScrbKD cells were seeded into hanging drop cultures and allowed to aggregate overnight. After trituration by passing the cell cluster 10 times through a 200-μl pipette tip, images were captured by phase-contrast microscopy using a 10× objective. (B) E-cadherin is not preferentially lost from the ScrbKD cells in suspension culture. (C) Quantification of the degree of aggregation shown in A. Data are presented as the area of the aggregated cells/number of individual nonaggregated cells and represent means of 10–12 images from triplicates of each sample ± SD. (D) Effect of βPIX silencing on cell aggregation.

Mentions: To determine whether cell–cell adhesion is compromised in the absence of Scrib, we first used an aggregation assay. Cells were trypsinized, triturated to break up clumps into individual cells, resuspended in fresh medium in a hanging drop beneath the lid of a tissue culture plate, and incubated for 18–20 h. Cell aggregation was then assessed microscopically. A dramatic loss of aggregation was apparent in cells expressing either pS-ScrbKD1 or 2 vectors, as compared with the control cells that were transfected with pS-Luc (Fig. 5, A and C). This effect was not a result of differential loss of E-cadherin in the cell suspensions as assessed by immunoblotting lysates from the suspended cell cultures (Fig. 5 B). Importantly, coexpression of human GFP-Scrib reversed the adhesion defect caused by the loss of endogenous SCRIB, proving that the effect of the shRNAs on adhesion is specifically mediated through destruction of the Scrib mRNA rather than through off-target effects (Fig. 5, A and C). We also tested for a possible role for βPIX on aggregation, using an shRNA directed against the canine gene (Fig. 4, A and B). However, loss of βPIX from the cells had no effect on the aggregation of control cells and did not reverse the loss of aggregation observed in the absence of Scrib (Fig. 5 D).


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)

Decreased adhesiveness of cells lacking Scrib. (A) 3 × 104 control and ScrbKD cells were seeded into hanging drop cultures and allowed to aggregate overnight. After trituration by passing the cell cluster 10 times through a 200-μl pipette tip, images were captured by phase-contrast microscopy using a 10× objective. (B) E-cadherin is not preferentially lost from the ScrbKD cells in suspension culture. (C) Quantification of the degree of aggregation shown in A. Data are presented as the area of the aggregated cells/number of individual nonaggregated cells and represent means of 10–12 images from triplicates of each sample ± SD. (D) Effect of βPIX silencing on cell aggregation.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Decreased adhesiveness of cells lacking Scrib. (A) 3 × 104 control and ScrbKD cells were seeded into hanging drop cultures and allowed to aggregate overnight. After trituration by passing the cell cluster 10 times through a 200-μl pipette tip, images were captured by phase-contrast microscopy using a 10× objective. (B) E-cadherin is not preferentially lost from the ScrbKD cells in suspension culture. (C) Quantification of the degree of aggregation shown in A. Data are presented as the area of the aggregated cells/number of individual nonaggregated cells and represent means of 10–12 images from triplicates of each sample ± SD. (D) Effect of βPIX silencing on cell aggregation.
Mentions: To determine whether cell–cell adhesion is compromised in the absence of Scrib, we first used an aggregation assay. Cells were trypsinized, triturated to break up clumps into individual cells, resuspended in fresh medium in a hanging drop beneath the lid of a tissue culture plate, and incubated for 18–20 h. Cell aggregation was then assessed microscopically. A dramatic loss of aggregation was apparent in cells expressing either pS-ScrbKD1 or 2 vectors, as compared with the control cells that were transfected with pS-Luc (Fig. 5, A and C). This effect was not a result of differential loss of E-cadherin in the cell suspensions as assessed by immunoblotting lysates from the suspended cell cultures (Fig. 5 B). Importantly, coexpression of human GFP-Scrib reversed the adhesion defect caused by the loss of endogenous SCRIB, proving that the effect of the shRNAs on adhesion is specifically mediated through destruction of the Scrib mRNA rather than through off-target effects (Fig. 5, A and C). We also tested for a possible role for βPIX on aggregation, using an shRNA directed against the canine gene (Fig. 4, A and B). However, loss of βPIX from the cells had no effect on the aggregation of control cells and did not reverse the loss of aggregation observed in the absence of Scrib (Fig. 5 D).

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