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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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Effects of E-cadherin depletion and expression of an E-cadherin–α-catenin fusion protein. (A) Cells were transfected with an shRNA targeted against canine E-cadherin. Lysates were immunoblotted for E-cadherin and Scrib. Equal protein concentrations were loaded and normalized using anti-tubulin. (B) Quantification of the motility of cells transfected with Luc or EcadKD shRNAs using Boyden chamber assay as described in Fig. 3. (C) Morphology of cells depleted of E-cadherin. Control and EcadKD cells were grown on six-well plates at low density for 3 d. Images were obtained by phase-contrast microscopy using a 10× objective. (D) Expression of an Ecad–αcat and Ecad–GFP fusion in MDCK cells. The fusion proteins and endogenous E-cadherin were detected by immunoblot with anti–E-cadherin antibodies. (E) Effects of the Ecad–αcat and Ecad–GFP fusions on motility of cells depleted of Scrib. Migration through filters was quantified as in Fig. 3. (F) Aggregation assay. 3 × 104 cells were seeded into hanging drop cultures and allowed to aggregate for overnight. After trituration with a 200-μl pipet tip, images were captured by phase-contrast microscopy using a 10× objective. (G) Quantification of the aggregation assay. Data are presented as the area of the aggregated cells/number of individual nonaggregated cells per field and represent means of 10–12 fields from triplicates of each sample ± SD.
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fig8: Effects of E-cadherin depletion and expression of an E-cadherin–α-catenin fusion protein. (A) Cells were transfected with an shRNA targeted against canine E-cadherin. Lysates were immunoblotted for E-cadherin and Scrib. Equal protein concentrations were loaded and normalized using anti-tubulin. (B) Quantification of the motility of cells transfected with Luc or EcadKD shRNAs using Boyden chamber assay as described in Fig. 3. (C) Morphology of cells depleted of E-cadherin. Control and EcadKD cells were grown on six-well plates at low density for 3 d. Images were obtained by phase-contrast microscopy using a 10× objective. (D) Expression of an Ecad–αcat and Ecad–GFP fusion in MDCK cells. The fusion proteins and endogenous E-cadherin were detected by immunoblot with anti–E-cadherin antibodies. (E) Effects of the Ecad–αcat and Ecad–GFP fusions on motility of cells depleted of Scrib. Migration through filters was quantified as in Fig. 3. (F) Aggregation assay. 3 × 104 cells were seeded into hanging drop cultures and allowed to aggregate for overnight. After trituration with a 200-μl pipet tip, images were captured by phase-contrast microscopy using a 10× objective. (G) Quantification of the aggregation assay. Data are presented as the area of the aggregated cells/number of individual nonaggregated cells per field and represent means of 10–12 fields from triplicates of each sample ± SD.

Mentions: If both the adhesion defect and the increased motility observed in response to Scrib silencing are caused by decreased E-cadherin activity, one would predict that depletion of E-cadherin would produce the same phenotype. We therefore expressed a shRNA targeted against the canine E-cadherin in MDCK cells and achieved a >50% reduction in E-cadherin expression (Fig. 8 A). Interestingly, cells depleted of E-cadherin migrated through filters significantly faster than the control (Fig. 8 B). Moreover, these cells were larger and more fibroblastic in appearance than control cells when plated at low densities (Fig. 8 C), just as observed for the Scrib KD cells. Based on these data, we conclude that both the morphological changes and increased motility in cells depleted of Scrib can be ascribed to a failure of the E-cadherin to form normal trans-adhesive interactions.


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)

Effects of E-cadherin depletion and expression of an E-cadherin–α-catenin fusion protein. (A) Cells were transfected with an shRNA targeted against canine E-cadherin. Lysates were immunoblotted for E-cadherin and Scrib. Equal protein concentrations were loaded and normalized using anti-tubulin. (B) Quantification of the motility of cells transfected with Luc or EcadKD shRNAs using Boyden chamber assay as described in Fig. 3. (C) Morphology of cells depleted of E-cadherin. Control and EcadKD cells were grown on six-well plates at low density for 3 d. Images were obtained by phase-contrast microscopy using a 10× objective. (D) Expression of an Ecad–αcat and Ecad–GFP fusion in MDCK cells. The fusion proteins and endogenous E-cadherin were detected by immunoblot with anti–E-cadherin antibodies. (E) Effects of the Ecad–αcat and Ecad–GFP fusions on motility of cells depleted of Scrib. Migration through filters was quantified as in Fig. 3. (F) Aggregation assay. 3 × 104 cells were seeded into hanging drop cultures and allowed to aggregate for overnight. After trituration with a 200-μl pipet tip, images were captured by phase-contrast microscopy using a 10× objective. (G) Quantification of the aggregation assay. Data are presented as the area of the aggregated cells/number of individual nonaggregated cells per field and represent means of 10–12 fields from triplicates of each sample ± SD.
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Related In: Results  -  Collection

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fig8: Effects of E-cadherin depletion and expression of an E-cadherin–α-catenin fusion protein. (A) Cells were transfected with an shRNA targeted against canine E-cadherin. Lysates were immunoblotted for E-cadherin and Scrib. Equal protein concentrations were loaded and normalized using anti-tubulin. (B) Quantification of the motility of cells transfected with Luc or EcadKD shRNAs using Boyden chamber assay as described in Fig. 3. (C) Morphology of cells depleted of E-cadherin. Control and EcadKD cells were grown on six-well plates at low density for 3 d. Images were obtained by phase-contrast microscopy using a 10× objective. (D) Expression of an Ecad–αcat and Ecad–GFP fusion in MDCK cells. The fusion proteins and endogenous E-cadherin were detected by immunoblot with anti–E-cadherin antibodies. (E) Effects of the Ecad–αcat and Ecad–GFP fusions on motility of cells depleted of Scrib. Migration through filters was quantified as in Fig. 3. (F) Aggregation assay. 3 × 104 cells were seeded into hanging drop cultures and allowed to aggregate for overnight. After trituration with a 200-μl pipet tip, images were captured by phase-contrast microscopy using a 10× objective. (G) Quantification of the aggregation assay. Data are presented as the area of the aggregated cells/number of individual nonaggregated cells per field and represent means of 10–12 fields from triplicates of each sample ± SD.
Mentions: If both the adhesion defect and the increased motility observed in response to Scrib silencing are caused by decreased E-cadherin activity, one would predict that depletion of E-cadherin would produce the same phenotype. We therefore expressed a shRNA targeted against the canine E-cadherin in MDCK cells and achieved a >50% reduction in E-cadherin expression (Fig. 8 A). Interestingly, cells depleted of E-cadherin migrated through filters significantly faster than the control (Fig. 8 B). Moreover, these cells were larger and more fibroblastic in appearance than control cells when plated at low densities (Fig. 8 C), just as observed for the Scrib KD cells. Based on these data, we conclude that both the morphological changes and increased motility in cells depleted of Scrib can be ascribed to a failure of the E-cadherin to form normal trans-adhesive interactions.

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