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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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Related in: MedlinePlus

Suppression of Scrib expression reduces E-cadherin–mediated adhesion. (A) In vitro E-cadherin binding assay. Different concentrations of recombinant E-cadherin extracellular domain were coated on plates. Control (Luc) and ScrbKD cells were collected in suspension and allowed to adhere to the plates for 60 min. Images were captured for nonwashed plates (to give total cell numbers), and plates were subjected to washing (to determine attached cell numbers). (B) Quantification of cell attachment to E-cadherin ectodomain. Data represent means of 10–12 images from triplicates of each condition ± SD.
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fig6: Suppression of Scrib expression reduces E-cadherin–mediated adhesion. (A) In vitro E-cadherin binding assay. Different concentrations of recombinant E-cadherin extracellular domain were coated on plates. Control (Luc) and ScrbKD cells were collected in suspension and allowed to adhere to the plates for 60 min. Images were captured for nonwashed plates (to give total cell numbers), and plates were subjected to washing (to determine attached cell numbers). (B) Quantification of cell attachment to E-cadherin ectodomain. Data represent means of 10–12 images from triplicates of each condition ± SD.

Mentions: To determine whether the aggregation defect is mediated through E-cadherin or some other cell adhesion protein, we assayed the ability of the MDCK cells to attach to a surface coated with the extracellular domain of E-cadherin. Cells were disassociated using an EGTA solution (with no trypsin), centrifuged and resuspended in fresh medium, and added to 96-well plates coated with the ectodomain of E-cadherin. After 60 min, the plates were washed and remaining attached cells were counted. Results are shown in Fig. 6. Almost no cells attached to the plates in the absence of the E-cadherin ectodomain, demonstrating that during the 60-min incubation period integrin-mediated attachment is negligible. Control cells attached efficiently, and attachment was proportional to the amount of E-cadherin ectodomain on the plate (Fig. 6 B). Importantly, loss of Scrib caused a substantial drop (approximately threefold) in cell attachment, demonstrating that E-cadherin homophilic adhesion is compromised in the absence of Scrib. Addition of an arginine–glycine–aspartic acid peptide to block integrin-mediated adhesion had no significant effect (unpublished data).


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 reduces E-cadherin–mediated adhesion. (A) In vitro E-cadherin binding assay. Different concentrations of recombinant E-cadherin extracellular domain were coated on plates. Control (Luc) and ScrbKD cells were collected in suspension and allowed to adhere to the plates for 60 min. Images were captured for nonwashed plates (to give total cell numbers), and plates were subjected to washing (to determine attached cell numbers). (B) Quantification of cell attachment to E-cadherin ectodomain. Data represent means of 10–12 images from triplicates of each condition ± SD.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Suppression of Scrib expression reduces E-cadherin–mediated adhesion. (A) In vitro E-cadherin binding assay. Different concentrations of recombinant E-cadherin extracellular domain were coated on plates. Control (Luc) and ScrbKD cells were collected in suspension and allowed to adhere to the plates for 60 min. Images were captured for nonwashed plates (to give total cell numbers), and plates were subjected to washing (to determine attached cell numbers). (B) Quantification of cell attachment to E-cadherin ectodomain. Data represent means of 10–12 images from triplicates of each condition ± SD.
Mentions: To determine whether the aggregation defect is mediated through E-cadherin or some other cell adhesion protein, we assayed the ability of the MDCK cells to attach to a surface coated with the extracellular domain of E-cadherin. Cells were disassociated using an EGTA solution (with no trypsin), centrifuged and resuspended in fresh medium, and added to 96-well plates coated with the ectodomain of E-cadherin. After 60 min, the plates were washed and remaining attached cells were counted. Results are shown in Fig. 6. Almost no cells attached to the plates in the absence of the E-cadherin ectodomain, demonstrating that during the 60-min incubation period integrin-mediated attachment is negligible. Control cells attached efficiently, and attachment was proportional to the amount of E-cadherin ectodomain on the plate (Fig. 6 B). Importantly, loss of Scrib caused a substantial drop (approximately threefold) in cell attachment, demonstrating that E-cadherin homophilic adhesion is compromised in the absence of Scrib. Addition of an arginine–glycine–aspartic acid peptide to block integrin-mediated adhesion had no significant effect (unpublished data).

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