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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 in MDCK cells causes a delay in tight junction assembly. (A) MDCK II cells were transiently transfected with either pS-Luciferase vector (Luc) as a control or three pSUPER constructs targeting different sequences of canine Scrib mRNA (ScrbKD). Equal amounts of proteins were analyzed by SDS-PAGE and immunoblot. (B) Control and ScrbKD MDCK cells grown at high density were fixed and stained for Scrib and occludin. (C) Control and ScrbKD cells were subjected to a calcium switch and then fixed and stained for ZO-1 at the indicated times after readdition of calcium. Magnified details of the junctions are shown in inverted black-and-white scale to highlight the defects caused by loss of Scrib.
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fig1: Suppression of Scrib expression in MDCK cells causes a delay in tight junction assembly. (A) MDCK II cells were transiently transfected with either pS-Luciferase vector (Luc) as a control or three pSUPER constructs targeting different sequences of canine Scrib mRNA (ScrbKD). Equal amounts of proteins were analyzed by SDS-PAGE and immunoblot. (B) Control and ScrbKD MDCK cells grown at high density were fixed and stained for Scrib and occludin. (C) Control and ScrbKD cells were subjected to a calcium switch and then fixed and stained for ZO-1 at the indicated times after readdition of calcium. Magnified details of the junctions are shown in inverted black-and-white scale to highlight the defects caused by loss of Scrib.

Mentions: Several target sequences were selected from the partial canine Scrib gene and used to construct pSUPER vectors for the expression of small hairpin RNAs (shRNAs). Of the three sequences tested, two efficiently knocked down Scrib expression when expressed by transient transfection in MDCK II cells (Fig. 1 A). Immunofluorescence microscopy revealed Scrib to be associated with the lateral membranes in mammalian epithelial cells, and staining was substantially reduced by transfection with the ScrbKD1 or 2 vectors (Fig. 1 B). Surprisingly, however, the tight junctions appeared to be intact in these cells, as assessed by occludin staining (Fig. 1 B) or ZO-1 staining (not depicted). Moreover, confocal sections of cells stained for the apical marker gp135 revealed no loss of apical/basal polarization in cells expressing reduced Scrib levels (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200506094/DC1), and cysts grown in Matrigel appeared to be polarized normally (not depicted). These results suggest that depletion of Scrib does not disrupt tight junction assembly. However, when the transfected cells were subjected to a calcium switch and stained for ZO-1, a short delay in junction assembly was observed (Fig. 1 C). ZO-1 accumulated rapidly at the cell–cell contacts in both the control and Scrib knockdown (KD) cells but, in the absence of Scrib, the fusion of the ZO-1 lines into a continuous band encircling each cell was delayed. The defect was particularly noticeable at vertices where several cell boundaries meet. By 20 h after calcium switch, however, the ZO-1 staining in the cells lacking Scrib was indistinguishable from that in the control cells.


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 in MDCK cells causes a delay in tight junction assembly. (A) MDCK II cells were transiently transfected with either pS-Luciferase vector (Luc) as a control or three pSUPER constructs targeting different sequences of canine Scrib mRNA (ScrbKD). Equal amounts of proteins were analyzed by SDS-PAGE and immunoblot. (B) Control and ScrbKD MDCK cells grown at high density were fixed and stained for Scrib and occludin. (C) Control and ScrbKD cells were subjected to a calcium switch and then fixed and stained for ZO-1 at the indicated times after readdition of calcium. Magnified details of the junctions are shown in inverted black-and-white scale to highlight the defects caused by loss of Scrib.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Suppression of Scrib expression in MDCK cells causes a delay in tight junction assembly. (A) MDCK II cells were transiently transfected with either pS-Luciferase vector (Luc) as a control or three pSUPER constructs targeting different sequences of canine Scrib mRNA (ScrbKD). Equal amounts of proteins were analyzed by SDS-PAGE and immunoblot. (B) Control and ScrbKD MDCK cells grown at high density were fixed and stained for Scrib and occludin. (C) Control and ScrbKD cells were subjected to a calcium switch and then fixed and stained for ZO-1 at the indicated times after readdition of calcium. Magnified details of the junctions are shown in inverted black-and-white scale to highlight the defects caused by loss of Scrib.
Mentions: Several target sequences were selected from the partial canine Scrib gene and used to construct pSUPER vectors for the expression of small hairpin RNAs (shRNAs). Of the three sequences tested, two efficiently knocked down Scrib expression when expressed by transient transfection in MDCK II cells (Fig. 1 A). Immunofluorescence microscopy revealed Scrib to be associated with the lateral membranes in mammalian epithelial cells, and staining was substantially reduced by transfection with the ScrbKD1 or 2 vectors (Fig. 1 B). Surprisingly, however, the tight junctions appeared to be intact in these cells, as assessed by occludin staining (Fig. 1 B) or ZO-1 staining (not depicted). Moreover, confocal sections of cells stained for the apical marker gp135 revealed no loss of apical/basal polarization in cells expressing reduced Scrib levels (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200506094/DC1), and cysts grown in Matrigel appeared to be polarized normally (not depicted). These results suggest that depletion of Scrib does not disrupt tight junction assembly. However, when the transfected cells were subjected to a calcium switch and stained for ZO-1, a short delay in junction assembly was observed (Fig. 1 C). ZO-1 accumulated rapidly at the cell–cell contacts in both the control and Scrib knockdown (KD) cells but, in the absence of Scrib, the fusion of the ZO-1 lines into a continuous band encircling each cell was delayed. The defect was particularly noticeable at vertices where several cell boundaries meet. By 20 h after calcium switch, however, the ZO-1 staining in the cells lacking Scrib was indistinguishable from that in the control cells.

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