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Numb regulates cell-cell adhesion and polarity in response to tyrosine kinase signalling.

Wang Z, Sandiford S, Wu C, Li SS - EMBO J. (2009)

Bottom Line: Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT.Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration.Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

ABSTRACT
Epithelial-mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E-cadherin (E-cad) through its phosphotyrosine-binding domain (PTB) and thereby regulates the localization of E-cad to the lateral domain of epithelial cell-cell junction. Moreover, Numb engages the polarity complex Par3-aPKC-Par6 by binding to Par3 in polarized Madin-Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E-cad and Par3 and associates preferably with aPKC-Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.

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Numb knockdown led to aberrant localization of E-cadherin, β-catenin and F-actin. (A) Confocal Z-stack images of E-cad-GFP, E-cad(3A)-GFP and E-cad(3F)-GFP in transfected MDCKII cells, respectively. The panel labelled as ‘apical' corresponds to an x/y section image (x/y focal plane) taken at the subapical region of the monolayer of cells (at 4 μm below the apical surface) and the panel labelled as ‘basal' corresponds to the basolateral region (a section at 4 μm above the basal surface), respectively. The corresponding x/z focal plane image was shown below each x/y image set. Apical is at the top, whereas basal is at the bottom. The same convention was used throughout. Size bars represent 10 μm. GFP fluorescence is in green and nuclei are stained in blue with DAPI. (B) Confocal Z-stack images of E-cadherin (green) and F-actin (red) in the control MDCKII cells versus in the numb-shRNA or sr-Numb cell lines. In each panel, the E-cadherin immunostaining (green) images were shown on the left, F-actin staining (red) images in the middle and the overlaid images on the right. The corresponding x/z focal plane images were shown below the x/y images. (C) Confocal Z-stack images of β-catenin immunofluorescence in the control MDCKII cells versus in the numb-shRNA or sr-Numb cells shown as either x/y (top) or x/z (bottom) section. (D) Confocal x/z sections showing the apical translocation of E-cadherin in the control MDCKII cells versus in the numb-shRNA or sr-Numb cells. The apical surface was identified by co-staining for ZO-1. (E–G) Quantitative analysis of immunofluorescence images shown in (A), (B) and (C), respectively. Values shown are changes in fold of apical over basal immunofluorescence. *P<0.001, n=3 in (E); *P<0.001, **P<0.05, n=5 in (F); *P<0.01, n=3 in (G). The error bars represent mean±s.d.
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f3: Numb knockdown led to aberrant localization of E-cadherin, β-catenin and F-actin. (A) Confocal Z-stack images of E-cad-GFP, E-cad(3A)-GFP and E-cad(3F)-GFP in transfected MDCKII cells, respectively. The panel labelled as ‘apical' corresponds to an x/y section image (x/y focal plane) taken at the subapical region of the monolayer of cells (at 4 μm below the apical surface) and the panel labelled as ‘basal' corresponds to the basolateral region (a section at 4 μm above the basal surface), respectively. The corresponding x/z focal plane image was shown below each x/y image set. Apical is at the top, whereas basal is at the bottom. The same convention was used throughout. Size bars represent 10 μm. GFP fluorescence is in green and nuclei are stained in blue with DAPI. (B) Confocal Z-stack images of E-cadherin (green) and F-actin (red) in the control MDCKII cells versus in the numb-shRNA or sr-Numb cell lines. In each panel, the E-cadherin immunostaining (green) images were shown on the left, F-actin staining (red) images in the middle and the overlaid images on the right. The corresponding x/z focal plane images were shown below the x/y images. (C) Confocal Z-stack images of β-catenin immunofluorescence in the control MDCKII cells versus in the numb-shRNA or sr-Numb cells shown as either x/y (top) or x/z (bottom) section. (D) Confocal x/z sections showing the apical translocation of E-cadherin in the control MDCKII cells versus in the numb-shRNA or sr-Numb cells. The apical surface was identified by co-staining for ZO-1. (E–G) Quantitative analysis of immunofluorescence images shown in (A), (B) and (C), respectively. Values shown are changes in fold of apical over basal immunofluorescence. *P<0.001, n=3 in (E); *P<0.001, **P<0.05, n=5 in (F); *P<0.01, n=3 in (G). The error bars represent mean±s.d.

Mentions: To investigate whether the interaction between Numb and E-cad is required for proper junctional localization of the cadherin complex, we transfected MDCKII cells with expression constructs for E-cad-GFP, E-cad-(3F)-GFP and E-cad-(3A)-GFP, respectively. As expected, E-cad-GFP localized to cell–cell junctions in polarized cells. Of the two E-cad mutants examined, E-cad-(3F)-GFP localized to the lateral membrane domain as did the wt protein. In contrast, E-cad-(3A)-GFP, which was deficient in Numb binding, was detected at the apical region of the cell–cell junction (Figure 3A and E). This suggests that a physical binding between Numb and E-cad is necessary for the proper lateral membrane localization of the latter. To interrogate whether Numb is necessary for the localization of E-cad, we generated a stable clone of MDCKII in which Numb expression was ablated by a specific shRNA (Supplementary Figure S2A and B). Confocal immunofluorescence microscopy revealed that endogenous E-cad localized to the lateral membrane of cell–cell junction as expected. Depletion of Numb by shRNA caused a basal-to-apicolateral membrane translocation of E-cad immunofluorescence (Figure 3B). However, this phenotype was completely reversed by restoration of Numb expression using a construct encoding an shRNA-resistant form of Numb (i.e. srNumb, Figure 3B and F). It should be noted that the effect of Numb knockdown on E-cad localization was less dramatic than observed for the E-cad(3A) mutant, likely due to the effect of residual Numb in the numb-shRNA cells. Alternatively, additional factors besides Numb may be involved in controlling endogenous E-cad localization. As the AJ is linked to the actin cytoskeleton and the reorganization of actin network underpins the formation and maturation of cell–cell contacts (Kametani and Takeichi, 2007; Hartsock and Nelson, 2008), we compared F-actin staining patterns between the control and the numb-shRNA MDCK cells. Numb knockdown led to a significant increase in F-actin staining compared with wt cells or cells expressing srNumb, suggesting that Numb is involved directly or indirectly in regulating actin dynamics in these cells (Figure 3B and F). Furthermore, in both numb-shRNA and srNumb-expressing cells, actin stain overlapped significantly with the E-cad immunofluorescence (Figure 3B). To find out whether other AJ proteins are affected by Numb depletion, we immunostained for β-catenin, a key component of the AJ. As shown in Figure 3C and G, β-catenin underwent a similar basal-to-apicolateral translocation as E-cad in numb-shRNA cells. This defect, however, is rescued, by expression of srNumb in these cells. Together, these data show that Numb is necessary for the proper junctional localization of E-cad and β-catenin in polarized epithelial cells. It should be pointed out that Numb depletion did not affect the formation of TJs for cells polarized on transwell filters as indicated by the apical staining of ZO-1 in numb-shRNA cells (Figure 3D).


Numb regulates cell-cell adhesion and polarity in response to tyrosine kinase signalling.

Wang Z, Sandiford S, Wu C, Li SS - EMBO J. (2009)

Numb knockdown led to aberrant localization of E-cadherin, β-catenin and F-actin. (A) Confocal Z-stack images of E-cad-GFP, E-cad(3A)-GFP and E-cad(3F)-GFP in transfected MDCKII cells, respectively. The panel labelled as ‘apical' corresponds to an x/y section image (x/y focal plane) taken at the subapical region of the monolayer of cells (at 4 μm below the apical surface) and the panel labelled as ‘basal' corresponds to the basolateral region (a section at 4 μm above the basal surface), respectively. The corresponding x/z focal plane image was shown below each x/y image set. Apical is at the top, whereas basal is at the bottom. The same convention was used throughout. Size bars represent 10 μm. GFP fluorescence is in green and nuclei are stained in blue with DAPI. (B) Confocal Z-stack images of E-cadherin (green) and F-actin (red) in the control MDCKII cells versus in the numb-shRNA or sr-Numb cell lines. In each panel, the E-cadherin immunostaining (green) images were shown on the left, F-actin staining (red) images in the middle and the overlaid images on the right. The corresponding x/z focal plane images were shown below the x/y images. (C) Confocal Z-stack images of β-catenin immunofluorescence in the control MDCKII cells versus in the numb-shRNA or sr-Numb cells shown as either x/y (top) or x/z (bottom) section. (D) Confocal x/z sections showing the apical translocation of E-cadherin in the control MDCKII cells versus in the numb-shRNA or sr-Numb cells. The apical surface was identified by co-staining for ZO-1. (E–G) Quantitative analysis of immunofluorescence images shown in (A), (B) and (C), respectively. Values shown are changes in fold of apical over basal immunofluorescence. *P<0.001, n=3 in (E); *P<0.001, **P<0.05, n=5 in (F); *P<0.01, n=3 in (G). The error bars represent mean±s.d.
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f3: Numb knockdown led to aberrant localization of E-cadherin, β-catenin and F-actin. (A) Confocal Z-stack images of E-cad-GFP, E-cad(3A)-GFP and E-cad(3F)-GFP in transfected MDCKII cells, respectively. The panel labelled as ‘apical' corresponds to an x/y section image (x/y focal plane) taken at the subapical region of the monolayer of cells (at 4 μm below the apical surface) and the panel labelled as ‘basal' corresponds to the basolateral region (a section at 4 μm above the basal surface), respectively. The corresponding x/z focal plane image was shown below each x/y image set. Apical is at the top, whereas basal is at the bottom. The same convention was used throughout. Size bars represent 10 μm. GFP fluorescence is in green and nuclei are stained in blue with DAPI. (B) Confocal Z-stack images of E-cadherin (green) and F-actin (red) in the control MDCKII cells versus in the numb-shRNA or sr-Numb cell lines. In each panel, the E-cadherin immunostaining (green) images were shown on the left, F-actin staining (red) images in the middle and the overlaid images on the right. The corresponding x/z focal plane images were shown below the x/y images. (C) Confocal Z-stack images of β-catenin immunofluorescence in the control MDCKII cells versus in the numb-shRNA or sr-Numb cells shown as either x/y (top) or x/z (bottom) section. (D) Confocal x/z sections showing the apical translocation of E-cadherin in the control MDCKII cells versus in the numb-shRNA or sr-Numb cells. The apical surface was identified by co-staining for ZO-1. (E–G) Quantitative analysis of immunofluorescence images shown in (A), (B) and (C), respectively. Values shown are changes in fold of apical over basal immunofluorescence. *P<0.001, n=3 in (E); *P<0.001, **P<0.05, n=5 in (F); *P<0.01, n=3 in (G). The error bars represent mean±s.d.
Mentions: To investigate whether the interaction between Numb and E-cad is required for proper junctional localization of the cadherin complex, we transfected MDCKII cells with expression constructs for E-cad-GFP, E-cad-(3F)-GFP and E-cad-(3A)-GFP, respectively. As expected, E-cad-GFP localized to cell–cell junctions in polarized cells. Of the two E-cad mutants examined, E-cad-(3F)-GFP localized to the lateral membrane domain as did the wt protein. In contrast, E-cad-(3A)-GFP, which was deficient in Numb binding, was detected at the apical region of the cell–cell junction (Figure 3A and E). This suggests that a physical binding between Numb and E-cad is necessary for the proper lateral membrane localization of the latter. To interrogate whether Numb is necessary for the localization of E-cad, we generated a stable clone of MDCKII in which Numb expression was ablated by a specific shRNA (Supplementary Figure S2A and B). Confocal immunofluorescence microscopy revealed that endogenous E-cad localized to the lateral membrane of cell–cell junction as expected. Depletion of Numb by shRNA caused a basal-to-apicolateral membrane translocation of E-cad immunofluorescence (Figure 3B). However, this phenotype was completely reversed by restoration of Numb expression using a construct encoding an shRNA-resistant form of Numb (i.e. srNumb, Figure 3B and F). It should be noted that the effect of Numb knockdown on E-cad localization was less dramatic than observed for the E-cad(3A) mutant, likely due to the effect of residual Numb in the numb-shRNA cells. Alternatively, additional factors besides Numb may be involved in controlling endogenous E-cad localization. As the AJ is linked to the actin cytoskeleton and the reorganization of actin network underpins the formation and maturation of cell–cell contacts (Kametani and Takeichi, 2007; Hartsock and Nelson, 2008), we compared F-actin staining patterns between the control and the numb-shRNA MDCK cells. Numb knockdown led to a significant increase in F-actin staining compared with wt cells or cells expressing srNumb, suggesting that Numb is involved directly or indirectly in regulating actin dynamics in these cells (Figure 3B and F). Furthermore, in both numb-shRNA and srNumb-expressing cells, actin stain overlapped significantly with the E-cad immunofluorescence (Figure 3B). To find out whether other AJ proteins are affected by Numb depletion, we immunostained for β-catenin, a key component of the AJ. As shown in Figure 3C and G, β-catenin underwent a similar basal-to-apicolateral translocation as E-cad in numb-shRNA cells. This defect, however, is rescued, by expression of srNumb in these cells. Together, these data show that Numb is necessary for the proper junctional localization of E-cad and β-catenin in polarized epithelial cells. It should be pointed out that Numb depletion did not affect the formation of TJs for cells polarized on transwell filters as indicated by the apical staining of ZO-1 in numb-shRNA cells (Figure 3D).

Bottom Line: Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT.Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration.Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and the Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada.

ABSTRACT
Epithelial-mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E-cadherin (E-cad) through its phosphotyrosine-binding domain (PTB) and thereby regulates the localization of E-cad to the lateral domain of epithelial cell-cell junction. Moreover, Numb engages the polarity complex Par3-aPKC-Par6 by binding to Par3 in polarized Madin-Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E-cad and Par3 and associates preferably with aPKC-Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and beta-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell-cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell-cell adhesion and a sensor of HGF signalling or Src activity during EMT.

Show MeSH
Related in: MedlinePlus