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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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Related in: MedlinePlus

Numb knockdown reduced cell–cell adhesion and promoted cell migration. (A) Decreased adhesiveness in cells lacking Numb. Aggregation assay showed weaker cell–cell adhesion for the numb-shRNA cell line compared with MDCKII. However, this defect was rescued by re-expression of sr-Numb. The scale bar is set at 25 μm. (B) Quantification of cell aggregation rate shown in (A). Aggregating rate=(No−Nt)/No, where No is the number of (non-aggregated) cells used in the assay and Nt denotes the total number of particles (non-aggregated and aggregated cells) in the sample after pipetting. Data shown are representative of four independent experiments. (C) Numb knockdown promoted wound healing in MDCKII cells. Images were captured immediately after wounding or at 6 or 12 h afterwards. The scale bar is set at 50 μm. (D) Rate of wound healing for MDCKII, numb-shRNA or sr-Numb cells. The rate shown was based on 10 independent measurements. The error bars represent mean±s.d. (E) Rate of cell migration measured using a Boyden chamber. Data shown were based on six independent assays. The error bars represent mean±s.d. (F) Comparison of the rate of proliferation for the MDCKII, numb-shRNA and sr-Numb cells. Data shown represent three independent sets of experiments. The error bars represent mean±s.d.
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f6: Numb knockdown reduced cell–cell adhesion and promoted cell migration. (A) Decreased adhesiveness in cells lacking Numb. Aggregation assay showed weaker cell–cell adhesion for the numb-shRNA cell line compared with MDCKII. However, this defect was rescued by re-expression of sr-Numb. The scale bar is set at 25 μm. (B) Quantification of cell aggregation rate shown in (A). Aggregating rate=(No−Nt)/No, where No is the number of (non-aggregated) cells used in the assay and Nt denotes the total number of particles (non-aggregated and aggregated cells) in the sample after pipetting. Data shown are representative of four independent experiments. (C) Numb knockdown promoted wound healing in MDCKII cells. Images were captured immediately after wounding or at 6 or 12 h afterwards. The scale bar is set at 50 μm. (D) Rate of wound healing for MDCKII, numb-shRNA or sr-Numb cells. The rate shown was based on 10 independent measurements. The error bars represent mean±s.d. (E) Rate of cell migration measured using a Boyden chamber. Data shown were based on six independent assays. The error bars represent mean±s.d. (F) Comparison of the rate of proliferation for the MDCKII, numb-shRNA and sr-Numb cells. Data shown represent three independent sets of experiments. The error bars represent mean±s.d.

Mentions: As the above biochemical and immunofluorescence data identified a critical role for Numb in the localization of both the cadherin and the Par complexes, we speculated that lack of Numb might contribute to weakened cellular junctions, reduced cell–cell adhesion, and increased cell motility. To test this hypothesis, we carried out cell aggregation and migration assays on MDCKII, numb-shRNA and sr-Numb cells. Although the control and sr-Numb-expressing cells formed aggregates that were not easily dispersed by pipetting, the numb-shRNA cells dissociate readily with the same manipulations (Figure 6A and B). To interrogate whether Numb has a function in cell migration, we performed wounding assays on these cells, respectively. Compared with the control, Numb-knockdown promoted wound healing significantly (Figure 6C and D). Boyden chamber assays also showed a marked increase in the rate of migration for the numb-shRNA cells. Importantly, restoration of Numb expression in the sr-Numb cells returned the migration rate to the same level as the wt cells (Figure 6E). To gauge the effect of cell proliferation on promoting cell migration, we measured the growth rate for each cell type. Numb-knockdown led to increased cell proliferation and re-expression of sr-Numb brought the cells back to their normal rate of growth (Figure 6F). It should be noted, however, that the elevated rate of cell proliferation was insufficient to account for the dramatic increase in the rate of cell migration (i.e. at 48 h post-seeding; Figure 6E and F). Therefore, it is likely that the depletion of Numb had a dual effect on MDCKII cells: increased cell proliferation and enhanced cell migration because of weakened cell–cell adhesion.


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 reduced cell–cell adhesion and promoted cell migration. (A) Decreased adhesiveness in cells lacking Numb. Aggregation assay showed weaker cell–cell adhesion for the numb-shRNA cell line compared with MDCKII. However, this defect was rescued by re-expression of sr-Numb. The scale bar is set at 25 μm. (B) Quantification of cell aggregation rate shown in (A). Aggregating rate=(No−Nt)/No, where No is the number of (non-aggregated) cells used in the assay and Nt denotes the total number of particles (non-aggregated and aggregated cells) in the sample after pipetting. Data shown are representative of four independent experiments. (C) Numb knockdown promoted wound healing in MDCKII cells. Images were captured immediately after wounding or at 6 or 12 h afterwards. The scale bar is set at 50 μm. (D) Rate of wound healing for MDCKII, numb-shRNA or sr-Numb cells. The rate shown was based on 10 independent measurements. The error bars represent mean±s.d. (E) Rate of cell migration measured using a Boyden chamber. Data shown were based on six independent assays. The error bars represent mean±s.d. (F) Comparison of the rate of proliferation for the MDCKII, numb-shRNA and sr-Numb cells. Data shown represent three independent sets of experiments. The error bars represent mean±s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2712596&req=5

f6: Numb knockdown reduced cell–cell adhesion and promoted cell migration. (A) Decreased adhesiveness in cells lacking Numb. Aggregation assay showed weaker cell–cell adhesion for the numb-shRNA cell line compared with MDCKII. However, this defect was rescued by re-expression of sr-Numb. The scale bar is set at 25 μm. (B) Quantification of cell aggregation rate shown in (A). Aggregating rate=(No−Nt)/No, where No is the number of (non-aggregated) cells used in the assay and Nt denotes the total number of particles (non-aggregated and aggregated cells) in the sample after pipetting. Data shown are representative of four independent experiments. (C) Numb knockdown promoted wound healing in MDCKII cells. Images were captured immediately after wounding or at 6 or 12 h afterwards. The scale bar is set at 50 μm. (D) Rate of wound healing for MDCKII, numb-shRNA or sr-Numb cells. The rate shown was based on 10 independent measurements. The error bars represent mean±s.d. (E) Rate of cell migration measured using a Boyden chamber. Data shown were based on six independent assays. The error bars represent mean±s.d. (F) Comparison of the rate of proliferation for the MDCKII, numb-shRNA and sr-Numb cells. Data shown represent three independent sets of experiments. The error bars represent mean±s.d.
Mentions: As the above biochemical and immunofluorescence data identified a critical role for Numb in the localization of both the cadherin and the Par complexes, we speculated that lack of Numb might contribute to weakened cellular junctions, reduced cell–cell adhesion, and increased cell motility. To test this hypothesis, we carried out cell aggregation and migration assays on MDCKII, numb-shRNA and sr-Numb cells. Although the control and sr-Numb-expressing cells formed aggregates that were not easily dispersed by pipetting, the numb-shRNA cells dissociate readily with the same manipulations (Figure 6A and B). To interrogate whether Numb has a function in cell migration, we performed wounding assays on these cells, respectively. Compared with the control, Numb-knockdown promoted wound healing significantly (Figure 6C and D). Boyden chamber assays also showed a marked increase in the rate of migration for the numb-shRNA cells. Importantly, restoration of Numb expression in the sr-Numb cells returned the migration rate to the same level as the wt cells (Figure 6E). To gauge the effect of cell proliferation on promoting cell migration, we measured the growth rate for each cell type. Numb-knockdown led to increased cell proliferation and re-expression of sr-Numb brought the cells back to their normal rate of growth (Figure 6F). It should be noted, however, that the elevated rate of cell proliferation was insufficient to account for the dramatic increase in the rate of cell migration (i.e. at 48 h post-seeding; Figure 6E and F). Therefore, it is likely that the depletion of Numb had a dual effect on MDCKII cells: increased cell proliferation and enhanced cell migration because of weakened cell–cell adhesion.

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