Numb partially overlaps with E-cadherin components at cell–cell contacts. (A) Epithelial MCF7 cells were stained with Numb and cadherin components. The insets in the left panels are magnified in the right panels. Bar, 10 μm. (B) Schematic representation of Numb. The domain organization of Numb and its fragments is represented. PTB, phosphotyrosine-binding; PRR, proline-rich region; DPF, α-adaptin-binding motif; NPF, Eps15-binding motif. (C) GFP-fused Numb was expressed in MCF7 cells and stained with E-cadherin. The insets in the left panels are magnified in the right panels. Bar, 10 μm. All results are representative of more than three experiments.

Numb is required for effective E-cadherin internalization. (A) Blocking of the antibodies remaining at the cell surface. See Materials and Methods for details. Bar, 10 μm. (B) Internalization of antibody-labeled E-cadherin in MCF7 cells. See Materials and Methods for details. Bar, 5 μm. (C) Internalization of E-cadherin in MCF7 cells transfected with the indicated siRNA. Inverted images are shown here (internalized E-cadherin is evident as black dots). The broken lines represent the cell margins, and the asterisks indicate cell-free space. The images represent the projection views of several confocal sections from the apical to basal membrane. Bar, 10 μm. (D) Quantification of (C) as described in Materials and Methods. All results are representative of more than three experiments. *, **, ***, **** = P < 0.001.

Numb is essential for proper cell adhesion and basolateral localization of E-cadherin and p120 in polarized MDCKII cells. (A) Representation of various MCF7 colonies. The colonies were classified as compact (>90% of cells in the colony have cell–cell contacts), loose (50–90% cell contacts), or scattered (<50% cell contacts). Bar, 40 μm. (B) Quantification of the colony classification in MCF7 cells transfected with indicated siRNA. Knockdown of Numb or endocytic proteins increased the fraction of tightly associated colonies. (C) The model epithelial cell shows that “apical” corresponds to a 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 “basal” corresponds to the basolateral region (a section 4 μm above the basal surface). The corresponding x/z focal plane image is shown to the left of each x/y image set. Apical is at the top, whereas basal is at the bottom. The same convention is used throughout. Polarized MDCKII cells were stained with Numb, E-cadherin, and p120. Bar, 10 μm. (D) MDCKII cells transfected with the indicated siRNA were stained with E-cadherin and phalloidin. Bar, 10 μm. (E) MDCK 3D cysts were stained on day 3 with Numb, E-cadherin, p120, and phalloidin. Phalloidin was used as an apical marker. Bar, 10 μm. (F) MDCK 3D cysts transfected with indicated siRNA were stained on day 3. Bar, 10 μm. All results are representative of more than three experiments.

aPKC regulates Numb localization and the interaction of Numb with E-cadherin and p120 by phosphorylation. (A) Schematic representation of the phosphomimetic and phosphodeficient Numb mutants. (B) Localization of phosphorylated Numb in polarized MDCKII cells. Bar, 10 μm. (C) Purified GST proteins were incubated with recombinant aPKCζ in the presence or absence of ATP and then mixed with the MCF7 cell lysate. (D) Immunoprecipitation of Numb mutants from transfected cells. The asterisk indicates a nonspecific band.

Numb controls E-cadherin endocytosis through p120 with aPKC, and Numb is involved in polarized E-cadherin trafficking. (A) Schematic representation of E-cadherin. E-cadherin consists of a signal peptide, extracellular cadherin domains 1–5 (EC1-5), transmembrane domain (TMD), and a cytoplasmic tail that contains a juxtamembrane domain (JMD) and cytoplasmic β-catenin-binding domain (CBD). In the E-cadherin cytoplasmic region, several motifs are prominent. The dileucine motif binds to cargo proteins, such as α-adaptin. NVYYY contains phosphorylated tyrosine residues that bind to Numb and Hakai. GGG is a core component that binds to p120 (Miyashita and Ozawa, 2007; Ishiyama et al., 2010). (B) The epithelial cell is highly polarized with an apical surface and basal membrane. E-cadherin composes adherens junctions and maintains epithelial polarity. E-cadherin endocytosis is essential for maintaining adherens junctions and cell polarity. By interacting with E-cadherin and p120, Numb plays a significant role in E-cadherin endocytosis, and its interactions are regulated through Numb phosphorylation by aPKC. Phosphorylated Numb cannot interact with E-cadherin and p120 and exists in the cytosol. There are two possible ways that Numb mediates E-cadherin endocytosis. First, Numb binds to p120 and mediates internalization of the entire E-cadherin complex containing p120. Alternatively, Numb binds directly to the NVYYY motif in E-cadherin, which is usually masked by p120, and internalizes p120-unbound E-cadherin. With these mechanisms, Numb controls E-cadherin endocytosis through p120 and aPKC. (C) Polarized E-cadherin trafficking by Numb establishes adherens junctions and epithelial cell polarity. The PAR complex with Cdc42, which localizes to tight and adherens junctions, has also recently emerged as an important regulator of endocytosis at adherens junctions (Balklava et al., 2007; Georgiou et al., 2008; Leibfried et al., 2008). Numb is phosphorylated by aPKC and thus loses its ability to mediate E-cadherin endocytosis. As a result, aPKC prevents E-cadherin endocytosis and may stabilize cell–cell adhesion at adherens junctions. This polarized endocytosis could account for the restricted distribution of E-cadherin at the lateral membrane.

Numb directly binds to E-cadherin and p120. (A) MCF7 cells were lysed, and the supernatants were incubated with the indicated antibodies. The immunocomplexes were subjected to immunoblot analysis with the indicated antibodies. CHC, clathrin-heavy-chain. (B) Beads coated with purified proteins were incubated with the MCF7 cell lysate, followed by immunoblot analysis with the indicated antibodies. The GST fusion proteins were visualized by Coomassie Brilliant Blue (CBB) staining. The asterisks indicate each intact band. α-Adaptin was used as a positive control. (C) Purified GST-fused p120 or E-cadherin cytoplasmic region was incubated with the MCF7 lysate, followed by immunoblot analysis with the indicated antibodies. The GST fusion proteins were visualized by CBB staining. The asterisks indicate each intact band. (D) Purified His-fused Numb proteins were mixed with the beads coated with purified GST-p120 and GST-E-cadherin cytoplasmic region. All results are representative of more than three experiments.

Numb is responsible for E-cadherin endocytosis. (A) Endocytosis of E-cadherin was attenuated in the Numb- or endocytic protein-depleted MCF7 cells. (B) Quantification of internalized E-cadherin. The ratio of internalized to total E-cadherin after a 30-min incubation was set to 1. (C) Precipitation of internalized E-cadherin after acid wash to strip the antibody remaining at the cell surface. Numb and other endocytic proteins were coprecipitated with internalized E-cadherin. All results are representative of more than three experiments.

Numb controls E-cadherin endocytosis by interacting with the C-terminal region of p120. (A) Schematic representation of p120. The domain organization of p120 and its fragments is represented. (B) Immunoprecipitation of p120 fragments from the transfected MCF7 cells. The asterisks indicate each intact band. (C) Immunostaining of GFP-p120 mutants in polarized MDCKII cells. The insets in the panels are magnified in the right panels. Bar, 10 μm. (D) Immunoprecipitation of Numb from MCF7 cells expressing indicated GFP fusions. The asterisks indicate each GFP fusions. (E) Overexpression and rescue experiments with p120 fragments and mutants in E-cadherin endocytosis. MCF7 cells were transfected with the indicated GFP-p120 mutants, siRNA, and sr-p120 constructs. The ratio of total intensity of internalized to Cell Area (μm²) of scramble + GFP-GST was set to 1. *, P < 0.05; **, P < 0.01. Expression level of each construct was biochemically measured and displayed in Supplemental Figure 7. All results are representative of more than three 3 experiments.

aPKC attenuates E-cadherin endocytosis. (A) Knockdown of aPKC in MCF7 cells by siRNA. Depletion of aPKC impaired Numb phosphorylation. (B) Quantification of (A). (C) Inhibition of aPKC accelerated E-cadherin endocytosis in MCF7 cells. The cells were either transfected with aPKC-targeting siRNA or treated with an aPKC inhibitor (aPKC pseudosubstrate) and subjected to an endocytosis assay for E-cadherin as in Figure 3. *, **, P < 0.001. (D) Categorization of colonies in the aPKC-depleted MCF7 cells. (E) Rescue experiments with Numb fragments and mutants in E-cadherin endocytosis. MCF7 cells were transfected with the indicated siRNA and siRNA-resistant (sr) Numb constructs. The ratio of total intensity of internalized to cell area (μm²) of scramble + GFP-GST was set to 1. *, P < 0.01. Expression level of each construct was biochemically measured and displayed in Supplemental Figure 7. All results are representative of more than three experiments.