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. 2013 May 17;288(20):14018-31.
doi: 10.1074/jbc.M113.454439. Epub 2013 Mar 25.

E-cadherin Polarity Is Determined by a Multifunction Motif Mediating Lateral Membrane Retention Through ankyrin-G and Apical-Lateral Transcytosis Through Clathrin

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Free PMC article

E-cadherin Polarity Is Determined by a Multifunction Motif Mediating Lateral Membrane Retention Through ankyrin-G and Apical-Lateral Transcytosis Through Clathrin

Paul M Jenkins et al. J Biol Chem. .
Free PMC article

Abstract

We report a highly conserved motif in the E-cadherin juxtamembrane domain that determines apical-lateral polarity by conferring both restricted mobility at the lateral membrane and transcytosis of apically mis-sorted protein to the lateral membrane. Mutations causing either increased lateral membrane mobility or loss of apical-lateral transcytosis result in partial mis-sorting of E-cadherin in Madin-Darby canine kidney cells. However, loss of both activities results in complete loss of polarity. We present evidence that residues required for restricted mobility mediate retention at the lateral membrane through interaction with ankyrin-G, whereas dileucine residues conferring apical-lateral transcytosis act through a clathrin-dependent process and function in an editing pathway. Ankyrin-G interaction with E-cadherin is abolished by the same mutations resulting in increased E-cadherin mobility. Clathrin heavy chain knockdown and dileucine mutation of E-cadherin both cause the same partial loss of polarity of E-cadherin. Moreover, clathrin knockdown causes no further change in polarity of E-cadherin with dileucine mutation but does completely randomize E-cadherin mutants lacking ankyrin-binding. Dileucine mutation, but not loss of ankyrin binding, prevented transcytosis of apically mis-sorted E-cadherin to the lateral membrane. Finally, neurofascin, which binds ankyrin but lacks dileucine residues, exhibited partial apical-lateral polarity that was abolished by mutation of its ankyrin-binding site but was not affected by clathrin knockdown. The polarity motif thus integrates complementary activities of lateral membrane retention through ankyrin-G and apical-lateral transcytosis of mis-localized protein through clathrin. Together, the combination of retention and editing function to ensure a high fidelity steady state localization of E-cadherin at the lateral membrane.

Keywords: Cell Adhesion; Cell Biology; Cell Junctions; Cell Polarity; Molecular Cell Biology.

Figures

FIGURE 1.
FIGURE 1.
Selective knock-out of large spliced variants of ankyrin-G in mice results in reduced lateral membrane height but retention of E-cadherin. A, shown is a schematic depiction of the Cre-mediated ankyrin-G null mouse strategy. LoxP sites (green), FRT sites (red), and a PGK-neo cassette (white) were inserted on either side of exons 22 and 23 of mouse ANK3. FLP-mediated recombination was used to remove the PGK-Neo cassette. Cre-mediated excision of exons 22–23 was performed by crossing the floxed mice with the β-actin promoter-driven Cre mouse line. B, the total number of ankyrin-G pups born based on genotype (n = 138 for +/+; n = 259 for +/−; n = 3 for −/−). C, left, shown is Ponceau staining of membranes from wild-type (+/+) or ankyrin-G-null (−/−) littermates from p1 brains or kidneys. Right, shown are Western blots (IB) of wild type (+/+) or ankyrin-G-null (−/−) littermates from p1 brains or kidneys using an antibody that recognized the ankyrin-G C terminus. D, shown are representative images of neonatal mouse kidney collecting duct epithelial cells and trachea epithelial cells stained with anti-E-cadherin to mark the lateral membrane (green) and anti-ankyrin-G (red). Bars represent 10 μm. E, shown is quantification of neonatal mouse kidney collecting duct epithelial and trachea epithelial lateral membrane height from wild-type (+/+) or ankyrin-G-null (−/−) littermates. *, p < 0.0001 compared with wild-type (Student's t test, n = 66 for wild-type collecting duct and 84 for ankyrin-G-null collecting duct; n = 33 for wild-type trachea and 26 for ankyrin-G-null trachea).
FIGURE 2.
FIGURE 2.
Lateral membrane biogenesis in MDCK cells requires ankyrin-G but not clathrin. A, shown are representative XZ projections of MDCK cells stably expressing inducible shRNA against ankyrin-G (left), luciferase (middle), or clathrin (right). Cells were plated at high confluence 2 days after induction of shRNA expression and fixed at the indicated times. E-cadherin, marking the height of the lateral membrane, is shown in green. The arrowhead marks apically mislocalized E-cadherin. Bars represent 5 μm. B, shown are representative XY images of MDCK cells stably expressing inducible shRNA against ankyrin-G (left), luciferase (middle), or clathrin (right). Cells were plated at high confluence 2 days after induction of shRNA expression and fixed at the indicated times. Ankyrin-G is shown in green. Bars represent 5 μm. C, shown is the average MDCK lateral membrane height for cells stably expressing inducible shRNA against ankyrin-G (red triangle), luciferase (black circle), or clathrin (blue squares). Average data are shown from 50 cells per time point. D, quantification of Western blot for ankyrin-G (red triangles) or clathrin (blue squares) demonstrate efficient silencing of both ankyrin-G and clathrin. Each time point was normalized to the respective Luc shRNA control. E, shown is a Western blot of ankyrin-G in either ankyrin-G shRNA MDCK cells (left) or Luc shRNA MDCK cells (right) at 8, 16, or 24 h post-plating. GAPDH is used as a loading control. F, shown is a Western blot of clathrin in either clathrin (CHC) shRNA MDCK cells (left) or Luc shRNA MDCK cells (right) at 8, 16, or 24 h post-plating. GAPDH was used as a loading control. G, shown are Western blots of luciferase shRNA or ankyrin-G shRNA MDCK cells. Labeling with ankyrin-G antibodies demonstrates that only the 220-kDa isoform of ankyrin-G is affected by this shRNA. Migration rate (MR) is shown in kDa. Antibodies against clathrin heavy chain demonstrate up-regulation of clathrin in ankyrin-G shRNA cells (1.00 ± 0.07 for Luc shRNA and 1.43 ± 0.14 for ankyrin-G shRNA; n = 3 for each condition; p < 0.05). GAPDH antibodies were used for the loading control.
FIGURE 3.
FIGURE 3.
Identification of a highly conserved, independently evolved motif. A, shown is a schematic depiction of the location of protein interaction sites on cytoplasmic domains of different members of the cadherin superfamily. The transmembrane segment is depicted as a gray box. Conserved putative ankyrin-G binding motif is shown as a yellow box. The p120 site is shown as a red ellipse. The β-catenin binding site is shown as a green ellipse. The plakoglobin-binding site is shown as a magenta box. Cadherin repeats are shown as blue ellipses, and desmoglein repeats are shown as pink circles. B, alignment of the proposed ankyrin-G-binding site shows conservation of critical binding residues (yellow) both across species and throughout other members of the cadherin super family. The dileucine motif is marked in cyan. Conservative substitutions are shown in green. Non-conservative substitutions are shown in gray.
FIGURE 4.
FIGURE 4.
Clathrin and ankyrin-G cooperate in localization of E-cadherin to MDCK cell lateral membranes. A, left, shown is a schematic representation of E-cadherin. Cadherin repeats (light blue), transmembrane segment (gray), ankyrin-G-binding site (yellow), p120 site (red), and β catenin site (green). Ankyrin-G binding sequences are shown below for wild-type (top), Poly(A) (2nd row), LL-AA (3rd row), and Poly(A) + LL-AA mutant (bottom) E-cadherin. Residues critical for ankyrin-G binding are shown in red, and the dileucine motif is highlighted in yellow. Right, membrane recruitment of ankyrin-G in HEK293 cells is shown. Cells were either transfected with ankyrin-G alone (control, white) or plus Wild-type (black), Poly(A) (blue), LL-AA (green), or Poly(A) + LL-AA (red) E-cadherin. Line fluorescence intensity analysis was performed on a single plane, and peak pixel intensity on the membrane was compared with average pixel intensity in cytoplasm. *, p < 0.05 compared with control, Poly(A), and Poly(A) + LL-AA mutant (one way ANOVA followed by the Tukey post hoc test, n = 10 cells for each condition). B, MDCK cells stably expressing an inducible clathrin shRNA were transfected with wild-type (top), Poly(A) (2nd row), LL-AA (3rd row), or Poly(A) + LL-AA (bottom) E-cadherin in the absence (+clathrin (+CHC)) or presence (−clathrin) of doxycycline induction of clathrin shRNA expression. XZ projections shown on the left. The bar represents 10 μm. XY projection of apical planes are shown on the right. C, shown is quantification of the apical mean pixel intensity in the absence (+clathrin) or presence (−clathrin) of doxycycline induction of clathrin shRNA expression. Mean pixel intensity of a three-dimensional region of interest containing the apical membrane was quantified and compared with a three-dimensional region of interest containing the lateral membrane. *, p < 0.05 compared with WT + clathrin. #, p < 0.05 compared with Poly(A) +clathrin (one-way ANOVA followed by the Tukey post hoc test, n = 5–8 cells for each condition). D, shown is a Western blot of MDCK cells stably expressing either luciferase shRNA (Luc, left) or clathrin shRNA (right) demonstrating silencing of clathrin (top) with GAPDH used as a loading control (bottom).
FIGURE 5.
FIGURE 5.
Ankyrin binding activity of neurofascin is necessary but not sufficient for high fidelity apical-basal polarity in MDCK cells. A, MDCK cells stably expressing an inducible clathrin shRNA were transfected with wild-type neurofascin (NF, top) or FIGQY-A neurofascin (NF-YA, bottom) in the absence (+clathrin (CHC)) or presence (−clathrin) of doxycycline induction of clathrin shRNA expression. XZ projections are shown on the left. The bar represents 10 μm. B, shown is an XY projection of apical planes from cells in panel A. C, shown is quantification of apical mean pixel intensity in the absence (+clathrin) or presence (−clathrin) of doxycycline induction of clathrin shRNA expression. Mean pixel intensity of a three-dimensional region of interest containing the apical membrane was quantified and compared with a three-dimensional region of interest containing the lateral membrane. *, p < 0.05 compared with WT + clathrin (one-way ANOVA followed by Tukey post hoc test, n = 5 cells for each condition). D, shown are representative images of membrane recruitment of ankyrin-G-GFP (green) by wild-type V5–186-kDa neurofascin (WT-NF) but not V5-FIGQY-A neurofascin (NF-YA). The V5 signal is shown in red. The merged image is shown on the right. The bar represents 10 μm.
FIGURE 6.
FIGURE 6.
Poly(A) E-cadherin demonstrates increased mobility within the MDCK lateral membrane. A, representative images of FRAP experiments in MDCK cells transiently expressing Wild-type (top), Poly(A) (middle), and LLAA (bottom) E-cadherin-GFP are shown before (Prebleach), immediately after (Bleach), or 100 s after high laser power illumination of the bleach region of interest (yellow square). The bar represents 5 μm. B, kymographs are from cells in panel A. A line was drawn along the lateral membrane, and fluorescence intensity was quantified along that line from time 0 (top) to 300 s (bottom). The bleach region of interest is denoted by a black bar. Time of bleach is marked by an arrowhead. The fluorescence intensity scale is shown below. C, shown are FRAP recovery curves for wild type (black), Poly(A) (blue), LL-AA (green), and Poly(A) + LL-AA mutant (red) E-cadherin-GFP. Curves represent single exponential best fits of the average data from seven cells per condition. D, shown is mobile fraction of FRAP recovery for recovery curves from panel C. *, p < 0.05 compared with WT and LL-AA E-cadherin (one-way ANOVA followed by Tukey post hoc test, n = 7 cells per condition).
FIGURE 7.
FIGURE 7.
Dileucine/alanine mutation abolishes editing of apically mislocalized E-cadherin in MDCK cells. A, a model of apical E-cadherin internalization assay is shown. Confluent, filter-grown MDCK cells were transfected with E-cadherin containing an extracellular V5 epitope and a cytoplasmic GFP epitope. Cells were then labeled on the apical surface with primary antibody (black circle) on ice for 1 h followed by extensive washing. Cells were warmed to 37 °C for the indicated times to allow trafficking of E-cadherin from the apical membrane. Cells were then fixed, permeabilized, and labeled with secondary antibodies tagged with Alexa568 (red circles). B, shown are representative XZ projections of MDCK cells stained with antibodies to endogenous E-cadherin. Total E-cadherin is marked with mouse anti-E-cadherin antibodies (green). Apically labeled antibody to the extracellular epitope of E-cadherin (rat anti-E-cadherin) is shown in red. Bars represent 10 μm. C, shown is quantification of the amount of apical V5 labeling of V5-E-cadherin and mutants normalized to total GFP expression. *, p < 0.05 compared with wild-type. #, p < 0.05 compared with both Poly(A) and LL-AA mutants (one way ANOVA followed by Tukey post hoc test; n = 5 cells per condition). D, shown is the quantification of the fraction of apically labeled E-cadherin remaining on the apical surface. Mean pixel intensity of a three-dimensional region of interest containing the apical membrane was quantified and compared with a three-dimensional region of interest containing the lateral membrane. *, p < 0.05 compared with both wild-type and Poly(A) E-cadherin (two-way ANOVA followed by Tukey post hoc test, n = 5 cells for point). E, shown are representative XZ projections of MDCK cells transfected with wild-type (top), Poly(A) (2nd row), LL-AA (3rd row), or Poly(A) + LL-AA mutant (bottom) E-cadherin warmed to 37 °C for 0 min (left) or 120 min (right). Total E-cadherin-GFP is marked with anti-GFP antibodies and is shown in green (left). Apically labeled anti-V5 to the extracellular epitope of E-cadherin is shown in red (right). Bars represent 5 μm.
FIGURE 8.
FIGURE 8.
Schematic model of complementary roles of the E-cadherin polarity motif in lateral membrane retention and apical membrane retrieval. A, ribbon diagrams based on crystal structures from ankyrin-G, p120, and β-catenin (21, 46, 47) show the prevalence of the solenoid structure in binding of the unstructured cytoplasmic domain of E-cadherin. B, shown is a model for E-cadherin localization. The dileucine motif (red star) engages the clathrin system for internalization of apically mislocalized protein. In contrast, protein localized to the lateral membrane can interact with ankyrin-G (green rectangle) and thus the underlying spectrin/actin cytoskeleton, p120 (blue ellipse), and or β-catenin (yellow rectangle). Interaction with p120, and perhaps ankyrin-G, will sterically hinder access to the dileucine motif and minimize clathrin-mediated endocytosis of properly localized protein. More work remains to be done to determine if ankyrin-G binding precludes binding of p120. Length of domains shown are based on the crystal structure for E-cadherin ectodomain (48) and using 152 amino acids at 3.6 Å per amino acid for cytoplasmic domain.

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