Research ArticleBIOPHYSICS

Dynamic spectrin/ankyrin-G microdomains promote lateral membrane assembly by opposing endocytosis

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Science Advances  11 Sep 2015:
Vol. 1, no. 8, e1500301
DOI: 10.1126/sciadv.1500301
  • Fig. 1 Ankyrin-G/βII-spectrin microdomains sample the entire lateral membrane through microtubule-dependent localized movement.

    (A) Schematic depicting the en face visualization of the columnar epithelial cell lateral membrane. Representative deconvolved en face (right) image from MDCK cells stained with antibodies against endogenous ankyrin-G. Scale bar, 2.5 μm. Region of interest depicts size of area analyzed in (B). (B) Representative deconvolved en face confocal images of live MDCK cells transfected with ankyrin-G–GFP shown at time 0 (green) or 5 min (red) for control cells (top) or cells treated for 3 hours with nocodazole (Nocod.) (100 ng/ml) (second row) or 1 μM latrunculin A (LatA) (third row). Cells fixed with paraformaldehyde (bottom) were used as an imaging control. Summed coverage at 0 and 5 min shown on the right. Scale bar, 1 μm. (C) Quantification of percent lateral membrane coverage from cells in (B). One-way analysis of variance (ANOVA) followed by Tukey post hoc test; *P < 0.05 compared to control, n = 4 to 9 cells per condition.

  • Fig. 2 Ankyrin-G co-patterns with βII-spectrin in lateral membrane microdomains that are distinct from clathrin and its cargoes.

    (A) Representative deconvolved en face confocal images of MDCK cell lateral membranes stained for endogenous ankyrin-G (AnkG) (green). βII-spectrin–GFP, βII-spectrin Y1874A–GFP, clathrin heavy chain (CHC), LDLR (LDLR-GFP), and transferrin receptor–yellow fluorescent protein (TfR-YFP) are shown in red. Positive displacement from the mean colocalization is shown in yellow. Merged image is on the right. Scale bars, 2.5 μm. (B) Quantification of Pearson coefficient of colocalization from (A).

  • Fig. 3 Disruption of ankyrin-G/βII-spectrin interaction or silencing of ankyrin-G increases bulk endocytosis from the epithelial lateral membrane.

    (A) Schematic for tunable expression of a dominant-negative fragment consisting of the ankyrin-binding domain (ABD) of βII-spectrin that disrupts the interaction between βII-spectrin and ankyrin-G. (B) (Left) Dose-response curve for increase in DsRed expression as determined by Western blot in response to 24-hour addition of indicated doses of Shield-1. (Right) Time course of Western blot levels of FKBP-DsRed-ABD in cells treated with 1 μM Shield-1. a.u., arbitrary units. (C) Representative X-Y confocal images of FITC-transferrin uptake (top, green) from the basolateral membrane of MDCK cells in the absence (left) or presence (middle) of the ankyrin-binding domain dominant negative, or in the presence of a control that lacks ankyrin-binding activity (right). DsRed-tagged dominant-negative expression is shown on the bottom (white). Scale bars, 20 μm. Cell borders are shown in white. WT, wild type. (D) Quantification of FITC-transferrin uptake from (A) in the absence (white) or presence (black) of the ankyrin-binding domain dominant negative, or in the presence of a control that lacks ankyrin-binding activity (red). One-way ANOVA, Tukey post hoc test; *P < 0.05, n = 86 to 120.

  • Fig. 4 Depletion of ankyrin-G or βII-spectrin causes a marked increase in lipid uptake from the lateral membrane, which is reversed by dynasore treatment.

    (A) Representative X-Y images of MDCK cells demonstrating internalization of CellMask (red) in the presence of luciferase (Luc) shRNA (left), ankyrin-G shRNA (middle), or ankyrin-G shRNA plus 80 μM dynasore (right). Arrowheads mark internalized vesicles. Higher magnification of yellow region of interest (ROI) is shown below. Scale bar, 10 μm. (B) Representative X-Y images of MDCK cells demonstrating internalization of CellMask (red) in the presence of luciferase shRNA (left), βII-spectrin shRNA (middle), or βII-spectrin plus 80 μM dynasore (right). Arrowheads mark internalized vesicles. Higher magnification of yellow region of interest is shown below. Scale bar, 10 μm.

  • Fig. 5 Silencing of clathrin heavy chain in ankyrin-G–depleted cells restores lateral membrane height.

    (A) Representative X-Z projections of confocal stacks of MDCK cells expressing shRNA to luciferase (Luc, top), ankyrin-G (second row), clathrin heavy chain (CHC, third row), or both ankyrin-G and CHC (bottom) stained for E-cadherin (green). Arrowheads mark apical mislocalization of E-cadherin. Scale bar, 10 μm. (B) Quantification of lateral membrane height from (A). One-way ANOVA followed by Tukey post hoc test; *P < 0.05 compared to Luc shRNA. n = 60 to 65. (C) Western blot of MDCK cells stably expressing inducible shRNA against ankyrin-G, CHC, both ankyrin-G and CHC, or luciferase as a control. Cells were preinduced with doxycycline for 24 hours, trypsinized, and replated for an additional 24 hours in medium containing doxycycline. Ankyrin-G shown on top, with loss of the 210- and 150-kD isoforms. The 120/110-kD isoforms, which are not targeted by this shRNA sequence, are spared. CHC levels are shown on bottom.

  • Fig. 6 Loss of lateral membrane caused by silencing of ankyrin-G or βII-spectrin is reversed by inhibiting endocytosis.

    (A) Representative X-Z projections of confocal stacks of MDCK cells expressing shRNA to ankyrin-G or βII-spectrin, with or without treatment with the endocytosis inhibitor dynasore. Cells stained for E-cadherin (green). Scale bar, 10 μm. (B) Quantification of lateral membrane height from (A). One-way ANOVA followed by Tukey post hoc test; *P < 0.05 compared to without dynasore. n = 50 to 75.

  • Fig. 7 Model of lateral membrane domain organization.

    (A) Representations of relative sizes of lipid rafts, mesoscale domains, the spectrin/ankyrin complex, junctional actin, and a clathrin coat. Note: size of junctional actin based on size of erythrocyte junctional actin. (B) Schematic representation of an ankyrin-G/spectrin lattice overlaid onto an image of spectrin/ankyrin-G lateral membrane domains (white areas). Note that where clathrin pits are in close proximity, they will appear as a fused patch at the 250-nm resolution of the light microscope. The remaining plasma membrane is indicated by gray stipple. Scale bar, 250 nm. The extended configuration of spectrin tetramers is based on the negative-stained images or erythrocytes obtained in low ionic strength (8). However, platinum replica imaging of native erythrocyte membranes indicates that spectrin exists in a much more compressed configuration with correspondingly smaller gaps (19).

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/8/e1500301/DC1

    Fig. S1. Ankyrin-G patterns into micrometer-scale domains in mouse kidney epithelial lateral membranes.

    Fig. S2. Latrunculin A and nocodazole treatments effectively depolymerize the MDCK microtubule and actin cytoskeleton, respectively.

    Fig. S3. Stabilization of microtubules or actin does not affect dynamics of ankyrin-G microdomains or lateral membrane coverage.

    Fig. S4. Microtubules colocalize with ankyrin-G microdomains at discrete sites and are required for lateral membrane biogenesis.

    Movie S1. Live imaging of ankyrin-G–GFP on the lateral membrane of control MDCK cells.

    Movie S2. Live imaging of ankyrin-G–GFP on the lateral membrane of paraformaldehyde-fixed MDCK cells.

    Movie S3. Live imaging of βII-spectrin–GFP on the lateral membrane of MDCK cells treated with latrunculin A.

    Movie S4. Live imaging of ankyrin-G–GFP on the lateral membrane of MDCK cells treated with latrunculin A.

    Movie S5. Live imaging of ankyrin-G–GFP on the lateral membrane of MDCK cells treated with nocodazole.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Ankyrin-G patterns into micrometer-scale domains in mouse kidney epithelial lateral membranes.
    • Fig. S2. Latrunculin A and nocodazole treatments effectively depolymerize the MDCK microtubule and actin cytoskeleton, respectively.
    • Fig. S3. Stabilization of microtubules or actin does not affect dynamics of ankyrin-G microdomains or lateral membrane coverage.
    • Fig. S4. Microtubules colocalize with ankyrin-G microdomains at discrete sites and are required for lateral membrane biogenesis.
    • Legends for movies S1 to S5

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Live imaging of ankyrin-G–GFP on the lateral membrane of control MDCK cells.
    • Movie S2 (.avi format). Live imaging of ankyrin-G–GFP on the lateral membrane of paraformaldehyde-fixed MDCK cells.
    • Movie S3 (.avi format). Live imaging of βII-spectrin–GFP on the lateral membrane of MDCK cells treated with latrunculin A.
    • Movie S4 (.avi format). Live imaging of ankyrin-G–GFP on the lateral membrane of MDCK cells treated with latrunculin A.
    • Movie S5 (.avi format). Live imaging of ankyrin-G–GFP on the lateral membrane of MDCK cells treated with nocodazole.

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