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First published online 1 August 2006
doi: 10.1242/jcs.03027

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Regulation of epithelial wound closure and intercellular adhesion by interaction of AF6 with actin cytoskeleton

Institute of Medical Virology, University of Zurich, Gloriastrasse 30, CH-8006 Zurich, Switzerland

View larger version (18K): [in a new window]   Fig. 1. Domain structure of AF6, afadin and canoe. (A) Domain structure of the three AF6 isoforms (AF6i1, AF6i2 and AF6i3), the two isoforms of its rat homologue afadin (long l-afadin and short s-afadin) and its Drosophila homologue canoe. Aa positions of individual AF6 domains are annotated. RA1,2: Ras-associating domain 1 and 2; FHA: forkhead associated domain; DIL: dilute domain; PDZ: PSD-95-Dlg-1-ZO-1 domain; pro: proline-rich domain. (B) Western blot analysis of HEK293 whole-cell lysate, using anti-AF6 antibody, showing the endogenous AF6i3 protein (left) and the endogenous plus the overexpressed AF6i1 protein (right).

View larger version (39K): [in a new window]   Fig. 2. Interaction of the AF6i3 C-terminus (AF6i3 CT) with F-actin. (Aa) Schematic representation of the AF6i3 CT constructs used in F-actin co-sedimentation assay (GST-tagged) and immunofluorescence (HA-tagged). (Ab,c) F-actin co-sedimentation assay performed with the GST fusion protein of (b) the whole AF6i3 CT (aa 1588-1743) or (c) the two C-terminal fragments (aa 1588-1666 and 1667-1743). F-actin-containing pellet (P) and the supernatant (SN) were analyzed on a denaturing protein gel and stained with Coomassie Blue. GST-fusion proteins or GST alone (arrows) and actin (arrowhead) are annotated. (B) Colocalization of the HA-tagged AF6i3-CT proteins (anti-HA antibody, green) and F-actin (red) in HeLa cells is depicted in merged pictures (right column). Bar, 20 µm. (C) Colocalization of endogenous (AF6; anti-AF6 antibody) and overexpressed full-length AF6i3 protein (myc-AF6i3; anti-myc antibody) (green) with F-actin (red). Bar, 20 µm.

View larger version (80K): [in a new window]   Fig. 3. AF6i3 knockdown in MCF10A cells accelerates epithelial wound closure. (A) (left) Western blot analysis of whole-cell lysate from MCF10A cells stably expressing AF6 shRNA (AF6i3 knockdown) or control shRNA (control). (Right) Immunofluorescence images of cell monolayers. Bar, 20 µm. (B) Light-microscopy images of control and AF6i3-knockdown cells immediately after wounding (0 h; top) and 13 hours later (13 h; bottom). Bar, 0.5 mm. (C) Graphic representation of the covered wound area 13 hours after wounding. Values for ±s.d. were derived from three independent experiments. Significant differences (p) were determined with the paired two-tailed Student's t-test.

View larger version (48K): [in a new window]   Fig. 4. Time-lapse analysis reveals increased directionality of migration and impaired intercellular adhesion in AF6i3-knockdown cells. (A) Time-lapse images showing the wound closure of AF6i3-knockdown and control cells at the time of wounding (0 h) and 10 hours later (see also supplementary material Movies 1 and 2). Red lines mark the boundary of the wound at time point 0 seconds. Bar, 0.1 mm. (B) Representative trajectories of cells during wound healing assay shown in A, followed from time 0 for 10 hours. Bar, 0.1 mm. (C) Graphic representation of the directionality of migration, defined by the D/T ratio, which represents the direct distance from the cell track start to the end point (D) divided by the total path length (T). Data are derived from three independent experiments. (D) Wound margin of migrating AF6i3-knockdown and control cells 10 hours after wounding. green, anti-E-cadherin antibody; red, phalloidin-TRITC. Bar, 10 µm. (E) Western blot analysis of whole-cell lysates during wound healing assay.

View larger version (48K): [in a new window]   Fig. 5. AF6i3 knockdown reduces E-cadherin-dependent adhesion in dissociation assay. (A) Light-microscopy images of cells trypsinised in the presence of 1 mM Ca2+ (top) or 1 mM EDTA (bottom) and dissociated by pipetting. Bar, 0.125 mm. (B) Graphic representation of the extent of cell dissociation, represented by index NTC/NTE. NTC and NTE correspond to the total particle number after treatment of cells in the presence of Ca2+ or EDTA, respectively. Values for ±s.d. were derived from three independent experiments.

View larger version (48K): [in a new window]   Fig. 6. Increased directionality of migration in AF6i3-knockdown cells is due to reduced intercellular adhesion. (A) AF6i3-knockdown and control cells were seeded at a low cell density (top), stimulated with EGF and tracked for two hours, resulting in corresponding trajectories (bottom). Start points of all trajectories were set to the same origin cross-point. Ten representative trajectories per sample are shown. Bar, 0.1 mm. (B) Graphic representation of the directionality of migration for cells shown in A, determined as described for Fig. 4C. Values for ±s.d. were derived from four independent experiments. (C-F) Wound healing assay of control cells in the presence or absence of HECD-1 antibodies, which block E-cadherin trans-interaction. (C) Wound margin 10 hours after wounding. Green, anti-E-cadherin antibody; red, phalloidin-TRITC. Bar, 10 µm. (D) Graphic representation of wound healing assay. Covered wound area 13 hours after wounding of control cell monolayers is depicted. Values for ±s.d. were derived from three independent experiments. (E) Representative trajectories of control cells in the presence or absence of HECD-1 antibody during wound healing assay, tracked for 10 hours. Bar, 0.1 mm. (F) Graphic representation of directionality of migration for cell tracks shown in E, determined as described for Fig. 4C.

View larger version (59K): [in a new window]   Fig. 7. AF6i3 deletion mutants in wound healing assay: a crucial role of the F-actin-binding site. (A) Schematic representation of AF6i1, AF6i3 and AF6i3 deletion mutants, AF6i3RA1 and AF6i3PDZ (top). Expression levels of shRNA-resistant AF6 constructs stably expressed in AF6i3-knockdown cells (bottom). (B) Graphic representation of wound closure 13 hours after wounding of AF6i3-knockdown cell monolayers reconstituted with mutant constructs as indicated. Values for ±s.d. were derived from three independent experiments. (C) Light microscopy pictures of control and AF6i3-knockdown cells without or with reconstitution by AF6i1 and AF6i3 immediately after wounding (0 h) and 13 hours later. Bar, 0.5 mm. (D) Immunofluorescence pictures of confluent cell monolayers of cells shown in C. Bar, 20 µm. (E) Western blot analysis of detergent-soluble supernatant (S) and the insoluble residue (R) of AF6i3-knockdown cell monolayers reconstituted with AF6i1 or AF6i3.

View larger version (43K): [in a new window]   Fig. 8. AF6i3 knockdown results in reduced association of E-cadherin with the actin cytoskeleton and with p120-catenin during wound closure. (A) Western blot analysis showing the Triton-insoluble fraction of AF6i3-knockdown and control cells prior to migration (0 h) and 6 hours after wounding. (B) Graphic representation of western blot analysis shown in A. Values were normalized to the values for control cells. Values for ±s.d. were derived from three independent experiments. (C) Coimmunoprecipitation of E-cadherin and ß-catenin with p120-catenin in AF6i3-knockdown and control cells 6 hours after wounding. Anti-Flag antibody was used as a negative control. (D) Graphic representation of coimmunoprecipitation shown in C. Values were normalized to the values for control cells. Values for ±s.d. were derived from three independent experiments. (E) Wound healing assay of AF6i3-knockdown and control cells in the presence of indicated inhibitors. Graphic representation of covered wound area 13 hours after wounding is depicted. Values for ±s.d. were derived from three independent experiments. (F) Western blot analysis of AF6i3-knockdown and control cell lysates during wound healing assay. One representative experiment out of three is shown.

View larger version (80K): [in a new window]   Fig. 9. AF6i3 knockdown decelerates cell-cell contact formation during Ca2+ switch assay. (A) Endogenous AF6i3 protein during Ca2+ switch assay. Cell monolayers (2 mM Ca2+) were incubated at 2 µM Ca2+ for 4 hours to disturb cell junctions (2 µM Ca2+ / 4h). Reformation of cell-cell contacts 2 hours and 6 hours after switch to 2 mM Ca2+ is depicted. Bar, 20 µm. (B) ß-catenin staining of AF6i3-knockdown (bottom row) and control cells (top row) during Ca2+ switch assay. Bar, 50 µm. (C) Graphic representation of the percentage of cell-cell contacts that stained positive for indicated junctional proteins 2 hours after switch to 2 mM Ca2+ in AF6i3-knockdown and control cells. Values for ±s.d. were derived from three independent experiments. (D) Length distribution of ß-catenin-positive cell-cell contacts 2 hours after switch to 2 mM Ca2+ for cells shown in B. Lengths of all ß-catenin-positive cell-cell contacts from three independent experiments were measured (n indicates the exact number) and grouped into 13 categories. Graphs represent the percentage of cell-cell contacts within each length category. (E) MCF10A AF6i3-knockdown and control cells grown in culture medium, 18 hours after seeding of singularized cells at equal sub-confluent density. Bar, 0.5 mm. (F) Graphic representation of Fig. 9E. The average number of cells per confluent cell group is depicted. Values for ±s.d. were derived from three independent experiments.

View larger version (68K): [in a new window]   Fig. 10. AF6i1 and AF6i3 protein in Ca2+ switch assay: crucial role of the F-actin binding site. (A) Anti-ß-catenin staining of control cells, AF6i3-knockdown cells and knockdown cells reconstituted with AF6i1 or AF6i3 construct, 2 hours after switching to 2 mM Ca2+. Bar, 30 µm. (B) Graphic representation of the percentage of ß-catenin-positive cell-cell contacts shown in A. Values for ±s.d. were derived from three independent experiments. Total numbers of counted cell-cell contacts are indicated (n). Similar results were obtained for E-cadherin, nectin-1 and -catenin. (C) Length distribution of ß-catenin-positive cell-cell contacts shown in A, determined as described for Fig. 9D.

View larger version (96K): [in a new window]   Fig. 11. AF6i3 C-terminus in Ca2+ switch assay. (A) Cell monolayers of the control cells and control or AF6i3-knockdown cells expressing the HA-tagged AF6i3 CT prior to assay (top) and 2 hours after switch to high Ca2+ concentration (bottom). Green, anti-AF6 antibody; red, anti-HA antibody. Bar, 20 µm. (B) Cells shown in A 2 hours after switch to high Ca2+ concentration, stained with anti-ß-catenin antibody. Bar, 20 µm.