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First published online 10 October 2006
doi: 10.1242/jcs.03208

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Gliotactin and Discs large form a protein complex at the tricellular junction of polarized epithelial cells in Drosophila

Department of Zoology, University of British Columbia, Vancouver, V6T 1Z3, Canada

View larger version (82K): [in a new window]   Fig. 1. Gliotactin is found at TCJs in numerous larval tissues. (A,B) Epidermal cells stained with Gliotactin at the TCJ. Gliotactin (green) is concentrated at the TCJ (arrows) compared with Dlg (red) in A or Coracle (red) in B, which label the entire SJ domain. (C,D) Tracheal cells labeled with Gliotactin at the TCJ between tracheal cells. Gliotactin (green) and Coracle (red) co-staining shows the concentration of Gliotactin at the TCJ (arrows). (E) Salivary gland cells labeled with Gliotactin (green) and Neuroglian (red). Gliotactin at the TCJ has a punctate pattern (arrow). (F) Peripodial cells from the wing imaginal disc labeled with Gliotactin (green) and Dlg (red) showing the ribbon like structure of the TCJ (arrows) which extend over several microns. (G,H) Columnar epithelial cells from wing imaginal discs labeled with Gliotactin (green) and Dlg (red) in G and the Na+/K+ ATPase subunit (red) in H. Despite their small size, the TCJ is distinguishable from the rest of the septate junction domain (arrows). Bars, 15 µm (A,B,D-H); 20 µm (C).

View larger version (68K): [in a new window]   Fig. 2. Gliotactin is distributed with Dlg in a range of TCJs. (A) Gliotactin (green) and Neuroglian (red) have only a small degree of overlap at the TCJ in epidermal cells. The merged image is followed by Neuroglian (red channel) and then Gliotactin (green channel) staining in grayscale. At the TCJ the degree of Neuroglian staining is far less than Gliotactin (arrows). Gliotactin can be seen in the SJ domain though at much lower levels than at the TCJ. (B) Gliotactin (green) and Dlg (red) overlap in the TCJ of epidermal cells. Dlg, like Gliotactin, has a greater degree of labeling at the TCJ (arrows). (C) Coracle (green) and Neuroglian (red) overlap in epidermal cells. The merged image is followed by Coracle (green channel) and Neuroglian (red channel) in grayscale. Both proteins show a distinct morphology with ruffled edges (asterisks) in these cells, perhaps reflecting the pleated nature of these septate junctions. (D) Gliotactin compared with Dlg and Neuroglian at the epidermal cell TCJ. For each set of panels the merged image (Gliotactin, green; Dlg, red; Neuroglian, blue) is followed by: Gliotactin (green) and Dlg (red), Neuroglian (green) and Dlg (red). On the bottom row: Dlg (red channel), Gliotactin (green channel) and Neuroglian (blue channel) are in grayscale to show the individual patterns. Gliotactin and Dlg have a greater degree of staining at the TCJ compared with Neuroglian (arrows). Dlg is distinct in pattern from Neuroglian at the SJ, where Neuroglian has a ruffled morphology (asterisks) compared with Dlg. (E,F) Gliotactin compared with Dlg and Neuroglian at the peripodial cell TCJ. For each set of panels the merged image (Gliotactin, green; Dlg, red; Neuroglian, blue) is followed by: Gliotactin (green) and Dlg (red), Neuroglian (green) and Dlg (red). Dlg (red channel), Gliotactin (green channel) and Neuroglian (blue channel) are in grayscale to show the individual patterns. Gliotactin and Dlg have a greater degree of overlap at the ribbon-like TCJ compared with Neuroglian (compare arrows in panels).

View larger version (43K): [in a new window]   Fig. 3. Endogenous and HA-tagged Gliotactin in protein complexes. (A) Gliotactin is in a Ca2+-dependent protein complex with Dlg but not with Neurexin IV. A Gliotactin mAb was used to immunoprecipitate Gliotactin from membrane preparations isolated from adult flies. The blots were probed with antibodies to Dlg, Neurexin IV and Gliotactin as indicated. The small arrows indicate the major Dlg isoforms isolated (120 kDa, 100 kDa and 87 kDa). 1, membrane preparation; 2, mouse IgG control 0.1 µM Ca2+; 3, Gli mAb no Ca2+; 4, Gli mAb 0.05 µM Ca2+; 5, Gli mAb 0.1 µM Ca2+; 6, Gli mAb 0.2 µM Ca2+. (B) GST pull down using the Gliotactin C-terminal domain recapitulates the immunoprecipitation assays. A GST fusion to the intracellular domain of Gliotactin (GST-GliCter) was used to pull down proteins from adult membrane preparations. The blot was probed with an antibody to Dlg and Neurexin IV. The small arrows indicate the major Dlg isoforms isolated (120 kDa, 100 kDa and 87 kDa). 1, membrane preparation; 2, GST alone 0.1 µM Ca2+; 3, GliCter no Ca2+; 4, GliCter 0.1 µM Ca2+; 5, GliCter 0.2 µM Ca2+. (C) Gliotactin C-terminal domain fails to bind directly to Dlg. A GST fusion to the intracellular domain of Gliotactin (GliCter) and to the intracellular domain lacking the last three amino acids (GliPDZ) were used to pull down in vitro translated Dlg. A GST-Shaker C-terminal fusion was used as the positive control. The blot was probed with an antibody to Dlg to detect the in vitro translated product. 1, in vitro translated input; 2, GST alone; 3, GliCter; 4, GliPDZ; 5, ShakerCter. (D) GST pull down of Dlg is independent of the PDZ-binding motif. GST-GliCter and GST-GliPDZ were used to isolate protein complexes from adult membrane preparations plus or minus Ca2+. The blot was probed with an antibody to Dlg and the interaction with Dlg was Ca2+ dependent but independent of the PDZ-binding motif. Similar isoforms were brought down with both forms of Gliotactin (arrows). 1, GST alone no Ca2+; 2, GliCter no Ca2+; 3, GliPDZ no Ca2+; 4, GST alone 0.1 µM Ca2+; 5, GliCter 0.1 µM Ca2+; 6, GliPDZ 0.1 µM Ca2+.

View larger version (66K): [in a new window]   Fig. 4. Gliotactin somatic clones in the wing disc have a normal distribution of septate junction proteins. (A-F) Somatic clones of Gliotactin null mutant in columnar epithelial cells of the wing disc. (A) Clone labeled with Neurexin IV (green) and Dlg (red); mutant cells lack GFP (blue) to show the location of the clone (outlined). (B,C) Side views to show the normal protein distribution in wild-type versus mutant cells. Dlg (B, red) and GFP (B, blue) or Dlg alone (B'). Neurexin IV (C, green) and GFP (C, blue) or Neurexin IV alone (C'). The dashed lines indicate the position of Gliotactin null cells. (D) Dlg (red channel from A) shown in grayscale. Arrowheads indicate the location of the TCJ. (E) Neurexin IV (green channel from A) shown in grayscale. (F) Somatic clones of Gliotactin showing the absence of Gliotactin staining in null clones. (G-I) Somatic clones of Gliotactin mutant cells in peripodial cells of the wing imaginal disc. (G) A clone labeled with Dlg shows normal distribution of Dlg at the TCJ. Compare arrowheads indicating the TCJ in the clone with wild-type cells. (H) Clone of Gliotactin mutant cells labeled with Neuroglian. (I) Clone labeled with E-cadherin. Bars, 15 µm (A-E, G-I); 40 µm (F).

View larger version (39K): [in a new window]   Fig. 5. Wild-type and PDZ Gliotactin are localized correctly to the TCJ. Embryonic epithelia were stained with anti-Gliotactin (green) or anti-HA (green) to show the distribution of wild-type protein or the different transgene proteins. Neurexin IV (red) was used to label the SJ domain. Each panel has en face and longitudinal views to show the distribution of Gliotactin to the TCJ. Arrows indicate the extent of the apical versus basal boundaries of the epidermis. (A) Wild-type embryonic epidermis showing the normal distribution of Gliotactin (green) and Neurexin IV (red). (B) Diagrams of the different Gliotactin constructs used including the untagged full-length (wt), HA-tagged full-length (HA or GliHA) and HA-tagged mutant lacking the last three amino acids (PDZHA). The different domains of the Gliotactin protein and location of the HA tag are indicated. The HA-tagged constructs generated proteins of the expected size when expression was driven in embryos with daughterless-GAL4. (C-F) Expression of transgenic Gliotactin in embryonic epidermis using the daughterless-GAL4 driver line. An antibody to HA was used to detect the expression of the tagged Gliotactin proteins (green) and Neurexin IV (red) was used to mark the SJ domain. (C) Expression of full length Gliotactin (GliHA) in a Gliotactin null background. The full-length protein is highly concentrated at the TCJ. (D) Expression of a full-length Gliotactin (GliHA) in a wild-type background. When overexpressed Gliotactin spreads around the periphery of the cell throughout the SJ domain. The majority of the Gliotactin protein remains in the plane of the SJ domain (arrowhead) with a small amount of basolateral spread (between the two arrows). (E) Expression of GliotactinPDZ (GliPDZ) in a Gliotactin null background. The GliotactinPDZ protein is also highly concentrated at the TCJ. The arrows indicate the extent to which Gliotactin expression spreads in a basolateral direction. (F) Expression of GliotactinPDZ (GliPDZ) in a wild-type background. The GliotactinPDZ protein spreads throughout the SJ domain. The arrows indicate the extent that Gliotactin expression spreads in a basolateral direction. (G) Overexpressed Gliotactin forms a Ca2+-dependent complex with Dlg and other SJ proteins. An anti-HA antibody was used to isolate the tagged full-length Gliotactin from embryos. Membrane preparations were isolated from 12- to 24-hour embryos containing the daughterless-GAL4 driving the UAS-Gliotactin HA-tagged transgene. The blots were probed with antibodies to Dlg, Neurexin, Coracle and Gliotactin. 1, membrane preparation; 2, IgG serum control; 3, HA mAb no Ca2+; 4, HA mAb 0.1 µM Ca2+; 5, HA mAb 0.5 µM Ca2+. (H) Gliotactin mAb was used to immunoprecipitate Gliotactin from wild-type embryos and the blot was probed with an antibody to Neurexin IV. Note the lack of Neurexin IV association with Gliotactin in the wild type versus Gliotactin overexpressing embryos. 1, membrane preparation; 2, IgG control; 3, Gli mAb no Ca2+; 4, Gli mAb 0.1 µM Ca2+; 5, Gli mAb 0.2 µM Ca2+.

View larger version (82K): [in a new window]   Fig. 6. Ectopic expression of Gliotactin in wing imaginal discs. (A,A') Patched-GAL4 driving full-length Gliotactin. (A) When Gliotactin (green) is overexpressed it is found throughout the cell membrane. There is a reduction in Dlg (red) staining intensity compared with neighbouring wild-type cells. (A') At higher magnification, the Gliotactin overexpressing cells adjacent to wild-type cells clearly show the reduced levels of Dlg compared with non-expressing cells (arrows). The Gliotactin channel (green) was collected at saturating levels of Gliotactin so that the boundary cells could be better assayed. A' has been digitally scaled to 200% of A. (B,B') Apterous-GAL4 driven overexpression of full length Gliotactin. (B) Overexpression of Gliotactin (green) with apterous-GAL4 also results in a reduction of Dlg staining (red). (B') A longitundinal x-axis projection of Gliotactin (red) and Dlg (green) and panels showing each individual channel in the LUT scale to indicate intensity of staining. Overexpressed Gliotactin is throughout the cell membrane with the greatest concentration around the apical/SJ region. Dlg expression in these regions is reduced throughout the cell and at the SJ (arrow). The Dlg levels in the overlying peripodial cell layer are not affected (arrowhead) because this cell layer does not express apterous-GAL4. The fire LUT scale ranges from purple (lowest) to white (highest) intensity. (C) Overexpression of GliotactinPDZ (green) has no effect on Dlg (red) levels. GliotactinPDZ spreads around the cell but fails to downregulate Dlg. (D) Overexpression of full-length Gliotactin (green) had no effect on Coracle (red) levels. (E-F) Overexpression of Gliotactin in embryonic epidermis using 69B-GAL4. (E) Overexpression of full-length Gliotactin (green) had little effect on Dlg (red) levels. (F) Similarly, overexpression of GliotactinPDZ (green) had little effect on Dlg (red). Bars, 15 µm.

View larger version (117K): [in a new window]   Fig. 7. Loss but not overexpression of Dlg affects Gliotactin localization. (A,B) Gliotactin is mislocalized in Dlgm52 mutant imaginal discs. Imaginal discs were isolated from second instar (A) and third instar (B) larvae homozygous from Dlgm52 and stained for Gliotactin (green), Dlg (red) and E-cadherin (blue). Arrows in A indicate puncta of staining remaining. (C,D) Gliotactin localization is normal when Dlg is overexpressed. Dlg-A fused to GFP was driven to high levels in the imaginal disc using patched-GAL4. Gliotactin (green) and Spectrin (red) were used to highlight the TCJ and cell boundaries, respectively. DlgGFP (blue) was localized to the TCJ in peripodial cells (C, arrows) and to the SJ domain in columnar epithelia (D). DlgGFP did spread down the SJ domain when overexpressed (D) but had no effect on cell morphology or Gliotactin localization. Bars, 15 µm.