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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⁺.

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