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Fig. 2. Heterozygous phenotypes and rescue of homozygotes in Drosophila epithelial transport assay. Three lines carrying recessive alleles of vha55 (A, vha55^7e1; B, vha55¹⁴; C, vha55^SzA9) were out-crossed to wild-type flies, and fluid secretion rates by the heterozygotes (blue circles) were compared with wild-type tubules (black squares). Homozygous mutants that had been rescued by ubiquitous (heat-shock-GAL4 driven) expression of vha55::GFP were also tested (red diamonds). After resting rates were recorded, the tubules were maximally stimulated by the addition of the diuretic neuropeptides Capa-1 (Kean et al., 2002) and drosokinin (Terhzaz et al., 1999), both at 10^-7 M, at 30 min. Data are expressed as mean ± s.e.m. (n=8). (D-F) The VHA55::GFP fusion protein localises correctly to the apical domain of Malpighian tubules in transgenic Drosophila. Immunocytochemistry of wild-type tubule with anti-VHA55 (D), negative control with primary antibody blocked with excess antigenic peptide (E) and direct epifluorescence view of a transgenic tubule with VHA55::GFP expression directed to the principal cells (F). The tubule is arranged as a simple epithelium, with wide, shallow cells wrapped around the central lumen. The apical localisation is established by the bulging of the V-ATPase fluorescence to the apical, rather than basal, side of the nuclei (visible as black holes in D&E, counterstained with DAPI in F). Bars, 10 µm. A confocal stack of the ICC is provided as Movie 1 in supplementary material.

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