Figure 1.
Figure 1. Expression of mutant β-III–spectrin causes a dosage-dependent and progressive neurodegenerative eye phenotype. (A) Scanning EM images of adult fly eyes grown at 25°C. Insets show higher magnification images of ommatidial fields. Expression of the American SCA5 mutation in human β-III–spectrin in the eye results in a fused and disorganized ommatidia missing interommatidial bristles (II). Flies expressing the German SCA5 mutation in β-III–spectrin show a mild roughness of the eye (III), with a more severe phenotype in flies expressing two copies of the German SCA5 transgene (V). Flies expressing wild-type β-III–spectrin show only a mild ommatidial phenotype similar to gmr-GAL4 control flies (I and IV). Similar disruption of ommatidial organization was observed when the mutant, but not wild-type, spectrin transgenes were expressed using the neuronal-specific nina E-GAL4 driver. (B, right) Bar graph showing mean ratios of myc-tagged spectrin versus tubulin protein for control gmr-GAL4/+, gmr-GAL4/+; hSPWT/+, gmr-GAL4/hSPAM; gmr-GAL4/+; hSPGM/+, and homozygous gmr-GAL4/+; hSPGM/hSPGM flies. Animals expressing one copy of the hSPGM transgene have a significant reduction in β-III–spectrin expression (P = 0.025, n = 4). Data represent mean ± SEM; *, P = 0.025. (B, left) β-III–spectrin expression for the different genotypes was detected using an antibody to the myc epitope with tubulin as a loading control. (C–F) Histological sections of fly eyes. Longitudinal frontal sections (C–F) and tangential sections (panels C′–F′) taken through the retina of 10-d-old flies. Images show thinning of the retinal layer (arrow) and loss of retinal neurons (arrowhead) in flies expressing the American (E and E′) and German (F and F′) SCA5 mutations compared with a wild-type control (D and D′) and gmr-GAL4 driver flies (C and C′). (G–J″) Optical eye images taken at days 10, 20, and 30 illustrate the progression of the eye phenotype. Flies expressing wild-type human β-III–spectrin had external eye morphology similar to the gmr-GAL4 driver (G), and showed no changes in ommatidia organization and pigmentation during the first 30 d of adult life (H–H″). The eye phenotype of flies expressing the American mutant spectrin worsens considerably over time (I–I″). Flies expressing low levels of the German mutant spectrin have defects in pigmentation by day 30 (J″). (K and K′) Optical eye images taken at days 2 and 10 correspond to flies expressing two copies of the hSPGM transgene. The severity and progression of the rough eye phenotype worsens with increasing dosage of mutant German β-III–spectrin. (L) Protein extracts from flies expressing the wild-type human β-III–spectrin transgene (gmr-GAL4/+; hSPWT/+) were subjected to immunoprecipitation using the anti–fly α-spectrin monoclonal antibody 3A9. Precipitated immune complexes were then analyzed by Western blotting assays using either anti-myc or anti-fly β-spectrin antibodies. BSA was used as a control for the immunoprecipitation reaction. Bars: (C–F) 50 µm; (C′–F′): 5 µm; (G–K) 50 µm.

Expression of mutant β-III–spectrin causes a dosage-dependent and progressive neurodegenerative eye phenotype. (A) Scanning EM images of adult fly eyes grown at 25°C. Insets show higher magnification images of ommatidial fields. Expression of the American SCA5 mutation in human β-III–spectrin in the eye results in a fused and disorganized ommatidia missing interommatidial bristles (II). Flies expressing the German SCA5 mutation in β-III–spectrin show a mild roughness of the eye (III), with a more severe phenotype in flies expressing two copies of the German SCA5 transgene (V). Flies expressing wild-type β-III–spectrin show only a mild ommatidial phenotype similar to gmr-GAL4 control flies (I and IV). Similar disruption of ommatidial organization was observed when the mutant, but not wild-type, spectrin transgenes were expressed using the neuronal-specific nina E-GAL4 driver. (B, right) Bar graph showing mean ratios of myc-tagged spectrin versus tubulin protein for control gmr-GAL4/+, gmr-GAL4/+; hSPWT/+, gmr-GAL4/hSPAM; gmr-GAL4/+; hSPGM/+, and homozygous gmr-GAL4/+; hSPGM/hSPGM flies. Animals expressing one copy of the hSPGM transgene have a significant reduction in β-III–spectrin expression (P = 0.025, n = 4). Data represent mean ± SEM; *, P = 0.025. (B, left) β-III–spectrin expression for the different genotypes was detected using an antibody to the myc epitope with tubulin as a loading control. (C–F) Histological sections of fly eyes. Longitudinal frontal sections (C–F) and tangential sections (panels C′–F′) taken through the retina of 10-d-old flies. Images show thinning of the retinal layer (arrow) and loss of retinal neurons (arrowhead) in flies expressing the American (E and E′) and German (F and F′) SCA5 mutations compared with a wild-type control (D and D′) and gmr-GAL4 driver flies (C and C′). (G–J″) Optical eye images taken at days 10, 20, and 30 illustrate the progression of the eye phenotype. Flies expressing wild-type human β-III–spectrin had external eye morphology similar to the gmr-GAL4 driver (G), and showed no changes in ommatidia organization and pigmentation during the first 30 d of adult life (H–H″). The eye phenotype of flies expressing the American mutant spectrin worsens considerably over time (I–I″). Flies expressing low levels of the German mutant spectrin have defects in pigmentation by day 30 (J″). (K and K′) Optical eye images taken at days 2 and 10 correspond to flies expressing two copies of the hSPGM transgene. The severity and progression of the rough eye phenotype worsens with increasing dosage of mutant German β-III–spectrin. (L) Protein extracts from flies expressing the wild-type human β-III–spectrin transgene (gmr-GAL4/+; hSPWT/+) were subjected to immunoprecipitation using the anti–fly α-spectrin monoclonal antibody 3A9. Precipitated immune complexes were then analyzed by Western blotting assays using either anti-myc or anti-fly β-spectrin antibodies. BSA was used as a control for the immunoprecipitation reaction. Bars: (C–F) 50 µm; (C′–F′): 5 µm; (G–K) 50 µm.

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