Figure 5.
Figure 5. DHC64C and p150Glued mutant alleles enhance the dominant neurodegenerative phenotypes associated with the SCA5 mutations. (A) Mutations in human β-III–spectrin interact with the cytoplasmic dynein heavy chain DHC64C6-10 mutation to produce posterior paralysis. Larvae shown in A I and A II are heterozygous for the American SCA5 mutation in human β-III–spectrin (elav-GAL4–syt-GFP/+, hSPAM/+) and the DHC64C6-10 mutant allele, respectively, and exhibit normal locomotion. Double heterozygous animals (elav-GAL4–syt-GFP/+, hSPAM/+, DHC64C6-10/+) developed an abnormal crawling behavior (A III). (B) Fluorescent images show large syt-GFP accumulations within segmental nerves from double heterozygous mutant larvae (B III, elav-GAL4–syt-GFP/+, hSPAM/+, DHC64C6-10/+). Bar, 10 µm. (C) Quantification of bouton number shows a significant reduction in synapse size (number of synaptic boutons per surface area of muscle 6/7) in animals expressing the American SCA5 mutation in human β-III–spectrin that are also heterozygous for the DHC64C6-10 mutant allele of dynein (elav-GAL4–syt-GFP/+, hSPAM/+, DHC64C6-10/+). Numbers are normalized and compared with the elav-GAL4/+;hSPWT/+ control line in the graph. Data are mean ± SEM (error bars); ***, P < 0.001. (D–K) Scanning EM images of Drosophila eyes. Insets show higher magnification images of the ommatidial field. Recombinant lines expressing the American SCA5 mutation in β-III–spectrin (gmr-GAL4-hSPAM/CyO, 5D) or the German SCA5 mutation in fly β-spectrin (gmr-GAL4-FSPGM/CyO, 5E) show disorganization in the arrangement of the ommatidia and bristles of the adult eye. Note that the recombinant gmr-GAL4-hSPAM line showed reduced levels of transgene expression and produced a less severe eye phenotype, making it easy to distinguish changes in the phenotype. DHC64C6-10/+ flies have a wild-type eye (F) but, in combination with gmr-GAL4-hSPAM or gmr-GAL4-FSPGM mutant spectrin alleles, produce a more severe eye phenotype than either parent (G and H). Eyes expressing the dominant Gl1 mutation exhibit a rough eye phenotype, with disordered ommatidia (I). Similarly, the combination of the Gl1 dynactin mutant allele and gmr-GAL4-hSPAM or gmr-GAL4-FSPGM mutant spectrins results in a dominant enhancement of the eye degeneration. Eyes from double mutant flies are reduced in size and show a dramatic roughens of the eye surface (J and K). Bars in D also apply to F–I.

DHC64C and p150Glued mutant alleles enhance the dominant neurodegenerative phenotypes associated with the SCA5 mutations. (A) Mutations in human β-III–spectrin interact with the cytoplasmic dynein heavy chain DHC64C6-10 mutation to produce posterior paralysis. Larvae shown in A I and A II are heterozygous for the American SCA5 mutation in human β-III–spectrin (elav-GAL4–syt-GFP/+, hSPAM/+) and the DHC64C6-10 mutant allele, respectively, and exhibit normal locomotion. Double heterozygous animals (elav-GAL4–syt-GFP/+, hSPAM/+, DHC64C6-10/+) developed an abnormal crawling behavior (A III). (B) Fluorescent images show large syt-GFP accumulations within segmental nerves from double heterozygous mutant larvae (B III, elav-GAL4–syt-GFP/+, hSPAM/+, DHC64C6-10/+). Bar, 10 µm. (C) Quantification of bouton number shows a significant reduction in synapse size (number of synaptic boutons per surface area of muscle 6/7) in animals expressing the American SCA5 mutation in human β-III–spectrin that are also heterozygous for the DHC64C6-10 mutant allele of dynein (elav-GAL4–syt-GFP/+, hSPAM/+, DHC64C6-10/+). Numbers are normalized and compared with the elav-GAL4/+;hSPWT/+ control line in the graph. Data are mean ± SEM (error bars); ***, P < 0.001. (D–K) Scanning EM images of Drosophila eyes. Insets show higher magnification images of the ommatidial field. Recombinant lines expressing the American SCA5 mutation in β-III–spectrin (gmr-GAL4-hSPAM/CyO, 5D) or the German SCA5 mutation in fly β-spectrin (gmr-GAL4-FSPGM/CyO, 5E) show disorganization in the arrangement of the ommatidia and bristles of the adult eye. Note that the recombinant gmr-GAL4-hSPAM line showed reduced levels of transgene expression and produced a less severe eye phenotype, making it easy to distinguish changes in the phenotype. DHC64C6-10/+ flies have a wild-type eye (F) but, in combination with gmr-GAL4-hSPAM or gmr-GAL4-FSPGM mutant spectrin alleles, produce a more severe eye phenotype than either parent (G and H). Eyes expressing the dominant Gl1 mutation exhibit a rough eye phenotype, with disordered ommatidia (I). Similarly, the combination of the Gl1 dynactin mutant allele and gmr-GAL4-hSPAM or gmr-GAL4-FSPGM mutant spectrins results in a dominant enhancement of the eye degeneration. Eyes from double mutant flies are reduced in size and show a dramatic roughens of the eye surface (J and K). Bars in D also apply to F–I.

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