Fig. 5.

Flow cytometric analysis of dissociated cells from whole zebrafish can be used to rapidly quantify insoluble EGFP ataxin-3 particles and screen compounds for the effect on proteinopathy. (A) Representative scatterplots of flow cytometric populations from dissociated 6-day-old zebrafish revealed changes in the number of detected detergent-insoluble particles despite similar numbers of nuclei. (B,C) Zebrafish expressing human mutant ataxin-3 displayed similar numbers of RedDot-stained nuclei but significantly more detergent-insoluble ataxin-3 particles at 2 days of age. (D) Examination of insoluble particle size revealed similar-sized particles across genotypes. (E,F) At 6 dpf, cells from dissociated zebrafish showed similar numbers of nuclei but more EGFP ataxin-3 particles were detected in cells obtained from zebrafish expressing human mutant ataxin-3 compared to non-transgenic siblings and zebrafish larvae expressing human wild-type ataxin-3. (G) Detergent-insoluble particles were a similar size in cells obtained from zebrafish expressing wild-type human ataxin-3 and mutant human ataxin-3. (H) Treatment with the autophagy inhibitor chloroquine (3 mM) increased the number of detergent-insoluble particles detected. (I) Treatment with the autophagy inducer calpeptin decreased the number of detergent-insoluble particles in 2-dpf zebrafish expressing mutant ataxin-3. Data are mean±s.e.m. n=2-9 experimental replicates. *P<0.05; **P<0.01; ***P<0.001; ns, not significant (one-way ANOVA with Tukey's post-hoc test or Student's t-test, paired, two-tailed). AU, arbitrary units; RFU, relative fluorescence units.