(A) Longevity curves from adults expressing the noted versions of ataxin-3 in all fly glia, throughout development and in adults. These are the same lines as in Figure 1. P values are from log-rank tests with Holm-Bonferroni adjustment. P values in italics are not statistically significant. There is no statistical difference for 100% death between flies expressing ‘Intact’ versus ‘Inactive’, even though 50% lethality is different. This is because each vial with flies is treated as one individual and for statistical purposes the day when all flies are dead is considered, not the rate of death over the course of time for each vial. (B) Negative geotaxis assay comparing motility of flies not expressing any pathogenic ataxin-3 versus flies expressing pathogenic ataxin-3 with mutated VBM in all glial cells. Driver, as in panel (A), was repo-Gal4. No statistical differences are observed at any time point between the groups based on two-tailed Student’s t-tests. We selected to conduct this assay for the VCP-binding mutations since in longevity curves we noticed no significant differences in the longevity of flies not expressing pathogenic ataxin-3 compared to ones expressing the SCA3 protein with the VCP-binding mutation. Note: The ‘Intact’ line shown here is a different transgenic line than the one we generated for the polyQ studies concerning the UIMs that are shown in Figure 2 and beyond. Those lines (Figure 2 and on) have a ‘CAGCAA’ repeat for reasons summarized in main text, compared to a pure 'CAG' repeat for all lines shown in Figure 1 and in this figure, which were generated for prior work. The lines generated for Figure 2 also have an optimized Kozak sequence. As we showed earlier (Johnson et al., 2019), the ‘CAGCAA’ repeat-harboring ‘Intact’ line is at higher protein levels and is more toxic than the pure 'CAG' repeat shown here. For all the work in this study, isogenic sets are compared only to one another, that is, none of the lines shown here and in Figure 1 is directly compared to lines in Figure 2 and beyond, since these lines were conceptualized as isogenic sets from the beginning. However, we remind the reader that all ataxin-3 lines are inserted into the same chromosomal site of Drosophila, attP2, in the same orientation and as a single copy number.