Figure 2. ATXN3 is essential for the restoration of degradative compartments after lysosome damage.

(A) Knockout-rescue assays for Gal3 clearance. HeLa ATXN3 KO and parental cells expressing indicated constructs were mock or LLOMe-treated (1 mM) for 1 h before washout. Cells were fixed at the indicated time points, and Gal3-positive lysosomes were immuno-stained. Note that Gal3-decorated lysosomes persisted in ATXN3 KO cells, which was rescued by re-expression of ATXN3 WT but not of the catalytically inactive ATXN3-C14A or the p97-binding deficient VBM mutant (VBM*). Scale bar, 5 µm. (B) Quantification of (A). n = 2 biologically independent experiments with >15 cells quantified per condition per experiment. Error bars, S.E.M. Two-way ANOVA with Tukey’s multiple comparison test was used to test significance. (C) HeLa cells were treated with indicated siRNAs, and Gal3 clearance was monitored after lysosomal damage was assayed as in (A). Scale bar, 15 µm. (D) Quantification of (C). n = 4 biologically independent experiments with >30 cells quantified per condition per experiment. Two-way ANOVA with Tukey’s multiple comparison test was used to test significance. The graph shows mean ± S.D. (E) Induced degradation of ATXN3 compromises clearance of damaged lysosomes. The ATXN3 gene was tagged with the FKBP12^F36V tag in U2OS cells. ATXN3 degradation was induced by dTAG^VHL treatment, and Gal3 clearance was assessed. Scale bar, 15 µm. See Fig. EV1I for degradation verification. (F) Quantification of (E), n = 3 biological replicates with >30 cells per condition per experiment. One-way ANOVA with Tukey’s multiple comparison test. The graph shows mean ± SD. Source data are available online for this figure.