Extended Data Fig. 7: Perturbation of tubulin acetylation by AtatRNAi does not affect central spindle asymmetry and symmetric endosome segregation.
(a-b) Validation of the AtatRNAi construct. (a) Notum of a fly expressing AtatRNAi under the control of the Pnr promoter after immunolabeling using Oregon Green-514 anti-β-tubulin and Atto 647N anti-K40 acetylated-α-tubulin antibodies (SDCM). White line: Pnr expression domain limit. Image corresponds to maximum intensity z-projection of epithelium by SDCM. (b) Quantification of the normalized K40 acetyl-α-tubulin/β-tubulin ratio at the central spindle in conditions shown in a (see methods). Mean±SEM. Statistics: two-sided Mann–Whitney test (p-value<0.0001; n: central spindle number). AtatRNAi leads to a marked decrease of K40-α-tubulin acetylation. (c) Dividing SOP of indicated genotype showing Jupiter–GFP (top row) or Jupiter–GFP with Rainbow RGB LUT applied (bottom row). Anterior/pIIb orientation as determined by the mRFP-Pon signal. Image corresponds to maximum intensity z-projection of the central spindle (SDCM). (d) Quantification of the effects seen in c: pIIb enrichment (see methods) in indicated genotypes. Median±95% confidence interval; n: number of central spindles analysed. Statistics: two-sided Student t-test. The control dataset is the same as the one presented in Fig. 1d, shown here for convenience. Central spindle asymmetry is not affected upon Atat depletion. (e) Dividing SOP showing Sara endosomes (iDelta20) and mRFP-Pon in the indicated genotype (pIIa cortex; dashed blue). Image corresponds to Maximum intensity z-projection of entire cells (SDCM). (f) Quantification of the effects seen in (e): iDelta20 percentage in pIIa after abscission in SOPs of indicated genotypes (mean ± SEM; Statistics: two-sided Student t-test; n: SOPs analysed. The control dataset is the same as the one presented in Fig. 2B, shown here for convenience. Sara endosome asymmetric segregation is not affected upon Atat depletion. (g) Summary of the AtatRNAi and Elp3RNAi phenotypes: Elp3RNAi phenotype cannot be due to a defect of α-tubulin acetylation. Scale bars: 10 µm (a); 2 µm (c); 5 µm (e). Source numerical data are available in source data.
