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. 2018 Dec 4;28(8):1227–1243. doi: 10.1093/hmg/ddy416

Figure 6.

Figure 6

α-tubulin R402C/H mutants in S. cerevisiae act in the dynein spindle positioning pathway. (A and B) Genetic interaction test of tub1-R403C (A) and tub1-R403H (B) mutants with bim1∆, a member of the compensatory spindle positioning pathway. Indicated strains were grown on rich media or rich media supplemented with benomyl (10 μg/ml), incubated at 30°C. Strains: WT, yJM2243, yJM2252; bim1∆, yJM2246, yJM2249; tub1-R403C, yJM2245; tub1-R403C bim1∆, yJM2244; tub1-R403H, yJM2251; tub1-R403H bim1∆, yJM2250. (C and D) Genetic interaction test of tub1-R403C (C) and tub1-R403H (D) mutants with dyn1∆. Indicated strains were grown on rich media or rich media supplemented with benomyl (10 μg/ml), incubated at 30°C. Strains: WT, yJM2256, yJM2261; dyn1∆, yJM2255, yJM2264; tub1-R403C, yJM2258; tub1-R403C dyn1∆, yJM2257; tub1-R403H, yJM2263; tub1-R403H dyn1∆, yJM2262. (E) Image of a WT cell expressing Dyn1-3GFP and spindle pole marker Spc110-DsRed. Arrowhead points to dynein localized to astral microtubule plus end. Arrows point to spindle pole bodies. (F) Quantification of GFP signal at microtubule plus ends. At least 39 dynein-plus-end foci were measured for each strain. Error bars are SEM. Strains: WT, yJM0307, yJM0308; tub1-R403C, yJM2202, yJM2203; tub1-R403H, yJM2204. (G) Image of a WT cell expressing Kip3-tdTomato and Spc110-GFP. Arrowhead points to Kip3 accumulated at astral microtubule plus end. Arrows point to spindle pole bodies. (H) Quantification of tdTomato signal at plus ends. At least 20 Kip3-plus-end foci were measured for each strain. Data are represented as mean ± SEM. Strains: WT, yJM2696, yJM2716; tub1-R403C, yJM2698, yJM2718; tub1-R403H, yJM2697, yJM2717. (I) Image of a WT cell expressing Kip2-mEmerald and Spc110-tdTomato. Arrowheads point to Kip2 accumulated at astral microtubule plus end. Arrows point to spindle pole bodies. (J) Quantification of mEmerald signal at plus ends. 40 Kip2-plus-end foci were measured for each strain. Data are represented as mean ± SEM. Strains: WT, yJM2693, yJM3177; tub1-R403C, yJM2694, yJM3178; tub1-R403H, yJM2695, yJM2715.