Tub3 optimizes spindle positioning by the Kar9 pathway. (a) Small-budded cells of the indicated genotype with properly positioned early mitotic spindles (category 1 on the left). (b) Preanaphase spindle positioning relative to the bud neck. Micrograph shows representative GFP-tubulin image with cell outline in white. (c) Small-budded cells with early mitotic spindle that display an astral microtubule oriented toward, or away from, the bud. Micrographs show cell (left) and GFP-tubulin image with cell outline in white (right) that are representative of categories shown in cartoons. (d) Astral microtubule length in small-budded cells. In panels a and c, bars show mean ± SEM from three trials; in panel a, for kar9Δ, n = 67, 64, and 83; for Tub1 only, n = 84, 82, and 94; for WT-Tub1, n = 57, 66, and 134; for WT-Tub3, n = 52, 79, and 100; for Tub3 only, n = 102, 116, and 116. In panel c, for Tub1 only, n = 65, 65, and 70; for WT-Tub1, n = 60, 60, and 58; for WT-Tub3, n = 62, 55, and 59; for Tub3 only, n = 66, 64, and 55. For panels b and d, lines and error bars show mean ± SD from three trials. In panel b, scatterplot shows individual cells observed in three trials with a total of 169, 205, 187, 180, and 165 for kar9Δ, Tub1 only, WT-Tub1, WT-Tub3, and Tub3 only, respectively. In panel d, scatterplot shows microtubules observed in three trials with a total of 193, 186, 193, and 207 for Tub1 only, WT-Tub1, WT-Tub3, and Tub3 only, respectively. **, P ≤ 0.01; and ***, P ≤ 0.001 by unpaired, two-tailed Student’s t test. Scale bars = 2 µm. aMT, astral microtubule.