Tub3 optimizes Kar9-dependent spindle positioning as observed in cells expressing nontagged tubulins solely from the two endogenous loci. Spindle positioning was monitored by visualizing SPBs (yeast spindle poles) with fluorescently tagged Cnm67-mRUBY2. (a) Number of small-budded cells of the indicated genotype with properly positioned early mitotic spindles (category 1 on left). Micrographs show cell (far left) and Cnm67-mRUBY2 image with cell outline in white (second from left) that are representative of categories shown in cartoons. Early mitotic spindles were classified as small-budded cells with duplicated but unseparated SPBs. (b) Preanaphase spindle position relative to the bud neck. Micrographs under cartoon show representative cell (lower) and Cnm67-mRUBY2 image with cell outline in white (upper). For panel a, bars show mean ± SEM from two trials; for kar9Δ, n = 67 and 92; Tub1 only, n = 86 and 40; WT, n = 118 and 132; and Tub3 only, n = 120 and 90. For panel b, line and error bars represent mean ± SEM, and scatterplot shows individual cells observed in two trials with a total of 101, 111, 175, and 167 for kar9Δ, Tub1 only, WT, and Tub3 only, respectively. *, P ≤ 0.05; ***, P ≤ 0.001 by unpaired, two-tailed Student’s t test. Scale bars = 2 µm.