PMC

Model of spindle organization in wild-type cells and β-CTT mutants. Spindle microtubules (black lines) emanate from SPBs (gray) to form antiparallel overlaps in the spindle center and attach to KTs (green). KT position is determined by a balance of outward forces (black arrows) generated by iMTs and inward forces (green arrows) generated by chromatin (green dashed lines). β-CTT mutants are defective for spindle stability and therefore predicted to generate weaker outward forces. This imbalance shortens the spindle and disrupts KT position.

β-CTT is important for chromosome segregation. (A) Schematic of chromosome loss assay. (B) Chromosome loss frequency per 1000 divisions. Values are based on the total number of half-sectored colonies divided by the total number of colonies analyzed from at least three separate experiments. Error bars are SE of proportion. *p = 0.01, **p < 0.0001, determined by chi-square text with Yates’ correction. WT, n = 14,866; tub2-430Δ, n = 16,299; tub1-442Δ, n = 8561; tub1-442Δ tub2-430Δ, n = 5504; ndc80-112Δ, n = 35,662; ndc80-112Δ tub2-430Δ, n = 15,479; dam1-1, n = 12816; and dam1-1 tub2-430Δ, n = 7405.

β-CTT regulates microtubule length and organization during spindle assembly. (A) Model of spindle microtubules based on a tomographic volume of a preanaphase WT cell. Green lines represent microtubules from one SPB, and pink lines represent microtubules from the other SPB. Scale bars, 200 nm. (B) Histogram of microtubule lengths in the WT cell modeled in A. (C) Model of spindle microtubules based on a tomographic volume of a preanaphase tub2-430∆ cell. Yellow lines represent microtubule fragments. (D) Histogram of microtubule lengths in the tub2-430∆ cell modeled in C. (E) Histograms of all microtubule lengths from two WT preanaphase spindles (black, n = 81 microtubules) and 4 tub2-430∆ preanaphase spindles (red, n = 195 microtubules). (F) Model of spindle microtubules based on a tomographic volume of an anaphase WT cell. (G) Histogram of microtubule lengths in the WT cell modeled in F. (H) Model of spindle microtubules based on a tomographic volume of an anaphase tub2-430∆ cell. (I) Histogram of microtubule lengths in the tub2-430∆ cell modeled in H.

β-CTT promotes normal cell cycle progression. (A) Normalized doubling times for WT and CTT mutant cells. Values are the mean from at least four separate experiments. Error bars are SEM. **p < 0.0001 determined by t test. (B) Duration of S/G2/M determined by measuring the time from bud emergence to separation in cells released from START. Dashed lines are the medians. WT, n = 617; tub2-430Δ, n = 561. (C) Time course of Pds1/securin levels in synchronized cells released from START. Cells expressing Pds1-13myc were collected at 15- min intervals, prepared for Western blots, and probed with myc antibodies. (D) Pds1-13myc signal at each time point normalized to t = 0. Values are averages from three experiments. Error bars are SEM.

Positioning bioriented sister centromeres. (A) Maximum intensity projections from 3D confocal images of WT and tub2-430∆ cells expressing CENIV-GFP and Spc110-tdTomato. Scale bars, 1 μm. Preanaphase cells containing a single focus of CENIV-GFP were classified as unseparated. (B) Preanaphase cells containing two foci of CENIV-GFP were classified as separated. (C) Percentage of preanaphase cells exhibiting separated CENIV. **p < 0.0001 determined by Fisher’s exact test. WT, n = 1420; tub2-430Δ, n = 857; mcm21Δ, n = 685; and mcm21Δ tub2-430Δ, n = 276. (D) Mean angle between separated CENIV foci and the SPB-SPB axis. Values are mean ± 95% confidence interval (CI). **p < 0.0001 determined by t test. WT, n = 61 cells; tub2-430Δ, n = 109; mcm21Δ, n = 131; and mcm21Δ tub2-430Δ, n = 70. (E) Mean distances between CENIV foci and the proximal SPBs, between separated CENIV foci, and between SPBs. Values are mean ±95% CI. *p < 0.01, **p < 0.0001, determined by t test.

Mapping the region of β-CTT that is important for chromosome segregation. (A) Amino acid sequence alignment of WT and mutant β-CTT. The acidic patch is shaded gray. (B) Chromosome loss frequency per 1000 divisions. *p = 0.0001 and **p < 0.0001 determined by chi-square test with Yates’ correction. WT, n = 14,866; tub2-430Δ, n = 16,299; tub2-438Δ, n = 20,492; tub2-445Δ, n = 23,932; tub2-polyQ438Δ, n = 17,792; tub2-polyQ445Δ, n = 15,776; tub2-polyQ, n = 6946; and tub2::CTT^TUBB3, n = 15,607. (C) Normalized doubling times. (D) Sequence logo for β-tubulin residues 426–445, created from amino acid sequences from 24 β-tubulins (Supplemental Table S1). (E) Genetic interactions with SAC mutants. Tenfold dilution series of strains indicated at left were spotted to rich medium or rich medium supplemented with 10 μg/ml benomyl and grown at 30°C.

β-CTT promotes KT positioning. (A) Maximum intensity projections from 3D confocal images of WT cells expressing Nuf2-GFP and Spc110-DsRed. Scale bars, 1 μm. (B) Maximum intensity projections from 3D confocal images of tub2-430∆ cells expressing Nuf2-GFP and Spc110-DsRed. (C) Maximum intensity projections from 3D confocal images of dam1-765 cells expressing Nuf2-GFP and Spc110-DsRed. (D) Volumetric distribution of Nuf2-GFP signal. Yellow bars denote the mean. The p value was determined by t test. Strains: WT, n = 101; tub2-430Δ, n = 117. (E) Sum of intensities of Nuf2-GFP in cells analyzed in three dimensions. (F) Distribution of spindle lengths in asynchronous populations of cells. (G) Proportion of cells exhibiting two peaks of Nuf2-GFP signal as a function of spindle length. Error bars are SE of proportion. (H–J) Distributions of Nuf2-GFP signal measured from the center of the spindle toward the spindle poles and sorted into bins according to spindle lengths. Intensity is internally normalized to the total GFP intensity of each cell. (K) Kymograph of Nuf2-GFP in WT and tub2-430∆ cells. Horizontal lines represent time points at 2.5-min intervals, with pixel values representing the sum of 15 pixels perpendicular to the spindle axis. Arrow shows Nuf2-GFP collapsing into a single lobe in the tub2-430∆ cell.