Volume 26, Issue 11, Pages 2875-2889.e3 (March 2019) Interphase Microtubules Safeguard Mitotic Progression by Suppressing an Aurora B- Dependent Arrest Induced by DNA Replication Stress  Guillaume Laflamme, Shannon Sim, Allen Leary, Mirela Pascariu, Jackie Vogel, Damien D’Amours  Cell Reports  Volume 26, Issue 11, Pages 2875-2889.e3 (March 2019) DOI: 10.1016/j.celrep.2019.02.051 Copyright © 2019 The Author(s) Terms and Conditions

Cell Reports 2019 26, 2875-2889.e3DOI: (10.1016/j.celrep.2019.02.051) Copyright © 2019 The Author(s) Terms and Conditions

Figure 1 A Subset of tub2 Mutants Are Sensitive to Genotoxic Stresses (A) Crystal structure of yeast α-tubulin (in red) and β-tubulin (in green) (PDB: 4U3J; Ayaz et al., 2014). The tub2 mutants tested are represented with different colors (tub2-401 [M233V, Y242C, and Q245L in magenta], tub2-104 [R241H in yellow], tub2-402 [R318W in cyan], tub2-311 [G93E in blue], tub2-150 [T238A, having an additional mutation V326A, in orange], tub2-C354S/A [in white], tub2-431 [deletion of C-terminal tail from amino acid 430 in green], and tub2-742 [combination of tub2-311 and tub2-431 mutations]). (B) 5-fold dilution series of yeast cells were spotted on solid medium, supplemented or not with HU, 4NQO, or MMS. Plates were incubated at 30°C. The smc5-3KE mutant strain was used as a control for DNA damage sensitivity. Cell Reports 2019 26, 2875-2889.e3DOI: (10.1016/j.celrep.2019.02.051) Copyright © 2019 The Author(s) Terms and Conditions

Figure 2 Cell Cycle Characterization of DNA Damage-Sensitive tub2 Mutants (A) 5-fold dilution series of yeast were spotted on rich media to evaluate growth at different temperatures. (B–D) Cell cycle progression of TUB2 (blue diamond), tub2-311 (red square), and tub2-431 (green triangle) yeast strains after synchronous release from an α factor-induced G1 arrest. After release, cells were cultured at 30°C, and samples of the cultures were taken at 15-min intervals to evaluate bud morphology (B), DNA content by flow cytometry (C), and spindle morphology (D). 100 cells were counted per condition. Representative experiment is shown, from 3 independent experiments. AS refers to asynchronous/exponential cultures of yeast in (C). Note that cells progress more rapidly and at a lower level of synchrony in the cell cycle that follows the first division, as previously observed (Hartwell and Unger, 1977; Singer and Johnston, 1983). (E) Spindle length distribution of TUB2 SPC42-GFP and tub2-311 SPC42-GFP cells grown at 25°C. Spindle length was measured as the distance between Spc42-GFP foci. Cells were imaged every 20 s for a total of 20 min. The mean spindle length of individual cells was measured over the 20-min imaging period. (F) Spindle length fluctuations of cells shown in (E), plotted as a function of mean spindle length. Length fluctuations (σ) were calculated as the standard deviation of spindle lengths. Statistical difference in spindle length fluctuations of TUB2 (n = 74 cells) and tub2-311 (n = 69 cells) are not significant (p = 0.1372; Bartlett’s test; distributions were tested for normality with a one-sample Kolmogorov-Smirnov test). Cell Reports 2019 26, 2875-2889.e3DOI: (10.1016/j.celrep.2019.02.051) Copyright © 2019 The Author(s) Terms and Conditions

Figure 3 tub2-311 cells Exhibit an Anaphase Entry Delay after HU Treatment (A) Flow cytometry analysis of cell cycle progression after HU release at 30°C. Cell cultures were arrested in G1 with an α factor, released in synchrony in 200 mM HU for 2 h. HU was washed off, and cells were resuspended in fresh medium. α factor was added to the cultures after HU release to prevent cell cycle re-entry. (B) Same as in (A), but cell cultures were released from G1-mediated α factor arrest into fresh medium at 30°C. (C) Cells shown in (A) were fixed with formaldehyde and stained with DAPI. The percentage of budded cells with undivided nuclei is shown. Representative experiment is shown, from three independent experiments. (D) Representative morphology of DAPI-stained TUB2 and tub2-311 mutant cells 150 min after HU release. Scale bar, 5 μm. (E) G1-arrested TUB2 and tub2-311 cells were released in HU for 2 h before being plated on solid YEPD medium at 30°C. Microcolony formation was monitored every 2 h for a total of 8 h. Error bars represent SEM from four independent experiments. (F) Representative cells of the indicated genotype at 0, 2, 4, 6, and 8 h after HU release. Scale bar, 10 μm. (G) Same as in (E), but 200 cells were plated on solid YEPD medium to monitor colony formation. Percentage of viability was calculated by dividing the number of colonies before and after HU treatment. Error bars represent SEM from four independent experiments. p = 0.0061; calculated by unpaired two-tailed t test. Cell Reports 2019 26, 2875-2889.e3DOI: (10.1016/j.celrep.2019.02.051) Copyright © 2019 The Author(s) Terms and Conditions

Figure 4 Anaphase Entry Delay of tub2-311 Mutant Cells Is Dependent on Pds1 (A) Pds1-HA protein level was monitored in TUB2 and tub2-311 cells after HU release at 30°C. Pgk1 was used as a loading control. The percentages of budded cells (i.e., budding index; BI) are shown below the immunoblots. (B) Pds1-AID protein level was monitored in TUB2 pds1-aid cells with auxin, tub2-311 pds1-aid cells with auxin, or ethanol and tub2-311 PDS1 cells with auxin. All strains express osTIR1 from the URA3 locus. G1-synchronized cells were released in synchrony in 200 mM of HU for 2 h at 30°C. HU was washed off, and cells were resuspended in fresh medium supplemented with 500 μM of auxin or 1% ethanol. Pgk1 was used as a loading control. (C) Cells shown in (B) were fixed with formaldehyde and stained with DAPI. The percentages of budded cells with undivided nuclei are shown. Representative experiment is shown, from three independent experiments. (D) Representative cells of the indicated genotypes 150 min after HU release. Scale bar, 5 μm. (E) Cells shown in (B) were analyzed by flow cytometry to evaluate DNA content of the yeast strains used in the time-course experiment. Cell Reports 2019 26, 2875-2889.e3DOI: (10.1016/j.celrep.2019.02.051) Copyright © 2019 The Author(s) Terms and Conditions

Figure 5 Anaphase Entry Delay of tub2-311 Mutant Cells Is Dependent on an Aurora B/Ipl1-Induced Checkpoint (A) Schematic representation of the time-course experiment. Asynchronous cultures were arrested in G1 using α factor at 25°C, a permissive temperature for ipl1-85. Cells were then released from the G1 arrest in fresh medium containing 200 mM HU, and allowed to grow for 2 h at 32°C, a condition that inactivates ipl1-85. HU was then washed off, and cells were resuspended in fresh medium. α factor was added to prevent cell cycle re-entry. (B) TUB2 IPL1, tub2-311 IPL1, TUB2 ipl1-85, and tub2-311 ipl1-85 cells were fixed with formaldehyde and stained with DAPI. The percentages of budded cells with undivided nuclei are shown. Representative experiment is shown, from three independent experiments. (C) Representative cells of the indicated genotypes 150 min after HU release. Scale bar, 5 μm. (D) Cells shown in (B) were analyzed by flow cytometry to evaluate DNA content of the yeast strains used in the time-course experiment. (E) tub2-311 is synthetically sick with ipl1-85. 5-fold dilution series of wild-type, TUB2, ipl1-85, tub2-311, and tub2-311 ipl1-85 yeast strains were spotted on solid medium and incubated at the indicated temperatures. Cell Reports 2019 26, 2875-2889.e3DOI: (10.1016/j.celrep.2019.02.051) Copyright © 2019 The Author(s) Terms and Conditions

Figure 6 Kinetochore-Microtubule Interaction Defects in tub2-311 Mutant Cells after HU Treatment (A) TUB2 MTW1-GFP and tub2-311 MTW1-GFP cells were arrested in G1 with α factor and released in synchrony in 200 mM HU for 2 h at 30°C. HU was washed off, and cells were resuspended in rich medium. α factor was added to prevent cell cycle re-entry. Cells were fixed and examined by microscopy to visualize Mtw1-GFP. 100 cells were scored based on their Mtw1-GFP morphology: single Mtw1-GFP dot in an unbudded cell, single Mtw1-GFP dot in a budded cell, two close Mtw1-GFP dots of equal intensity near the bud neck in a budded cell, or two Mtw1-GFP dots segregated in a telophase cell. Representative experiment is shown, from three independent experiments. (B) Representative cells of the indicated genotypes 60 min after HU release. Scale bar, 5 μm. (C) Cells shown in (A) were analyzed by flow cytometry to evaluate DNA content of the yeast strains used in the time-course experiment. (D) Centromeric DNA is replicated after HU release. TUB2 mcd1-1 and tub2-311 mcd1-1 cells carrying CENIV-tetO and tetR-GFP were arrested in G1 with α factor at 23°C, and released in parallel in 200 mM HU at 23°C and 37°C (i.e., permissive and restrictive temperatures for mcd1-1, respectively). HU was washed off, and cells were resuspended in fresh medium supplemented with nocodazole (30 μg/mL for 2 h at either 23°C or 37°C). Cells were fixed and analyzed by microscopy. 100 cells were counted for the presence of one or two GFP dots. Error bars represent SD from three independent experiments. (E) Fluorescence signals of TUB2 mcd1-1 (n = 133) and tub2-311 mcd1-1 (n = 130) cells showing two GFP dots at 37°C were quantified to confirm complete replication of tetO repeats. Distribution (minimum to maximum) of fluorescence-intensity differences for the separated GFP dots within a cell is shown. Statistical difference in fluorescence intensities in TUB2 mcd1-1 (n = 133 cells) and tub2-311 mcd1-1 (n = 130 cells) is not significant (p = 0.3101; Mann-Whitney U test; distributions were tested for normality by the D’Agostino-Pearson omnibus test). (F) TUB2 SPC42-GFP and tub2-311 SPC42-GFP cells were arrested in G1 with an α factor and synchronously released in medium containing 200 mM HU for 2 h at 30°C. HU was washed off, and cells were resuspended in rich medium. α factor was also added to prevent cell cycle re-entry. The distance between adjacent SPBs, which directly reflects spindle length, was measured in 100 cells. Cells in G1 (no spindle) were omitted from the analysis, whereas cells with SPBs too close to be fully resolved were attributed a spindle length value of 0. Percentages of budded cells are shown above the graph. Representative experiment is shown, from three independent experiments. Cell Reports 2019 26, 2875-2889.e3DOI: (10.1016/j.celrep.2019.02.051) Copyright © 2019 The Author(s) Terms and Conditions

Figure 7 Tension Defects at Centromeres of tub2-311 Mutant Cells after DNA Replication Stress (A) TUB2 SGO1-GFP SPC42-mKate2 and tub2-311 SGO1-GFP SPC42-mKate2 cells were arrested in G1 with α factor and synchronously released in medium containing 200 mM of HU for 2 h at 30°C. After washing off the HU, cells were allowed to grow in rich medium containing α factor (to prevent cell-cycle re-entry). Samples of cells were taken at the indicated times, fixed, and processed for Sgo1-GFP signal analysis. 100 cells were scored by microscopy for the presence of Sgo1-GFP dots. The distribution of GFP fluorescence intensities is shown for cells displaying Sgo1 dots. Representative experiment is shown, from three independent experiments. (B) Representative pre-anaphase cells from the experiment described in (A) at 60 min (for TUB2) and 90 min (for tub2-311) after HU release. Scale bar, 5 μm. (C) tub2-311 is synthetically lethal with deletion of CIN8 and MCM21. Diploid yeast strains carrying tub2-311 and cin8 or mcm21 were sporulated, and the viability of the resulting spores was determined after 3 days of growth on solid medium at 30°C. Three typical tetrads are shown per genotype. Co-segregation of the relevant alleles in individual spores is indicated by the white hexagons and was deduced using the HIS3 marker associated with the TUB2 alleles together with the URA3 and kanMX6 markers associated with deletions of CIN8 and MCM21, respectively. Cell Reports 2019 26, 2875-2889.e3DOI: (10.1016/j.celrep.2019.02.051) Copyright © 2019 The Author(s) Terms and Conditions