PMC

Differential requirements of SAC genes for growth of the γ-tubulin mutant. Approximately the same amount of cells freshly grown on a YE plate at 33° was streaked out on the same medium and incubated at 30° for 3 days. Strains used are HM123 (WT), W604 (gtb1), HM713 (mad2), YT578 (mad2 gtb1), YT406 (mad3), YT410 (mad3 gtb1), YT405 (bub1), YT409 (bub1 gtb1), YT407 (bub3), YT411 (bub3 gtb1), YT408 (mph1), and YT412 (mph1 gtb1).

Live analysis of sister-chromatid separation. Fission yeast cells carrying both Sid4-GFP and cen2-GFP were observed every 15 sec with 10 steps (0.2 μm) along the z-axis at each time point. Cells in late phase 1 or in phase 2 were chosen for observation. Distances (d1–d4), as defined in the figure (Sid4-GFP: green dots; cen2-GFP: orange stars), were measured and plotted. (A and B) Wild type (P172). (C–F) gtb1-93 (P171).

Immunoprecipitation analysis of the mutant Mad2 proteins. Strains containing mad2⁺-GFP (YT579), mad2-56-GFP (YT580), and mad2-64-GFP (YT581) were used. Immunoprecipitated proteins were detected by Western blot analysis using the indicated antibodies. IP, immunoprecipitated. WCE, whole-cell extracts. Protein size markers are on the right: open triangle (80 kDa) and closed triangle (50 kDa). See materials and methods for experimental details.

Genetic properties of new mad2 mutants. (A) The G-418 plate assay. Colonies grown on EMM2 plates from spores produced from crosses between P5 (kan^r linked to the gtb1 mutation) and W986 (mad2) carrying a plasmid with mad2⁺ (a), empty vector alone (b), mad2-56 (c), or mad2-64 (d) were replica plated onto YE with or without G418 (see text for details). (B) The TBZ-sensitivity assay. HM713 (mad2Δ) carrying a plasmid with the indicated mad2 alleles grown on EMM2 medium was streaked on YE plates with or without TBZ and incubated at 33° for 3 days.

Dynamic localization of Mad2-GFP in mitotic cells. (A–D) Wild type (W949). (E–H) gtb1 (W948). (A) Prophase. (B) Prometaphase. (C) Metaphase (phase 2). (D) Anaphase (phase 3). (E–G) Early anaphase to late anaphase. (H) Metaphase (phase 2): sequential images showing dynamic movement of intense Mad2-GFP dot along the spindle. Original images are available as movies in supplemental Figures 4 and 5 (wild type) and supplemental Figures 6 and 7 (gtb1) at http://www.genetics.org/supplemental/. Images were taken every 15 sec, nine images with 0.3-μm steps in the z-axis at each time point.

Timing of sister-chromatid separation of different chromosomes. Cells containing cen2-GFP and ade6-GFP as well as Sid4-GFP were observed in living cells every 15 sec. At each time point, 10 images were taken with a single z-axis step of 0.2 μm. P175 (WT) and P174 (gtb1) were used. In wild type, one of the sister-chromatid pairs was already separated in the fourth frame, but the other remained unseparated until the sixth frame. In the mutant, both pairs appeared to separate simultaneously (third frame from the top), but one of them (marked by green triangle) stopped separating for >1 min while the other (red triangle) continued to separate.

Separation of chromosomes on the mitotic spindle. Synchronized cells with GFP-tagged α-tubulin at 30° in EMM2 medium were fixed with methanol and stained with DAPI. In A–D, microtubules (green) and chromosomes (red) (color panels) and chromosomes in black-and-white images are shown. Cells containing a spindle >∼5 μm were chosen, and then the spindle length and the segregation figures of chromosomes were recorded. The figures were classified as equal (representative image as in A and in blue boxes in E–G), incomplete separation (B, red), lagging chromosome near the spindle center (C, pink) or close to the spindle pole (green), and unequal (D, yellow). Each box in E–G represents one cell. Strains used were YT417 (wild type), YT413 (gtb1-93), and YT415 (gtb1-93 mad2Δ). See supplemental Table 1 (at http://www.genetics.org/supplemental/) for strains.

Live observation of spindle dynamics. Spindle length was determined as the distance between two separating Sid4-GFP signals. Living cells were observed every 20 sec, and at each time point 12 images were taken at serial focal planes at 0.2-μm steps (A and B). These parameters were changed to 30 sec, 6 images, and 0.4-μm steps in C and D. The time of the onset of anaphase B (phase 3) was set as time 0 and all figures obtained for each strain are assembled in a single figure. (A) Wild type (strain P18, number of cells (n) = 21). (B) gtb1-93 (P168, n = 24). (C) mad2Δ (YT522, n = 10). (D) gtb1-93 mad2Δ (YT519, n = 14).

Activity of a single copy of the new mad2 alleles. mad2⁺ (#) indicates the normal wild-type mad2 allele, while mad2⁺, mad2-56, and mad2-64 indicate that these alleles were replaced with the mad2Δ allele together with the kan^r marker gene (see materials and methods). mad2Δ denotes the mad2 allele disrupted by insertion of the ura4⁺ gene, the kan^r marker was not used here. (A) TBZ sensitivity test. YE containing 20 μg/ml TBZ was used together with a control plate. Incubation was at 33° for 3 days. Strains used were HM123, HM713, YT547, YT549, and YT553. (B) Activity of mitotic arrest. Strains used were the same as in A. CBZ (50 μg/ml) was added to a logarithmic-phase culture and incubated for the indicated time periods. Percentage of cells containing hypercondensed chromosomes was measured. mad2⁺(#) (solid circles); mad2⁺ (open circles); mad2Δ (crosses); mad2-56 (open triangles); mad2-64 (open squares). More than 200 cells were scored for each strain. (C) Capability of different mad2 alleles to support the growth of gtb1 mutant. Incubation was at 33° or 30° for 3 days. Strains used were W604, YT578, YT555, YT556, and YT559. (D) Patterns of chromosome segregation in late anaphase. Strains with indicated mad2 alleles in the gtb1-93 background (same as in C) were incubated at 30° in YE for 1.5 hr after the treatment with HU and stained with DAPI. More than 170 mitoses were scored for each strain.