Expansion of the pole-to-pole distance in metaphase also occurs in the mis6 mutant. (A) One living mis6 mutant cell cultured at 36°C for 6 hr and expressing Sad1–GFP is shown. The number indicates minutes. Phase 2 lasts from 6 to 10 min. Bar, 10 μm. (B, left) Time course of the pole-to-pole distance in two mis6 mutant cells (#1, #2) and one wild-type cell (right). Distribution of length of the phase-2 spindle in 15 mis6 cells (solid bars) is shown with the wild-type control (open bar).

Mis12 is an essential conserved protein. (A) Sequences of S. cerevisiae, A. nidulans, and M. grisea are similar to the amino terminus of Mis12. (Top) Identical amino acids are boxed; similar ones are shaded. (Bottom) Alignment of Mis12 and Mtw1p. The predicted coiled–coil exists in the middle (amino acids 100–150); while the conserved regions are in the amino terminus (amino acids 1–88). (B) Gene disruption of mis12⁺ led to the missegregation phenotype with the large and small daughter nuclei. Gene-disrupted cells were stained by DAPI. Bar, 10 μm. (C) Identification of Mis12 protein by immunoblotting. The carboxyl terminus of the mis12⁺ gene was tagged with HA and integrated onto the chromosome of mis12-537 by homologous recombination (the promoter was native). The temperature-sensitive phenotype was rescued in the resulting integrant, which grew normally. Mis12 was detected at the expected molecular mass (lane 2). The band intensity increased in cells overproducing Mis12–HA by multicopy plasmid (lane 1), but the band was not detected in extracts carrying the vector (lane 3).

Requirement of Mis12 for maintaining the inner centromere. (A, top) Organization of the cen1 is schematized (Takahashi et al. 1992). Probes used for PCR primers and Southern hybridization are indicated by vertical and horizontal bars, respectively. (Bottom) Cells expressing the Mis12–HA by the integrated gene were immunoprecipitated after fixation with formaldehyde and glass bead breakage. Coprecipitated DNA was amplified by PCR with the primers of cnt1, imr1, otr2 (dh), and lys1 (Saitoh et al. 1997). These primer sequences (indicated by the vertical bars) were unique or repeated up to three times in the genome. Approximately the same level of PCR-made DNAs was obtained as control from the strain expressing Mis12–HA and the wild-type 972 (lanes 4,5). Precipitates of anti-HA yielded the PCR products of cnt1 and imr1 but not of otr2 or lys1 (lane 1). Lanes 2 and 3 are the control with beads alone or the wild-type 972 extract, respectively. (B) S. pombe cells expressing Mis12–HA and Mis6–Myc were immunoprecipitated by anti-HA antibody, and the materials precipitated (Ppt) and supernatant (Sup) were immunoblotted with anti-HA and anti-Myc antibodies. Mis6–Myc was not present in the precipitates. (C) mis12-537 mutant expressing the integrated Mis6–HA (lanes 2,5) and mis6-302 expressing Mis12–HA (lanes 8,11) were constructed, and their formaldehyde-fixed extracts were immunoprecipitated by anti-HA antibody. Lanes 1–3 and 7–9 are the PCR products after immunoprecipitation; lanes 4-6 and 10-12 are the PCR products from the whole extracts. Wild-type-expressing Mis6–HA (lanes 1,4), Mis12–HA (lanes 7,10), and the wild-type 972 without the HA tag (lanes 3,6,9,12) were used as control. Mis6–HA could bind to the inner centromere regions (cnt1 and imr1) in mis12 at 36°C, and vice versa. (D) Nuclear chromatin was prepared from wild-type and the mis12 mutant cultured at 36°C for 8 hr, and digested with micrococcal nuclease for 1, 2, 4, and 8 min, followed by agarose gel electrophoresis and Southern hybridization with the three DNA probes, otr1, imr1, and cnt1 (Saitoh et al. 1997). The ethidium-bromide staining patterns are shown at right with the size markers. The smeared nucleosome pattern in the inner centromere was abolished in mis12 mutant.

Interaction of mis12 and cold-sensitive mutations. (A) The double mutant between temperature-sensitive mis12 and cold-sensitive dis1 null produced small colonies at 22°C, whereas it failed to produce colonies at 33°C. (B) Two cold-sensitive suppressors (strains 163 and 165) isolated were able to grow at 36°C. They were stained by anti-Sad1 antibodies and the pole-to-pole distance in mitotic cells was measured. The number of cells are indicated as the frequency. The open and solid arrowheads indicated the sizes of wild-type and mis12 spindle at metaphase.

The metaphase spindle is expanded in mis12. (A) Two living mis12-537 cells expressing Sad1–GFP cultured at 36°C for 7–8 hr and observed at 0.5 min intervals by a confocal microscope. The number indicates minutes. Asymmetric nuclear division is evident for both cells. Bar, 10 μm. (B) A wild-type control. (C) Time-course of the pole-to-pole distance measured for three mis12 (#1–#3) and one wild-type (WT) cell. Three phases are clearly distinguished (see text). (D) The pole-to-pole distances in the spindle of phase 2 measured for 22 mis12 (solid bars) and 6 wild-type cells (open bar) are plotted. Frequencies are expressed as the number of living cells for which mitosis was analyzed. (E) Plot of duration of time for phase 2 measured for the same number of mis12 (shaded bars) and wild-type cells (open bars). (F) Two mis12 mutant cells cultured at 36°C for 5 hr (left) and 8 hr (right) and expressing the cen1–GFP. The number indicates minutes.

Impaired spindle elongation in mis4. (A) Time-lapse images of one mis4-242 cell expressing Sad1–GFP and cultured at 36°C for 4 hr. Bar, 10 μm. (B, left) Time course of the pole-to-pole distance in two mis4 mutant cells (#1, #2) at 36°C. The spindle formed and increased in length without interruption up to 4–5 μm, thereafter, the pole-to-pole distance increased very slowly. (Right) Distribution of the pole-to-pole distances measured for mis4 cells is shown (solid bar).

Mis12–GFP colocalizes with the centromeres. (A) Mis12–GFP was expressed by the single-copy-integrated gene and observed by fluorescence microscopy after Hoechst 33342 staining. The GFP signal was located as a dot on the periphery of the interphase nucleus (top). Hoechst 33342 stained (middle) and the merged images (bottom) are also shown. (B) Visualization of microtubules (TUB) and Mis12–GFP in methanol-fixed cells. Mis12–GFP expressed by multicopy plasmid with the native promoter was observed after cells were treated with methanol and stained with anti-tubulin antibodies. (C) Mis12–GFP (top) was expressed by multicopy plasmid in nda3-311 (defective in β-tubulin) cultured at 20°C for 8 hr. DAPI (middle) and the merged (bottom) images are also shown. (D) Time-lapse images of one living wild-type cell expressing Mis12–GFP by the single-copy integrated gene. The number indicates minutes. Bars, 10μm.

Missegregation occurs in mis12-537 after cells traverse the previous mitosis at 36°C. (A, top) mis12-537 cells stained by DAPI after 8 hr at 36°C. Bar, 10 μm. (Bottom) FISH was applied with the probe of the rDNA repeats in chromosome III. The FISH signal was not separated in the top and middle cells, but was separated in the bottom. (B) Asynchronously growing mis12 mutant cells (in the rich culture medium) were shifted to 36°C (most cells in G₂). Frequencies of mutant cells displaying unequal nuclear division reached 80% after 6 hr at 36°C. Cell viability decreased after 4 hr, whereas cell number ceased to increase after 8 hr. (C) Wild type and mis12-537 were arrested at G₁ by nitrogen starvation at 26°C in the synthetic medium and then released to the complete YPD medium at 36°C (0 hr). DNA contents determined by the FACScan are shown (left). The S phase, the first and the second mitoses occurred at 3, 5-6, and 8–9 hr after the release, respectively, in mutant cells. The heterogeneous DNA contents appeared after the second cell division (right). Cell viability score decreased after 8 hr, whereas the frequency of missegregation was very low in the first mitosis but high in the second mitosis. The cell number increase in the mutant culture ceased after 10 hr. (D) mis12-537 was arrested at G₁ by nitrogen starvation at 26°C in the synthetic medium, and shifted to 36°C in the same culture for 4 hr: Cells did not grow under the starved condition. Cells were then released to the complete YPD medium at 36°C (release at 4 hr). In the control culture (release at 0 hr), cells were shifted to the complete YPD at 36°C without 4-hr preincubation at 36°C. DNA contents determined by the FACScan are shown (left). The S phase and the first M phase occurred after 7 and 9–10 hr in the culture released at 4 hr, respectively, whereas cell viability decreased after 12 hr (in the second M phase). Chromosome missegregation (unequal mitosis percent) occurred in the second mitosis. All this occurred 4 hr earlier in the control culture released at 0 hr. (E) The cut4 mis12 double mutant was grown as described in text. In the first metaphase, the signals of Mis6–GFP were clustered in the middle as the control single cut4, whereas they were split into four or five in the second metaphase at 36°C. Bar, 10 μm.