Ark1 is required for the bi-orientation of sister chromatids in mitosis. (A) Wild-type (ark1+), ark1-T7, ark1-as2 or ark1-as3 cells having cen2-GFP were synchronized in S-phase using hydroxyurea. Wild-type and ark1-T7 cells were shifted to the restrictive temperature for ark1-T7 (34°C) when being released, whereas ark1-as2 and ark1-as3 were grown at 30°C, but 5 μM 1NM-PP1 was added to the cultures at release. Eighty to hundred minutes after the release, the cells were fixed. Tubulin was visualized by TAT-1 antibody staining or mCherry-Atb2 (tubulin). cen2 segregation was determined in a minimum of 100 anaphase cells. (B, C) Ark1-T7 mutants having cen2-GFP and expressing Sid4-GFP to label the spindle pole bodies (SPB) were followed by live-cell microscopy at the restrictive temperature (34°C). Exemplary kymographs are shown in panel C.

Ark1 is required for nuclear division during meiosis-I. (A) The position of metaphase-II spindles was evaluated in fixed cells. DNA was stained with Hoechst 33342. (B, C) The indicated strains were arrested after anaphase-I by the mes1 mutation. Cells were fixed and DNA was stained with DAPI. The number of nuclei was determined in at least 200 asci (C); exemplary cells are shown in panel B.

Ark1 is required for proper sister chromatid mono-orientation in meiosis-I independent of Moa1. (A) One of the homologous chromosomes 2 was marked by GFP in the indicated strains and the segregation pattern was determined during anaphase-I using CFP- or mCherry-Atb2 (tubulin) as a marker. (B) Cells from the indicated strains expressing Moa1-GFP and CFP-Atb2 (tubulin) were observed during metaphase-I. (C) Diploid strains of the indicated genotype were arrested in prophase-I by deletion of mei4+, and the amount of Moa1 at centromeres was determined by ChIP using a Moa1 antibody.

Ark1 is required for the correction of malattachment. (A) The indicated strains carrying the nda3-KM311 mutation were arrested in mitosis by incubation at 19°C for 6 h, and released by transfer to 32°C. Where indicated (+inhib.) 5 μM 1NM-PP1 were added to the culture 10 min before release. Cells were fixed with methanol 10 min after upshift to 32°C. Chromosome segregation was assessed by determining cen2-GFP localization on anaphase spindles, which were labeled by mCherry-Atb2 (tubulin). (B, C) nda3-KM311 ark1-as3 cells were arrested in mitosis as in panel A and released by transferring cells to a microscope stage kept at 32°C. 1NM-PP1 (5 μM) was added 5 min before release. Only cells that could be followed through mitosis starting from very short spindle length were considered. Cells in which the spindle was defective and those in which centromere 2 did not attach to the spindle were excluded from the analysis. A kymograph of a cell that showed chromosome 2 segregating with one SPB is shown in panel C. Example kymographs for all phenotypes as well as example kymographs from ark1-as3 cells released from the nda3-KM311 arrest without any inhibitor are shown in Supplementary Figure S2A. (D) The indicated strains marked with cen2-GFP and mCherry-Atb2 (tubulin) were followed by live-cell microscopy at the restrictive temperature (34°C). A centromere was considered to have ‘switched' when the trajectory of cen2-GFP from one pole to the other could be clearly observed (arrows). The number of ‘switches' per hour in a total of 2 h prometaphase observation time is given.

Ark1 is required for proper homolog bi-orientation in meiosis-I. (A) The indicated strains with both homologous chromosomes 2 marked by GFP were observed during anaphase-I using CFP-Atb2 (tubulin) as a marker. (B) The indicated strains, in which one of the homologous chromosomes 2 was marked by GFP, were fixed during meiosis by methanol. DNA was stained by Hoechst 33342 and the segregation of sister centromeres was determined in binucleated cells.

Sgo2 promotes mono-orientation similar to Ark1. (A) To assess the percentage of equational segregation (light gray bars), one of the homologous chromosomes 2 was marked by GFP in the indicated strains and the segregation pattern was judged in asci that had formed four spores. The percentage of lagging chromosomes or chromatids (dark gray bars) was determined by staining fixed cells with DAPI and the TAT-1 antibody recognizing tubulin. (B) Model for the chromosome segregation defects after Sgo2/Ark1 versus Moa1/Rec8 depletion. The centromeric region of one homolog is shown. In sgo2Δ or ark1 s.o. cells, the pair of sister kinetochores on one homolog tends to attach in a merotelic manner, that is, to both spindle poles, during metaphase-I. In anaphase, lagging homologs are common because of merotelic attachment. Eventually, both sister centromeres segregate to the same pole in most cases. If the protector Sgo1 is removed, sister centromeres segregate to opposite poles more frequently. Lagging chromatids are still observed after deletion of sgo1+, indicating merotelic attachment of single sister kinetochores. When moa1+ or rec8+ is deleted, sister kinetochores on one homolog lose their intimate connection. When rec12+ is deleted in addition to moa1+, sister chromatids segregate completely equationally in anaphase-I. Nevertheless, lagging chromosomes occur, but deleting sgo1+ abolishes their appearance.