Bub1 and Bub3 promote the conversion from monopolar to bipolar chromosome attachment independently of shugoshin

Abstract

The eukaryotic spindle assembly checkpoint (SAC) delays anaphase in the presence of chromosome attachment errors. Bub3 has been reported to be required for SAC activity in all eukaryotes examined so far. We find that Bub3, unlike its binding partner Bub1, is not essential for the SAC in fission yeast. As Bub3 is needed for the efficient kinetochore localization of Bub1, and of Mad1, Mad2 and Mad3, this implies that most SAC proteins do not need to be enriched at the kinetochores for the SAC to function. We find that Bub3 is also dispensable for shugoshin localization to the centromeres, which is the second known function of Bub1. Instead, Bub3, together with Bub1, has a specific function in promoting the conversion from chromosome mono-orientation to bi-orientation.

Figure 1

Bub3 is not essential for the SAC, but for the efficient localization of SAC proteins. (A) Cells expressing plo1–GFP and the β-tubulin nda3-KM311 allele were followed by live-cell microscopy at 17°C. The duration of prometaphase was determined by the presence of Plo1–GFP on the SPBs. Circles indicate cells in which the entire prometaphase took place within the recording time. Triangles indicate cells that had not exited prometaphase when recording was stopped; thus the actual time of prometaphase must be longer than this value. Kymographs of exemplary cells are shown in supplementary Fig S1A online. (B) Kymographs of exemplary mitotic cells that were followed by live-cell microscopy at 30°C using Sid4–mCherry to visualize SPBs. A quantitative analysis is shown in supplementary Fig S3 online. GFP, green fluorescent protein; SAC, spindle assembly checkpoint; SPB, spindle pole body.

Figure 2

Bub3 is required for correct chromosome segregation. (A) The duration of prometaphase at 30°C in bub3Δ, bub3Δ mad2Δ, mad2Δ and wild-type (wt) cells was determined by live-cell microscopy using Plo1–GFP as a marker. In all box-whisker graphs, the lines from top to bottom are: maximum value, 75th percentile, median, 25th percentile and minimum value. (B) Cells carrying cen2–GFP and mCherry–atb2(tubulin) were synchronized with HU, released from HU arrest and arrested in mitosis by treatment with the microtubule-destabilizing drug MBC for 3.5 h. After washout of MBC, segregation of chromosome 2 (cen2–GFP) was followed by live-cell microscopy at 20°C. Only those cells that were already in mitosis when recording was started were considered. Mis-segregation in unperturbed mitosis was similarly determined by live-cell microscopy at 20°C after HU release. GFP, green fluorescent protein; HU, hydroxyurea.

Figure 3

Bub3 and its interaction with Bub1 are required for chromosome bi-orientation in an Sgo2-independent manner. (A–C) Cells expressing GFP–atb2(tubulin) and the kinetochore marker mis6–mCherry were pre-synchronized and treated with MBC as described in Fig 2B. After washout of MBC, segregation of the chromosomes was monitored by using live-cell microscopy at 20°C. (B) Chromosomes that persisted close to an SPB for at least 10 min were followed and the time until they became bi-oriented was determined (open circles). Filled circles indicate chromosomes that apparently never achieved bi-orientation; triangles indicate chromosomes that had failed to achieve bi-orientation by the end of the recording. The total number of cells observed, the number of cells showing mono-oriented chromosomes for at least 10 min and the number of mono-oriented chromosomes are given in (C). (D) Exemplary cells from this experiment. Arrowheads indicate mono-oriented chromosomes. For the bub3Δ cell, correction of mono-orientation to bi-orientation can be seen at 33 min. In the bub1-ΔGLEBS cell, mono-orientation was never corrected and the cell entered anaphase after 37 min. In the sgo2Δ cell, bi-orientation was achieved after about 15 min. The sgo2Δ bub3Δ cell failed to bi-orient two chromosomes and delayed entry into anaphase for more than 1 h (supplementary Fig S4 online). (E) Living cells from the indicated strains expressing mCherry–atb2(tubulin) and sgo2–GFP were imaged by fluorescence microscopy. For each genotype, two independent strains were used. The maximum signal intensity in the nucleus was determined. Interphase or metaphase cells were identified by the characteristic microtubule signal. bub3Δ and bub1-ΔGLEBS cells often showed more than one Sgo2–GFP signal in mitosis (supplementary Fig S9 online). GFP, green fluorescent protein; SPB, spindle pole body; wt, wild type.

Figure 4

Possible pathways for the correction of chromosome mono-orientation. (A) When chromosomes are clustered close to the SPBs at the beginning of mitosis or when spindles are short, direct capture by a microtubule from the opposite SPB might be the predominant way of correcting mono-orientation (left). When spindles become elongated, further mechanisms might be needed (right). We propose that Bub1 and Bub3 are involved in moving mono-oriented chromosomes closer to the centre of the spindle either by promoting movement along pre-existing kinetochore microtubules or by modulating kinetochore–microtubule attachment of the mono-oriented chromosome to the proximal pole. (B) Kymograph of a bub3–GFP-expressing cell treated with MBC after release from HU-arrest as described in Fig 2B. An initially unattached chromosome was captured by microtubules, pulled towards an SPB and subsequently bi-oriented. Bub3–GFP localized to one or both kinetochores of this chromosome until the chromosome had achieved bi-orientation. GFP, green fluorescent protein; HU, hydroxyurea; SPB, spindle pole body.

Figure 5

Mono-orientation of chromosomes in bub3Δ cells can be rescued when mitotic spindles are short. (A) Model for the possible mechanism of rescue: when the mitotic spindle is long, microtubules from the opposite spindle pole cannot reach the mono-oriented chromosome, whereas they can if the spindle is short. A second, indirect mechanism of rescue (Fig 4A) is presumably not functional in bub3Δ cells. (B–D) Exemplary kymographs of (B) wild-type or (C,D) bub3Δ cells carrying cen2–GFP and expressing mCherry–atb2(tubulin) from the experiment described in Fig 2B. GFP, green fluorescent protein.