Figure 1.
Bub1p is a relatively stable component of fission yeast kinetochores, whereas the bulk of Mad3p rapidly exchanges.
(A) Bub1-GFP fluorescence recovery after photo-bleaching (FRAP): nda3 cells expressing Bub1-GFP were arrested in mitosis at 18°C and treated with anti-microtubule drugs (25 µg/ml carbendazim) to ensure the arrest was maintained. Specific GFP kinetochore signals were then photobleached with a laser, and images captured at intervals throughout the recovery period. The % fluorescence recovery and half-times indicated are the average of nine experiments. The recovery curve shown is representative, and the dashed line indicates the 50% post-bleach recovery level. (B) Mad3-GFP fluorescence recovery after photo-bleaching (FRAP): nda3 cells expressing Mad3-GFP were arrested in mitosis at 18°C and treated with anti-microtubule drugs (25 µg/ml carbendazim) to ensure the arrest was maintained. Specific GFP kinetochore signals were then photobleached with a laser, and images captured at intervals throughout the recovery period. The % fluorescence recovery and half-times indicated are the average of 5 experiments.
Figure 2.
Bub1-(1-586)-GFP-Taz1Myb is targeted to telomeres.
(A) A model of the Bub1-GFP-Taz scaffold protein (Bub1-Tel). (B) Field of bub1Δ cells expressing Bub1-Tel. (C) Anti-Bub1p immunoblot of extracts from strains expressing Bub1-GFP or Bub1-Tel. (D) Bub1-Tel co-localises with telomeres (Pot1-mRFP) and not kinetochores (Ndc80-CFP) in interphase cells. In mitotic cells the scaffold is recruited to both telomeres and kinetochores (data not shown). Scale bar is 5 microns.
Figure 3.
Bub1-Tel is sufficient to recruit both Bub3p and Mad3p.
(A) Bub1-Tel recruits and co-localises with Bub3-mCherry at telomeres. (B) Bub1-Tel recruits and co-localises with Mad3-tdTomato at telomeres. Scale bars are 5 µm. (C) Quantitation of the co-localisation observed between checkpoint proteins and telomeres (Pot1), or kinetochores (Ndc80). Full co-localisation was scored when all of the telomere (or kinetochore) foci observed in a given cell co-localised with the Bub1-Tel and either Bub3p (grey columns) or Mad3p (red columns). See supplementary data (Tables S1, S2, S3 and S4) for further details. (D) Speculative model of Bub1 scaffold action at a telomere (TEL). Note, due to low signal intensity, we have not yet demonstrated that Bub3p and Mad3p exchange rapidly at the telomeres.
Table 1.
Yeast strains