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Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes.

Rischitor PE, May KM, Hardwick KG - PLoS ONE (2007)

Bottom Line: Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors.Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1.We propose that the major checkpoint role for Bub1 is as a signalling scaffold.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold.

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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.
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pone-0001342-g001: 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.

Mentions: As mentioned above, there are a number of caveats with the published FRAP studies on the intracellular dynamics of spindle checkpoint proteins. Vertebrate studies have argued that Bub1-GFP is a relatively stable kinetochore component. Less than 20% recovery was observed after bleaching cell lines stably expressing YFP-Bub1 [10], and in cells transiently transfected with GFP-Bub1 56% recovered with a t1/2 of ∼30 seconds [8]. The fission yeast wild-type bub1+ gene has been C-terminally tagged with GFP, such that it is expressed from its own promoter at the endogenous locus, and a range of checkpoint and chromosome segregation assays demonstrate that it is fully functional [21]–[23]. To analyse the dynamics of Bub1-GFP at unattached kinetochores, we used a cold-sensitive tubulin mutant (nda3). These cells arrest early in mitosis, with no microtubules, unattached kinetochores and hyper-condensed chromosomes [24]. In addition, we treated these nda3 cells with anti-microtubule drugs (25 µg/ml carbendazim) to ensure the arrest was maintained. We carried out FRAP experiments and a representative example is shown (Fig. 1A). Analysis of the recovery profiles (see Supplementary material) showed that Bub1-GFP displayed 39 (±16) % recovery, and that the dynamic pool recovered with a half-time of ∼31 (+/−3) seconds (n = 9). This value is mid-way between the two published recovery profiles for vertebrate Bub1.


Bub1 is a fission yeast kinetochore scaffold protein, and is sufficient to recruit other spindle checkpoint proteins to ectopic sites on chromosomes.

Rischitor PE, May KM, Hardwick KG - PLoS ONE (2007)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2147072&req=5

pone-0001342-g001: 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.
Mentions: As mentioned above, there are a number of caveats with the published FRAP studies on the intracellular dynamics of spindle checkpoint proteins. Vertebrate studies have argued that Bub1-GFP is a relatively stable kinetochore component. Less than 20% recovery was observed after bleaching cell lines stably expressing YFP-Bub1 [10], and in cells transiently transfected with GFP-Bub1 56% recovered with a t1/2 of ∼30 seconds [8]. The fission yeast wild-type bub1+ gene has been C-terminally tagged with GFP, such that it is expressed from its own promoter at the endogenous locus, and a range of checkpoint and chromosome segregation assays demonstrate that it is fully functional [21]–[23]. To analyse the dynamics of Bub1-GFP at unattached kinetochores, we used a cold-sensitive tubulin mutant (nda3). These cells arrest early in mitosis, with no microtubules, unattached kinetochores and hyper-condensed chromosomes [24]. In addition, we treated these nda3 cells with anti-microtubule drugs (25 µg/ml carbendazim) to ensure the arrest was maintained. We carried out FRAP experiments and a representative example is shown (Fig. 1A). Analysis of the recovery profiles (see Supplementary material) showed that Bub1-GFP displayed 39 (±16) % recovery, and that the dynamic pool recovered with a half-time of ∼31 (+/−3) seconds (n = 9). This value is mid-way between the two published recovery profiles for vertebrate Bub1.

Bottom Line: Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors.Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1.We propose that the major checkpoint role for Bub1 is as a signalling scaffold.

View Article: PubMed Central - PubMed

Affiliation: The Wellcome Trust Centre for Cell Biology, Institute of Cell Biology, University of Edinburgh, Edinburgh, United Kingdom.

ABSTRACT
The spindle checkpoint delays anaphase onset until all chromosomes have attached in a bi-polar manner to the mitotic spindle. Mad and Bub proteins are recruited to unattached kinetochores, and generate diffusible anaphase inhibitors. Checkpoint models propose that Mad1 and Bub1 act as stable kinetochore-bound scaffolds, to enhance recruitment of Mad2 and Mad3/BubR1, but this remains untested for Bub1. Here, fission yeast FRAP experiments confirm that Bub1 stably binds kinetochores, and by tethering Bub1 to telomeres we demonstrate that it is sufficient to recruit anaphase inhibitors in a kinase-independent manner. We propose that the major checkpoint role for Bub1 is as a signalling scaffold.

Show MeSH
Related in: MedlinePlus