Abstract
The spindle assembly checkpoint detects errors in kinetochore attachment to the spindle including insufficient microtubule occupancy and absence of tension across bi-oriented kinetochore pairs. Here, we analyse how the kinetochore localization of the Drosophila spindle checkpoint proteins Bub1, Mad2, Bub3 and BubR1, behave in response to alterations in microtubule binding or tension. To analyse the behaviour in the absence of tension, we treated S2 cells with low doses of taxol to disrupt microtubule dynamics and tension, but not kinetochore-microtubule occupancy. Under these conditions, we found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does. Consistently, in mono-oriented chromosomes, both kinetochores accumulate BubR1 whereas Bub1 and Mad2 only localize at the unattached kinetochore. To study the effect of tension we analysed the kinetochore localization of spindle checkpoint proteins in relation to tension-sensitive kinetochore phosphorylation recognised by the 3F3/2 antibody. Using detergent-extracted S2 cells as a system in which kinetochore phosphorylation can be easily manipulated, we observed that BubR1 and Bub3 accumulation at kinetochores is dependent on the presence of phosphorylated 3F3/2 epitopes. However, Bub1 and Mad2 localize at kinetochores regardless of the 3F3/2 phosphorylation state. Altogether, our results suggest that spindle checkpoint proteins sense distinct aspects of kinetochore interaction with the spindle, with Mad2 and Bub1 monitoring microtubule occupancy while BubR1 and Bub3 monitor tension across attached kinetochores.
Highlights
IntroductionEukaryotic cells have evolved a surveillance mechanism known as the spindle assembly checkpoint (Murray, 1994), kinetochore attachment checkpoint (Rieder et al, 1994), chromosome distribution checkpoint (Nicklas, 1997) or the mitotic checkpoint
During mitosis, chromosome segregation is carefully monitored to prevent aneuploidy
We found that Mad2 and Bub1 do not accumulate at metaphase kinetochores whereas BubR1 does
Summary
Eukaryotic cells have evolved a surveillance mechanism known as the spindle assembly checkpoint (Murray, 1994), kinetochore attachment checkpoint (Rieder et al, 1994), chromosome distribution checkpoint (Nicklas, 1997) or the mitotic checkpoint. This checkpoint guarantees that anaphase onset is initiated only when all chromosomes are properly attached to microtubules and aligned at the metaphase plate. Homologues of many of these genes have been identified in Saccharomyces pombe, Drosophila melanogaster, Xenopus laevis, Mus musculus and Homo sapiens (reviewed by Musacchio and Hardwick, 2002). Antibody microinjection in tissue culture cells and expression of truncated proteins with a dominant negative phenotype, have shown that inhibition of a single protein inactivates the checkpoint (reviewed by Amon, 1999), allowing for sister chromatid separation in the absence of microtubules
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