The spindle assembly checkpoint (SAC) prevents anaphase until all kinetochores attach to the spindle. Each mammalian kinetochore binds many microtubules, but how many attached microtubules are required to turn off the checkpoint, and how the kinetochore monitors microtubule numbers, are not known and are central to understanding SAC mechanisms and function. To address these questions, here we systematically tune and fix the fraction of Hec1 molecules capable of microtubule binding. We show that Hec1 molecules independently bind microtubules within single kinetochores, but that the kinetochore does not independently process attachment information from different molecules. Few attached microtubules (20% occupancy) can trigger complete Mad1 loss, and Mad1 loss is slower in this case. Finally, we show using laser ablation that individual kinetochores detect changes in microtubule binding, not in spindle forces that accompany attachment. Thus, the mammalian kinetochore responds specifically to the binding of each microtubule and counts microtubules as a single unit in a sensitive and switch-like manner. This may allow kinetochores to rapidly react to early attachments and maintain a robust SAC response despite dynamic microtubule numbers.
During mitosis, the Bub1–Bub3 complex concentrates at kinetochores, the microtubule-coupling interfaces on chromosomes, where it contributes to spindle checkpoint activation, kinetochore-spindle microtubule interactions, and protection of centromeric cohesion. Bub1 has a conserved N-terminal tetratricopeptide repeat (TPR) domain followed by a binding motif for its conserved interactor Bub3. The current model for Bub1–Bub3 localization to kinetochores is that Bub3, along with its bound motif from Bub1, recognizes phosphorylated “MELT” motifs in the kinetochore scaffold protein Knl1. Motivated by the greater phenotypic severity of BUB-1 versus BUB-3 loss in C. elegans, we show that the BUB-1 TPR domain directly recognizes a distinct class of phosphorylated motifs in KNL-1 and that this interaction is essential for BUB-1–BUB-3 localization and function. BUB-3 recognition of phospho-MELT motifs additively contributes to drive super-stoichiometric accumulation of BUB-1–BUB-3 on its KNL-1 scaffold during mitotic entry. Bub1’s TPR domain interacts with Knl1 in other species, suggesting that collaboration of TPR-dependent and Bub3-dependent interfaces in Bub1–Bub3 localization and functions may be conserved.
more » « less- PAR ID:
- 10499166
- Publisher / Repository:
- DOI PREFIX: 10.1083
- Date Published:
- Journal Name:
- Journal of Cell Biology
- Volume:
- 223
- Issue:
- 7
- ISSN:
- 0021-9525
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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