Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes1,2. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules3. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B4,5. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules6. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanism that does not require Aurora B7-10. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

Summary Kinetochores are the macromolecular complexes that interact with microtubules to mediate chromosome segregation [1]. Accurate segregation requires that kinetochores make bioriented attachments to microtubules from opposite poles. Attachments between kinetochores and microtubules are monitored by the spindle checkpoint, a surveillance system that prevents anaphase until every pair of chromosomes makes proper bioriented attachments [2]. Checkpoint activity is correlated with the recruitment of checkpoint proteins to the kinetochore [1]. Mps1 is a conserved protein kinase that regulates segregation and the spindle checkpoint, but few of the targets that mediate its functions have been identified. Here, we show that Mps1 is the major kinase activity that co-purifies with budding yeast kinetochore particles and identify the conserved Spc105/KNL-1/Blinkin kinetochore protein as a substrate. Phosphorylation of conserved MELT motifs within Spc105 recruits the Bub1 protein to kinetochores and this is reversed by protein phosphatase I (PP1). Spc105 mutants lacking Mps1 phosphorylation sites are defective in the spindle checkpoint and exhibit growth defects. Together, these data identify Spc105 as a key target of the Mps1 kinase and show that the opposing activities of Mps1 and PP1 regulate the kinetochore localization of the Bub1 protein.

Chromosome segregation depends on kinetochores, the structures that mediate chromosome attachment to the mitotic spindle. We isolated mutants in IPL1, which encodes a protein kinase, in a screen for budding yeast mutants that have defects in sister chromatid separation and segregation. Cytological tests show that ipl1 mutants can separate sister chromatids but are defective in chromosome segregation. Kinetochores assembled in extracts from ipl1 mutants show altered binding to microtubules. Ipl1p phosphorylates the kinetochore component Ndc10p in vitro and we propose that Ipl1p regulates kinetochore function via Ndc10p phosphorylation. Ipl1p localizes to the mitotic spindle and its levels are regulated during the cell cycle. This pattern of localization and regulation is similar to that of Ipl1p homologs in higher eukaryotes, such as the human aurora2 protein. Because aurora2 has been implicated in oncogenesis, defects in kinetochore function may contribute to genetic instability in human tumors.

The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating Ipl1p allows cells overexpressing Mps1p to escape from mitosis and segregate their chromosomes normally. Therefore, the requirement for Ipl1p in the spindle checkpoint is not a consequence of kinetochore and/or spindle defects. The requirement for Ipl1p distinguishes two different activators of the spindle checkpoint: Ipl1p function is required for the delay triggered by chromosomes whose kinetochores are not under tension, but is not required for arrest induced by spindle depolymerization. Ipl1p localizes at or near kinetochores during mitosis, and we propose that Ipl1p is required to monitor tension at the kinetochore. The accurate propagation of genetic information depends on faithful chromosome segregation. Accurate chromosome segregation depends on the precise coordination of events in the chromosome cycle. When chromosomes replicate during S phase, linkage between the sister chromatids (cohesion) is established and must be maintained while chromosomes condense and align on the mitotic spindle. Chromosomes attach to the mitotic spindle by their kinetochores, specialized protein structures that are assembled on centromeric DNA sequences. Once all the chromosomes are correctly aligned on the mitotic spindle, sister chromatid cohesion must dissolve promptly at anaphase to allow the sister chromatids to segregate rapidly to opposite poles of the mitotic spindle. Defects in any of these steps can result in aneuploidy, a hallmark of tumor cells and some birth defects (Lengauer et al. 1997.The spindle checkpoint prevents cells from separating their sister chromatids until chromosome alignment is complete. The conserved components of the checkpoint include the Mad (Mad1-Mad3) proteins, Bub1 and Bub3, Mps1 (a protein kinase), and Cdc55 (Hoyt et al. 1991;Li and Murray 1991;Minshull et al. 1996;Weiss and Winey 1996;Wang and Burke 1997). A separate control, the Bub2-dependent checkpoint, monitors a second aspect of chromosome segregation, the delivery of DNA or a spindle pole body into the daughter cell (Alexandru et al. 1999;Fesquet et al. 1999;Fraschini et al. 1999;Li 1999). Spindle checkpoint defects are associated with genetic instability, and some human cancers contain mutant spindle checkpoint genes (Cahill et al. 1998;Takahashi et al. 1999) The spindle checkpoint monitors the interaction between kinetochores and microtubules. Spindle checkpoint proteins localize to kinetochores (Chen et al. 1996;Taylor and McKeon 1997;Bernard et al. 1998), and all known kinetochore and spindle defects that activate the checkpoint affect the i...

Deep learning for protein interactions The use of deep learning has revolutionized the field of protein modeling. Humphreys et al . combined this approach with proteome-wide, coevolution-guided protein interaction identification to conduct a large-scale screen of protein-protein interactions in yeast (see the Perspective by Pereira and Schwede). The authors generated predicted interactions and accurate structures for complexes spanning key biological processes in Saccharomyces cerevisiae . The complexes include larger protein assemblies such as trimers, tetramers, and pentamers and provide insights into biological function. —VV