Samejima et al. use quantitative proteomics to investigate how protein subcomplexes assemble into kinetochores on mitotic chromosomes.
Approximately 100 different proteins assemble on centromeric chromatin to form the kinetochores that attach chromosomes to spindle microtubules during mitosis. To better understand the assembly process, Samejima et al. used quantitative mass spectrometry to compare the composition of kinetochores on chromosomes isolated from cell lines lacking various kinetochore components.
The researchers identified groups of proteins whose incorporation into kinetochores was similarly affected in each of the mutant cell lines. These “cohorts” probably represent subcomplexes within the kinetochore and, while some corresponded to known protein complexes, others suggested new details about the kinetochore’s organization in situ. CENP-T, for example, which links the centromere-associated inner kinetochore to the microtubule-binding outer kinetochore, may form a complex with the inner kinetochore proteins CENP-N and CENP-L, because the incorporation of all three proteins into kinetochores was tightly coordinated. Similarly, the outer kinetochore RZZ complex, which is involved in mitotic checkpoint silencing, formed a cohort with three other proteins: Mad1, CENP-E, and Spindly.
Incorporation of the microtubule-binding Ndc80 complex was closely correlated with several different cohorts, suggesting it acts as a “hub” during kinetochore assembly. In fact, Samejima et al.’s data support the idea that there are two pools of Ndc80; one, associated with the microtubule-binding factors Mis12 and Knl1, that would help keep chromosomes attached to the mitotic spindle, and another associated with the putative RZZ/Mad1/CENP-E/Spindly complex, which might coordinate microtubule attachment with checkpoint silencing.
The researchers now want to confirm the physical interactions suggested by their proteomic data and to analyze how the composition of kinetochores is altered by microtubule attachment.