Kinetochores are macromolecular protein structures assembled on chromosomal loci called centromeres. They physically link centromeres to microtubules emanating from opposite spindle poles which is a prerequisite for the faithful segregation of sister chromatids in mitosis. Defects in kinetochore-microtubule attachments lead to aneuploidy, which is associated with tumorigenesis, congenital trisomies, and aging .
The main kinetochore functions that contribute to proper chromosome segregation can be assigned to distinct structural modules. The inner kinetochore is built of at least 16 subunits of the so called constitutive centromere associated network (CCAN) complex which provides a structural framework for the cell cycle regulated assembly of the outer kinetochore. The KMN (KNL1-, Mis12 and Ndc80) network forms a load-bearing microtubule-binding interface and facilitates chromosome movements. Kinetochore modules are also involved in two regulatory feedback mechanisms that are essential for the fidelity of chromosome segregation. An error correction mechanism positions the Aurora B kinase activity to discriminate between correct and improper microtubule attachments. Kinetochores are composed of ~60 proteins in S. cerevisiae and ~100 proteins in humans. Proteomic analyses of purified stable kinetochore subcomplexes and bioinformatic sequence analysis revealed that most of their subunits are conserved from yeast to humans. Although X-ray structures of the KMN network unveiled the operation of a key component, the structural analysis of native kinetochore complexes will reveal functional characteristics that cannot be deduced from studies of individual parts.
Ground breaking objectives and specific aims
Aim1. Molecular architecture of native kinetochores assembled on budding yeast centromeres
The CXMS analysis of native centromere-associated kinetochores purified from budding yeast minichromosomes will identify the protein-protein interactions of the CCAN complex by a comprehensive set of distance restraints within the whole ensemble of kinetochore proteins indicating how it is linked to distinct centromeric nucleosomes and how the kinetochore structure integrates the chromosomal passenger complex (CPC) and facilitates error correction.
Aim2. Molecular mechanisms of the cell cycle regulated assembly of the human kinetochore
I will perform a systematic analysis of the dynamics of phosphorylation levels, their kinase dependencies and of the changes in protein stoichiometries of soluble and nucleosome-associated kinetochore complexes isolated from different cell cycle stages. Monitoring the molecular changes in a time-resolved manner is crucial for understanding the tight temporal control of kinetochore assembly in mitosis.
Aim3. Epigenetic marks and topology of human nucleosome-associated complexes facilitating CENP-A replenishment at mitotic exit
The CXMS analysis of nucleosome-proximal and –distal associated complexes will delineate the architecture of human CCAN complexes. The determination of post-translational modifications and the topology of complexes that specifically assemble at centromeric nucleosomes at mitotic exit will elucidate how the spatial proximity to CENP-A nucleosomes marks histone H3 for replacement.
Conclusion of the action
The results of the ERC project showed that the interaction of the chromosomal passenger complex with the inner kinetochore positions the Ipl1/Aurora B kinase, which is important for accurate chromosome segregation. Our work further demonstrated the potential of the quantitative crosslinking and mass spectrometry technology for estimating protein affinities and characterizing mechanistic effects on protein assemblies upon post-translational modifications or cofactor binding.
