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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - MolStruKT (Molecular structure and cell cycle regulated assembly of the kinetochore)

Teaser

Summary

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 .

This research project investigates the architecture of endogenous centromere-assembled kinetochores and will elucidate how feedback control mechanisms are integrated into the kinetochore structure, detect the type and dynamics of modifications orchestrating the assembly of the human kinetochore and will identify the epigenetic marks and architecture of nucleosome-associated complexes mediating CENP-A replenishment at centromeric chromatin after mitosis. All three goals are central to the understanding of kinetochore function in chromosome segregation and represent a hybrid structural and mechanistic analysis of the most complex cellular protein structure to date.

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 (for KNL1-, Mis12 and Ndc80 complex) 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. A surveillance mechanism, called the spindle checkpoint, monitors the state of kinetochore-microtubule attachments and halts cell cycle progression until all attachment errors are corrected. 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. I have recently demonstrated that the analysis of a chromatin remodeler in complex with its nucleosome substrate by CXMS provides detailed mechanistic insights and establishes CXMS as a key technology for the topological analysis of native chromatin-assembled protein complexes.

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 complex

Work performed

Aim 1 has been split up into 3 projects which are all focusing on elucidating the differences between budding yeast and human kinetochore architectures. The first subproject (1.1) revealed a yeast specific architecture of the inner kinetochore assembled on the budding yeast point centromere which elucidated the kinetochore build-up that links a single nucleosome to a single microtubule and investigated the interaction of the kinetochore with the chromosomal passenger complex, a key regulator of chromosome segregation. Our characterization of the binding interface of the chromosomal passenger complex with the inner kinetochore COMA complex provided initial insights into the molecular understanding of the error correction mechanism which is crucial for the accurate segregation of chromosomes during cell division. This work is currently under review at eLife. I have summarized the key observations of this study in detail in the following paragraph.
The second subproject (1.2) highlights the cooperativity of outer kinetochore assembly supporting the load-bearing attachment of a single kinetochore unit to one microtubule. This work has been submitted to Nature communications. In the following paragraph I have summarized the key findings of this project in more detail.
The third subproject (1.3) investigates alternative pathways of kinetochore assembly where the outer kinetochore is recruited through previously unknown interactions to the nucleosome which could be part of an important kinetochore conformation ensuring the correct segregation of chromosomes.

In Aim 2 we study the effect of post-translational modifications for the kinetochore assembly at CENP-A containing nucleosomes. Here, we discovered the importance of a single phosphorylation site that contributes to the stabilization of the kinetochore structure in order to withstand microtubule pulling forces in mitosis. This project is part of our method development effort.

To provide molecular insights into the maintenance of centromeric chromatin (Aim 3) we have isolated nucleosome-associated complexes from different cell cycle stages. Their mass spectrometric analysis uncovered stage-specific protein complexes which are in good agreement with the literature and include promising candidates that so far have not been implicated in this process. We are currently following up on the candidates in order to assess their in vivo relevance for CENP-A incorporation and maintenance. These efforts include the biochemical characterization of CENP-A associated complexes as well as loss-of-function assays to study the effect on CENP-A levels at centromere chromatin.

Final results

We have used the crosslinking and mass spectrometry technique to identify so far unknown protein-protein interactions and characterized the physiological relevance of their interfaces in vivo. The in vitro and in vivo data together show that the C-terminus of Ctf19 is a primary Sli15 binding site within COMA, suggesting that Ctf19/Mcm21, linked to Cse4(CENP-A) nucleosomes through Ame1/Okp1, is important for positioning Ipl1 activity close to Cse4 nucleosomes independently of Bir1. The functional importance of the interaction of Sli15 with Ctf19 suggests that positioning of Ipl1 at the inner kinetochore may facilitate selective recognition and destabilization of erroneous microtubule attachments upon loss of tension. Our observations support a model that places COMA as at the centrepiece of kinetochore assembly in budding yeast and contributes to the molecular understanding of the fascinating question of how cells establish correct chromosome biorientation at the mitotic spindle (Figure).

The ongoing projects focus on elucidating the molecular mechanisms of how budding yeast kinetochores are stabilized on Cse4(CENP-A) nucleosomes and how the kinetochore assists in maintaining CENP-A levels at the centromere.