Periodic Reporting for period 4 - MolStruKT (Molecular structure and cell cycle regulated assembly of the kinetochore)
Periodo di rendicontazione: 2019-12-01 al 2020-11-30
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.
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.
The initial experiment of Aim 1 was the systematic XLMS analysis of endogenous budding yeast kinetochore complexes. A comprehensive map of about 400 inter-protein crosslinks showed the protein connectivity of the entire kinetochore structure from the centromeric nucleosome to the microtubule-binding site.
The work of Aim 1.1 was published in eLife (Fischböck-Halwachs et al., 2019) and investigated the binding interfaces of the inner kinetochore to the chromosomal passenger complex (CPC) and the role of this interaction in the error correction mechanism. The Sli15/Ipl1 core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affected chromosome segregation fidelity in Sli15 wild-type cells. This study showed molecular characteristics of the point-centromere kinetochore architecture and suggests a role for the inner kinetochore Ctf19 C-terminus in mediating accurate chromosome segregation.
The study of Aim 1.2 'C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast', was published in Cell Reports (Ghodgaonkar-Steger et al., 2020). We identified 225 inter-protein crosslinks by mass spectrometry on the outer kinetochore KMN network isolated from Saccharomyces cerevisiae that delineate its subunit connectivity. We showed that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association.
The project of Aim1.3 identified a previously unknown interaction of the centromere-associated CBF3 complex with the outer kinetochore in mitosis. Our results suggested that the CBF3 complex represents a mitosis-specific pathway of recruiting the outer kinetochore within the point-centromere kinetochore architecture that directly links the centromeric nucleosome to a single microtubule and enhances force transmission for chromosome segregation (submitted).
In Aim 2 I proposed to investigate the molecular mechanisms of the cell cycle regulated assembly of the human kinetochore. This plan was changed and two PhD students started to develop a quantitative XLMS workflow, which estimated binding affinities and facilitated monitoring the assembly of multi-subunit complexes and their stabilization through phosphorylation. This work demonstrated the potential of quantitative XLMS for characterizing mechanistic effects on protein assemblies upon post-translational modifications or cofactor interaction and for biological modelling (in revision).
The goal of the proposed work in Aim 3 had to be adapted. The mass spectrometric analysis of the initial large-scale pulldowns of CENP-A nucleosomes from HeLa cells synchronized from early G1 to late S-phase identified previously unknown cell cycle specific interactors. In follow-up experiments of this proteomics screen we investigated the role of a chromatin remodeler that might facilitate CENP-A incorporation in early G1 phase and of chromatin-associated factors guiding CENP-A redistribution to the leading and lagging strand during DNA replication. This study is still ongoing.
The work of Aim 1.1 was published in eLife (Fischböck-Halwachs et al., 2019) and investigated the binding interfaces of the inner kinetochore to the chromosomal passenger complex (CPC) and the role of this interaction in the error correction mechanism. The Sli15/Ipl1 core-CPC interacted with COMA in vitro through the Ctf19 C-terminus whose deletion affected chromosome segregation fidelity in Sli15 wild-type cells. This study showed molecular characteristics of the point-centromere kinetochore architecture and suggests a role for the inner kinetochore Ctf19 C-terminus in mediating accurate chromosome segregation.
The study of Aim 1.2 'C-Terminal Motifs of the MTW1 Complex Cooperatively Stabilize Outer Kinetochore Assembly in Budding Yeast', was published in Cell Reports (Ghodgaonkar-Steger et al., 2020). We identified 225 inter-protein crosslinks by mass spectrometry on the outer kinetochore KMN network isolated from Saccharomyces cerevisiae that delineate its subunit connectivity. We showed that a hub of three C-terminal MTW1 subunit motifs mediates the cooperative stabilization of the KMN network, which is augmented by a direct NDC80-SPC105 association.
The project of Aim1.3 identified a previously unknown interaction of the centromere-associated CBF3 complex with the outer kinetochore in mitosis. Our results suggested that the CBF3 complex represents a mitosis-specific pathway of recruiting the outer kinetochore within the point-centromere kinetochore architecture that directly links the centromeric nucleosome to a single microtubule and enhances force transmission for chromosome segregation (submitted).
In Aim 2 I proposed to investigate the molecular mechanisms of the cell cycle regulated assembly of the human kinetochore. This plan was changed and two PhD students started to develop a quantitative XLMS workflow, which estimated binding affinities and facilitated monitoring the assembly of multi-subunit complexes and their stabilization through phosphorylation. This work demonstrated the potential of quantitative XLMS for characterizing mechanistic effects on protein assemblies upon post-translational modifications or cofactor interaction and for biological modelling (in revision).
The goal of the proposed work in Aim 3 had to be adapted. The mass spectrometric analysis of the initial large-scale pulldowns of CENP-A nucleosomes from HeLa cells synchronized from early G1 to late S-phase identified previously unknown cell cycle specific interactors. In follow-up experiments of this proteomics screen we investigated the role of a chromatin remodeler that might facilitate CENP-A incorporation in early G1 phase and of chromatin-associated factors guiding CENP-A redistribution to the leading and lagging strand during DNA replication. This study is still ongoing.
The project resulted in two major contributions to the fields of cell cycle research and structural proteomics. Our findings on the role of the kinetochore structure in the error correction mechanism provided experimental evidence for the molecular understanding of how cells ensure accurate chromosome segregation, which is essential for the proliferation of all living organisms.
We developed a quantitative crosslinking and mass spectrometric technology and demonstrated its potential for biomedical applications in the characterization of protein therapeutics.
We developed a quantitative crosslinking and mass spectrometric technology and demonstrated its potential for biomedical applications in the characterization of protein therapeutics.