Accurate chromosome segregation crucially depends on the dynamic attachments between chromosomal kinetochores and spindle microtubules. Recent years brought tremendous progress in identifying molecular components of the kinetochores, but mechanistic studies of how these proteins interact with microtubules and enable chromosome motions are lagging behind. We propose to address this deficiency by using reductionist multiscale approaches and innovative assays that reconstitute physiological aspects of kinetochore-microtubule interactions in vitro. We have molecular tools, equipment and expertise to address in a quantitative and rigorous manner some of the most fundamental questions about mitotic chromosome segregation: (1) how the kinetochores convert their initial microtubule-wall binding into microtubule-end attachment, (2) how they subsequently hang onto the microtubule ends and move in conjunction with tubulin assembly/disassembly, (3) and how these mobile links persist under force.
In Aim 1 we will recreate these interactions using purified Ndc80 protein complex and strategically chosen assisting proteins. We recently reconstituted microtubule end conversion by Ndc80, assisted by a plus-end directed kinesin CENP-E. Different Ndc80 variants will be used to uncover the underlying mechanism, and additional kinetochore components will be added to reveal their relative impact. Our findings with purified components will be critically compared with the activity of native kinetochore complexes isolated from extracts of mitotic human cells (Aim 2). We have found that complexes associated with the kinetochore scaffold protein CENP-T can move at the dissembling microtubule ends. This essential achievement lays the groundwork for our functional assays, and subsequent identification and characterization of key kinetochore components for microtubule end coupling in human cells.
In Aim 3 we will use advanced laser tweezers techniques to critically compare the ability of purified proteins and complexes to move under pulling force, mimicking tension between sister kinetochores. The results from these studies will help us to construct an integrative view of the mechano-molecular coupling at human kinetochore, define the specific roles of key kinetochore proteins Ndc80, Ska1 and others, and reveal functional difference between kinetochore complexes assembled with different scaffolds. Little is known about functional behavior of human kinetochore proteins, and our research will undoubtedly provide novel insights into the fundamentals of kinetochore-microtubule interactions, and promote new discoveries in the cell division field.

Public Health Relevance

Errors in chromosome segregation lead to abnormal cell proliferation and genetic abnormalities that are strongly associated with human cancer, pregnancy loss, and developmental defects. The proposed research is relevant to public health because it will facilitate identification of the specific features of the force-transducing links between chromosomal kinetochores and microtubules, which may be exploited for a disruption of abnormal cell proliferation, thereby facilitating development of novel anti-cancer drugs. The proposed research is in line with NIH's mission to foster fundamental creative discoveries that would ultimately advance our capacity to protect and improve human health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Deatherage, James F
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University of Pennsylvania
Schools of Medicine
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Gudimchuk, Nikita; Tarasovetc, Ekaterina V; Mustyatsa, Vadim et al. (2018) Probing Mitotic CENP-E Kinesin with the Tethered Cargo Motion Assay and Laser Tweezers. Biophys J 114:2640-2652
Chakraborty, Manas; Tarasovetc, Ekaterina V; Grishchuk, Ekaterina L (2018) In vitro reconstitution of lateral to end-on conversion of kinetochore-microtubule attachments. Methods Cell Biol 144:307-327
Grishchuk, Ekaterina L (2017) Biophysics of Microtubule End Coupling at the Kinetochore. Prog Mol Subcell Biol 56:397-428
Monda, Julie K; Whitney, Ian P; Tarasovetc, Ekaterina V et al. (2017) Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces. Curr Biol 27:3666-3675.e6
Barisic, Marin; Silva e Sousa, Ricardo; Tripathy, Suvranta K et al. (2015) Mitosis. Microtubule detyrosination guides chromosomes during mitosis. Science 348:799-803
Zakharov, Pavel; Gudimchuk, Nikita; Voevodin, Vladimir et al. (2015) Molecular and Mechanical Causes of Microtubule Catastrophe and Aging. Biophys J 109:2574-2591
Zaytsev, Anatoly V; Mick, Jeanne E; Maslennikov, Evgeny et al. (2015) Multisite phosphorylation of the NDC80 complex gradually tunes its microtubule-binding affinity. Mol Biol Cell 26:1829-44
Vitre, Benjamin; Gudimchuk, Nikita; Borda, Ranier et al. (2014) Kinetochore-microtubule attachment throughout mitosis potentiated by the elongated stalk of the kinetochore kinesin CENP-E. Mol Biol Cell 25:2272-81
Zaytsev, Anatoly V; Sundin, Lynsie J R; DeLuca, Keith F et al. (2014) Accurate phosphoregulation of kinetochore-microtubule affinity requires unconstrained molecular interactions. J Cell Biol 206:45-59
Kononova, Olga; Kholodov, Yaroslav; Theisen, Kelly E et al. (2014) Tubulin bond energies and microtubule biomechanics determined from nanoindentation in silico. J Am Chem Soc 136:17036-45

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