Abstract
The kinetochore is the primary site of interaction between chromosomes and microtubules of the mitotic spindle during chromosome segregation. Kinetochores are composed of more than 100 proteins that transiently assemble during mitosis at a single epigenetically defined region on each chromosome, known as the centromere. Kinetochore assembly and activity must be tightly regulated to ensure proper microtubule interaction and faithful chromosome segregation. Kinetochore malfunction can result in chromosome segregation defects leading to aneuploidy and cell death. As such, cell free and reconstituted systems to analyze kinetochore formation and function are invaluable in probing the biochemical activities of kinetochores. In vitro approaches to studying kinetochores have enabled the manipulation of kinetochore protein structure, function, interactions, and regulation that are not possible in cells. Here we outline a cell-free approach for the assembly of centromeres and recruitment of functional kinetochores that enables their manipulation and analysis.
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References
Westhorpe FG, Straight AF (2013) Functions of the centromere and kinetochore in chromosome segregation. Curr Opin Cell Biol 25:334–340
Fukagawa T, Earnshaw WC (2014) The centromere: chromatin foundation for the kinetochore machinery. Dev Cell 30:496–508
Mitchison TJ, Kirschner MW (1985) Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding. J Cell Biol 101:755–765
Mitchison TJ, Kirschner MW (1985) Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J Cell Biol 101:766–777
Kingsbury J, Koshland D (1991) Centromere-dependent binding of yeast minichromosomes to microtubules in vitro. Cell 66:483–495
Akiyoshi B, Nelson CR, Ranish JA, Biggins S (2009) Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit. Genes Dev 23:2887–2899
Lechner J, Carbon J (1991) A 240 kd multisubunit protein complex, CBF3, is a major component of the budding yeast centromere. Cell 64:717–725
Sorger PK, Severin FF, Hyman AA (1994) Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro. J Cell Biol 127:995–1008
Guse A, Carroll CW, Moree B et al (2011) In vitro centromere and kinetochore assembly on defined chromatin templates. Nature 477:1–7
Guse A, Fuller CJ, Straight AF (2012) A cell-free system for functional centromere and kinetochore assembly. Nat Protoc 7:1847–1869
Murray AW (1991) Cell cycle extracts. In: Kay BK, Peng HB (eds) Xenopus laevis: practical uses in cell and molecular biology, vol 36, Methods in cell biology. Academic, New York, pp 581–605
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Miell, M.D.D., Straight, A.F. (2016). In Vitro Kinetochore Assembly. In: Chang, P., Ohi, R. (eds) The Mitotic Spindle. Methods in Molecular Biology, vol 1413. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3542-0_8
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DOI: https://doi.org/10.1007/978-1-4939-3542-0_8
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