title: In Vitro Kinetochore Assembly.
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.
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Beckman 409A, 404 Stanford Serra Mall, Stanford, CA, 94305, USA.
authors.forename: Matthew D D
authors.initials: MD
authors.lastname: Miell
authors.affiliation: Department of Biochemistry, Stanford University School of Medicine, Beckman 409A, 404 Stanford Serra Mall, Stanford, CA, 94305, USA. astraigh@stanford.edu.
authors.forename: Aaron F
authors.initials: AF
authors.lastname: Straight
mesh_terms: D000818:Animals; D059447:Cell Cycle Checkpoints; D002457:Cell Extracts; D002503:Centromere / Q000235:genetics / Q000378:metabolism; D000076247:Centromere Protein A / Q000235:genetics / Q000378:metabolism; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D018386:Kinetochores / Q000378:metabolism*; D008677:Metaphase; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008938:Mitosis; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D055503:Protein Multimerization; D008941:Spindle Apparatus / Q000378:metabolism; D014981:Xenopus
chemical_list: D002457:Cell Extracts; D000076247:Centromere Protein A; D002843:Chromatin; D006657:Histones; D009707:Nucleosomes
keywords: Centromere; Chromatin; In vitro assembly; Kinetochore; Xenopus egg extract