Reconstitution and Functional Analysis of Kinetochore Subcomplexes

被引：14

作者：

Gestaut, Daniel R. ^{[1
]}

Cooper, Jeremy ^{[3
]}

Asbury, Charles L. ^{[2
]}

Davis, Trisha N. ^{[1
]}

Wordeman, Linda ^{[2
]}

机构：

[1] Univ Washington, Dept Biochem, Seattle, WA 98195 USA

[2] Univ Washington, Dept Physiol & Biophys, Seattle, WA 98195 USA

[3] Appl Precis Inc, Res Engineer, Issaquah, WA 98027 USA

来源：

MICROTUBULES, IN VITRO: MICROTUBULES, IN VITRO | 2010年 / 95卷

关键词：

DAM1; COMPLEX; MICROTUBULE; MOVEMENT; PROTEIN; GLASS;

D O I：

10.1016/S0091-679X(10)95032-2

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

Kinetochores are multifunctional supercomplexes that link chromosomes to dynamic microtubule tips. Groups of proteins from the kinetochore are arranged into distinct subcomplexes that copurify under stringent conditions and cause similar phenotypes when mutated. By coexpressing all the components of a given subcomplex from a polycistronic plasmid in bacteria, many laboratories have had great success in purifying active subcomplexes. This has enabled the study of how the microtubule-binding subcomplexes of the kinetochore interact with both the microtubule lattice and dynamic microtubule tips. Here we outline methods for rapid cloning of polycistronic vectors for expression of kinetochore subcomplexes, their purification, and techniques for functional analysis using total internal reflection fluorescence microscopy (TIRFM).

引用

页码：641 / 656

页数：16

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