Zhong Zhixia, Wilson Katherine L, Dahl Kris Noel
Department of Chemical Engineering, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, USA.
Methods Cell Biol. 2010;98:97-119. doi: 10.1016/S0091-679X(10)98005-9.
The nucleus is bordered by a double bilayer nuclear envelope, communicates with the cytoplasm via embedded nuclear pore complexes, and is structurally supported by an underlying nucleoskeleton. The nucleoskeleton includes nuclear intermediate filaments formed by lamin proteins, which provide major structural and mechanical support to the nucleus. However, other structural proteins also contribute to the function of the nucleoskeleton and help connect it to the cytoskeleton. This chapter reviews nucleoskeletal components beyond lamins and summarizes specific methods and strategies useful for analyzing nuclear structural proteins including actin, spectrin, titin, linker of nucleoskeleton and cytoskeleton (LINC) complex proteins, and nuclear spindle matrix proteins. These components can localize to highly specific functional subdomains at the nuclear envelope or nuclear interior and can interact either stably or dynamically with a variety of partners. These components confer upon the nucleoskeleton a functional diversity and mechanical resilience that appears to rival the cytoskeleton. To facilitate the exploration of this understudied area of biology, we summarize methods useful for localizing, solubilizing, and immunoprecipitating nuclear structural proteins, and a state-of-the-art method to measure a newly-recognized mechanical property of nucleus.
细胞核由双层核膜包被，通过嵌入其中的核孔复合体与细胞质进行物质交换，并由其下方的核骨架提供结构支撑。核骨架包括由核纤层蛋白形成的核中间丝，为细胞核提供主要的结构和机械支撑。然而，其他结构蛋白也对核骨架的功能有贡献，并有助于将其与细胞骨架连接起来。本章综述了核纤层蛋白以外的核骨架成分，并总结了用于分析包括肌动蛋白、血影蛋白、肌联蛋白、核骨架与细胞骨架连接体（LINC）复合体蛋白以及核纺锤体基质蛋白在内的核结构蛋白的具体方法和策略。这些成分可定位于核膜或核内高度特异的功能亚域，并能与多种伙伴稳定或动态地相互作用。这些成分赋予核骨架功能多样性和机械弹性，其程度似乎可与细胞骨架相媲美。为便于探索这一尚未得到充分研究的生物学领域，我们总结了用于定位、溶解和免疫沉淀核结构蛋白的有用方法，以及一种测量细胞核新发现的力学性质的先进方法。
