Reconstitution of nuclear import in permeabilized cells.
Reconstitution of nuclear import in permeabilized cells.
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DOI：
10.1007/978-1-60327-461-6_11
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
Gerace, Larry
中科院分区：
文献类型：
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作者：
Cassany, Aurelia;Gerace, Larry
真核细胞的蛋白和RNA货物在细胞质和细胞核之间的运输是参与细胞调控的主要途径，它是由货物中的核运输序列和穿梭运输因子介导的。后者包括识别货物并携带它们通过核孔复合体（NPC）的受体（核丝蛋白）和调节核丝蛋白货物结合的小GTPase Ran。核输入可以在体外使用洋地黄苷渗透细胞进行研究，这些细胞缺乏穿梭运输因子。核输入可以用外源性细胞质或纯化的重组转运因子在渗透细胞中重建，并可以通过荧光标记货物的光镜或免疫荧光染色进行定量。在这里，我们描述了体外核导入渗透的哺乳动物细胞的程序，以及重组运输因子（进口蛋白α，进口蛋白β，进口蛋白7，运输蛋白，Ran, NTF2）和其他常用试剂的制备。该分析提供了表征核进口分子机制和研究特定货物进口要求的手段。
The trafficking of protein and RNA cargoes between the cytoplasm and the nucleus of eukaryotic cells, which is a major pathway involved in cell regulation, is mediated by nuclear transport sequences in the cargoes and by shuttling transport factors. The latter include receptors (karyopherins) that recognize the cargoes and carry them across the nuclear pore complex (NPC), and the small GTPase Ran, which modulates karyopherin–cargo binding. Nuclear import can be studied in vitro using digitonin-permeabilized cells, which are depleted of shuttling transport factors. Nuclear import can be reconstituted in the permeabilized cells with exogenous cytosol or with purified recombinant transport factors, and can be quantified by light microscopy of fluorescently labeled cargoes or by immunofluorescence staining. Here we describe procedures for in vitro nuclear import in permeabilized mammalian cells, and for the preparation of recombinant transport factors (importin α, importin β, importin 7, transportin, Ran, NTF2) and other reagents commonly used in the assay. This assay provides means to characterize the molecular mechanisms of nuclear import and to study the import requirements of specific cargoes.