Ultrastructural nuclear import assay.
Ultrastructural nuclear import assay.
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超微结构核输入测定。
DOI：
10.1016/j.ymeth.2006.07.012
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Yaseen,NabeelR
中科院分区：
文献类型：
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作者：
Zhong,Hualin;Shio,Helen;Yaseen,NabeelR
几十年来，电子显微镜 (EM) 一直被用来研究核运输机制。在核输入的早期研究中，将金缀合的核蛋白显微注射到细胞中，然后进行电镜观察。随着核孔复合物 (NPC) 的成分和核输入的可溶性介质的克隆和表征，金缀合抗体被用于通过免疫电镜对核转运机制的成分进行亚定位。此外，金缀合重组蛋白用于探测透化细胞或分离的核膜，并表征这些蛋白在 NPC 上的结合位点。最近，重组金缀合核蛋白被用于体外核输入测定，以帮助剖析核输入机制。我们使用这种超微结构核输入测定来研究转录因子 PU.1 的核输入。结果表明，这种导入需要能量，但与载体无关。在存在能量的情况下，金缀合的 PU.1 转移到 NPC 的核侧和细胞核内部。结合生化测定，这些结果表明这种转变涉及 PU.1 与 NUP153（一种位于 NPC 核侧的核孔蛋白）的 Ran 依赖性结合。在这里，我们详细描述了超微结构核输入测定中使用的方法，包括重组蛋白的制备、金缀合、体外核输入测定、电子显微镜和数据分析。
Electron microscopy (EM) has been used for several decades to study the mechanisms of nuclear transport. In early studies of nuclear import, gold-conjugated nuclear proteins were microinjected into cells and followed by EM. As the components of the nuclear pore complex (NPC) and soluble mediators of nuclear import were cloned and characterized, gold-conjugated antibodies were utilized to sublocalize the components of the nuclear transport machinery by immuno-EM. Further, gold-conjugated recombinant proteins were used to probe permeabilized cells or isolated nuclear envelopes and characterize binding sites for these proteins at the NPC. More recently, recombinant gold-conjugated nuclear proteins were used in in vitro nuclear import assays to help dissect the mechanisms of nuclear import. We have used this ultrastructural nuclear import assay to study the nuclear import of the transcription factor PU.1. The results showed that this import requires energy but is carrier-independent. In the presence of energy, gold-conjugated PU.1 shifted to the nuclear side of the NPC and the inside of the nucleus. In conjunction with biochemical assays, these results indicated that this shift involved Ran-dependent binding of PU.1 to NUP153, a nucleoporin situated at the nuclear side of the NPC. Here we describe in detail the methods used in the ultrastructural nuclear import assay including preparation of recombinant protein, gold conjugation, in vitro nuclear import assay, electron microscopy, and data analysis.