Deciphering the Structure and Function of Nuclear Pores Using Single-Molecule Fluorescence Approaches.
Deciphering the Structure and Function of Nuclear Pores Using Single-Molecule Fluorescence Approaches.
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DOI：
10.1016/j.jmb.2016.02.023
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发表时间：
2016-05-22
影响因子：
5.6
通讯作者：
Grünwald D
中科院分区：
文献类型：
--
作者：
Musser SM;Grünwald D
由于核孔复合体（NPC）在大分子运输和核质信息传递中起着核心作用，人们使用多种方法对其进行了详细研究。因此，其结构、一般功能以及在细胞生命周期中的作用等许多方面都已被充分了解。在过去十年中，荧光显微镜方法使得能够实时观察与核孔复合体相互作用并通过它的单个分子，从而能够以纳米精度研究新的问题。虽然最初的单分子研究主要集中在使用透化细胞的输入途径上，但最近已证明在活细胞中研究mRNA的输出是可行的。单分子分析能够解决其他方法难以或无法回答的问题，然而核质运输的复杂性要求解释必须基于坚实的遗传、生化和结构基础。此外，概念上简单的单分子实验在技术上仍然具有挑战性，特别是在信号强度、信噪比以及噪声、随机性和精度分析方面。我们讨论了最近通过单分子显微镜解决的核运输问题，评估了现有分析方法和数据的局限性，并确定了未来研究的未解决问题。我们预计单分子荧光（SMF）方法将继续应用于尚未解决的核质运输问题，并且为核孔复合体研究开发的方法可扩展到细胞内其他复杂系统和途径。
Due to its central role in macromolecular trafficking and nucleocytoplasmic information transfer, the nuclear pore complex (NPC) has been studied in great detail using a wide spectrum of methods. Consequently, many aspects of its architecture, general function and role in the life cycle of a cell are well understood. Over the last decade, fluorescence microscopy methods have enabled the real-time visualization of single molecules interacting with and transiting through the NPC, allowing novel questions to be examined with nanometer precision. While initial single molecule studies focused primarily on import pathways using permeabilized cells, it has recently proven feasible to investigate the export of mRNAs in living cells. Single molecule assays can address questions that are difficult or impossible to answer by other means, yet the complexity of nucleocytoplasmic transport requires that interpretation be based on a firm genetic, biochemical, and structural foundation. Moreover, conceptually simple single molecule experiments remain technically challenging, particularly with regards to signal intensity, signal-to-noise ratio, and the analysis of noise, stochasticity, and precision. We discuss nuclear transport issues recently addressed by single molecule microscopy, evaluate the limits of existing assays and data, and identify open questions for future studies. We expect that single molecule fluorescence (SMF) approaches will continue to be applied to outstanding nucleocytoplasmic transport questions, and that the approaches developed for NPC studies are extendable to additional complex systems and pathways within cells.