Department of Biological Sciences, Center for Photochemical Sciences, Bowling Green State University, Bowling Green, OH 43403, USA.
Integr Biol (Camb). 2012 Jan;4(1):10-21. doi: 10.1039/c1ib00041a. Epub 2011 Oct 24.
In eukaryotic cells, the bidirectional trafficking of proteins and genetic materials across the double-membrane nuclear envelope is mediated by nuclear pore complexes (NPCs). A highly selective barrier formed by the phenylalanine-glycine (FG)-nucleoporin (Nup) in the NPC allows for two transport modes: passive diffusion and transport receptor-facilitated translocation. Strict regulation of nucleocytoplasmic transport is crucial for cell survival, differentiation, growth and other essential activities. However, due to the limited knowledge of the native configuration of the FG-Nup barrier and the interactions between the transiting molecules and the barrier in the NPC, the precise nucleocytoplasmic transport mechanism remains unresolved. To refine the transport mechanism, single-molecule fluorescence microscopy methods have been employed to obtain the transport kinetics of individual fluorescent molecules through the NPC and to map the interactions between transiting molecules and the FG-Nup barrier. Important characteristics of nucleocytoplasmic transport, such as transport time, transport efficiency and spatial distribution of single transiting molecules in the NPC, have been obtained that could not be measured by either ensemble average methods or conventional electron microscopy. In this critical review, we discuss the development of various single-molecule techniques and their application to nucleocytoplasmic transport in vitro and in vivo. In particular, we highlight a recent advance from one-dimensional to three-dimensional single-molecule characterization of transport through the NPC and present a comprehensive understanding of the nucleocytoplasmic transport mechanism obtained by this new technical development (105 references).
在真核细胞中，蛋白质和遗传物质在双层核膜之间的双向运输是由核孔复合物（NPC）介导的。NPC 中由苯丙氨酸-甘氨酸（FG）-核孔蛋白（Nup）形成的高度选择性屏障允许两种运输模式：被动扩散和运输受体促进的易位。核质转运的严格调节对于细胞存活、分化、生长和其他基本活动至关重要。然而，由于对 FG-Nup 屏障的天然构象以及转运分子与 NPC 中屏障之间的相互作用的了解有限，精确的核质转运机制仍未解决。为了完善转运机制，已经采用单分子荧光显微镜方法来获得单个荧光分子通过 NPC 的转运动力学，并绘制转运分子与 FG-Nup 屏障之间的相互作用图。已经获得了核质转运的重要特征，例如转运时间、转运效率和 NPC 中单转运分子的空间分布，这些特征无法通过集合平均方法或传统电子显微镜来测量。在这篇评论中，我们讨论了各种单分子技术的发展及其在体外和体内核质转运中的应用。特别是，我们强调了从一维到 NPC 中转运的三维单分子表征的最新进展，并通过这一新技术发展获得了对核质转运机制的全面理解（105 篇参考文献）。
