Cardarelli Francesco
Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127 Pisa, Italy.
Comput Struct Biotechnol J. 2017 Apr 4;15:299-306. doi: 10.1016/j.csbj.2017.03.005. eCollection 2017.
Molecules are continuously shuttling across the nuclear envelope barrier that separates the nucleus from the cytoplasm. Instead of being just a barrier to diffusion, the nuclear envelope is rather a complex filter that provides eukaryotes with an elaborate spatiotemporal regulation of fundamental molecular processes, such as gene expression and protein translation. Given the highly dynamic nature of nucleocytoplasmic transport, during the past few decades large efforts were devoted to the development and application of time resolved, fluorescence-based, biophysical methods to capture the details of molecular motion across the nuclear envelope. These methods are here divided into three major , according to the differences in the way they report on the molecular process of nucleocytoplasmic transport. In detail, the first class encompasses those methods based on the perturbation of the fluorescence signal, also known as ensemble-averaging methods, which average the behavior of many molecules (across many pores). The second class comprises those methods based on the localization of fluorescently-labelled molecules and tracking of their position in space and time, potentially across pores. Finally, the third class encompasses methods based on the statistical analysis of spontaneous fluorescence fluctuations out of the equilibrium or stationary state of the system. In this case, the behavior of molecules is probed in presence of many similarly-labelled molecules, without dwelling on any of them. Here these three classes, with their respective pros and cons as well as their main applications to nucleocytoplasmic shuttling will be briefly reviewed and discussed.
分子不断穿梭于将细胞核与细胞质分隔开来的核膜屏障。核膜并非仅仅是扩散的屏障，而是一个复杂的过滤器，为真核生物提供了对基因表达和蛋白质翻译等基本分子过程的精细时空调控。鉴于核质运输的高度动态性质，在过去几十年中，人们投入了大量精力来开发和应用基于荧光的时间分辨生物物理方法，以捕捉分子穿过核膜运动的细节。根据它们报告核质运输分子过程的方式差异，这些方法在此分为三大类。具体而言，第一类包括基于荧光信号扰动的方法，也称为系综平均方法，该方法对许多分子（穿过许多核孔）的行为进行平均。第二类包括基于荧光标记分子的定位以及对其在空间和时间上位置的跟踪（可能穿过核孔）的方法。最后，第三类包括基于对系统平衡或稳态之外的自发荧光波动进行统计分析的方法。在这种情况下，在存在许多类似标记分子的情况下探测分子的行为，而不关注其中任何一个分子。这里将简要回顾和讨论这三类方法，以及它们各自的优缺点和在核质穿梭中的主要应用。
