Abstract
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 classes, 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 single fluorescently-labelled molecules and tracking of their position in space and time, potentially across single 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 single 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.
Highlights
In eukaryotic cells, the cytoplasm and the nucleus are spatially separated by a double membrane, the nuclear envelope (NE)
The overall shape of the pore is known since pioneering studies, among others, were conducted yeast by electron microscopy (EM) [2] and on Dictyostelium discoideum by cryo-electron tomography [3]: the pore is a channel-like structure of about 40–90 nm in length and 40–75 nm in width, showing an asymmetric structure with flexible protein filaments extending out from the pore into the cytoplasmic environment, and an open basket-like structure extending to about 75 nm into the nucleus
Our results suggested that active export/ import and active export/passive diffusion fluxes must be largely uncoupled, and that a mechanism of differential gating at the nuclear pore complexes (NPCs) level must exist
Summary
The cytoplasm and the nucleus are spatially separated by a double membrane, the nuclear envelope (NE). The first comprises perturbation-based approaches, such as Fluorescence Recovery After Photobleaching (FRAP) By these methods, the characteristic time of molecular transport across the entire NE can be measured by averaging the behavior of many molecules, across many pores. The recently developed spatial extension of FCS, named pair correlation function (pCF) approach, was proven to be suited to study the shuttling of molecules across the NE [27] and other sub-cellular structures (e.g. chromatin territories [28,29,30]) This approach builds on the dual-foci FCS concept [31] and combines FCS and SPT potentialities into a new method in which the time needed for each molecule to be found in a given point in space that is different from the position at time zero can be extracted [32]. A glimpse into the future directions in the field will be provided
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More From: Computational and Structural Biotechnology Journal
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