The central channel of the nuclear pore complex (NPC) is occupied by non-structured polypeptides with a high content of Phe-Gly (FG) motifs. This protein-rich environment functions as an entropic barrier that prevents the passage of molecules, as well as the binding sites for karyopherins, to regulate macromolecular traffic between the nucleoplasm and the cytoplasm. In this study, we expressed individual Nups fused with a crowding-sensitive probe (GimRET) to determine the spatial distribution of protein-rich domains within the central channel in vivo, and characterize the properties of the entropic barrier. Analyses of the probe signal revealed that the central channel contains two protein-rich domains at both the nucleoplasmic and cytoplasmic peripheries, and a less-crowded central cavity. Karyopherins and other soluble proteins are not the constituents of the protein-rich domains. The time-lapse observation of the post-mitotic reassembly process also revealed how individual protein-rich domains are constructed by a sequential assembly of nucleoporins.

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The nuclear pore complex (NPC) of eukaryotic cells regulates macromolecular traffic between the cytoplasm and the nucleoplasm. The central channel of the pore is thought to form a crowded hydrophobic environment, due to the high content of Phe-Gly (FG) motifs in pore-forming subunits (Nups), which determines the selectivity of the pore. Here, we analyzed the spatiotemporal formation of crowded environments within the NPC by utilizing a crowding-sensitive fluorescent protein probe (GimRET), which was constructed by fusing ECFP with YFP carrying a single amino acid insertion (YFP-G1). The fluorescent properties of GimRET exhibited sensitivity to high concentrations of protein (>~100 mg/mL). When GimRET-fused Nups were expressed in HeLa cells. The FRET signal in the nuclear envelop revealed that the extent of crowding is different among Nups. Nups located in the outer rim of the pore (Nups50, 153, 214 and 358) exist in a highly crowded environment, whereas Nups located in the middle channel (Nups54, 58 and 62) exhibited a minimal crowding, suggesting that a large entropic barrier exits at both ends of the pore. We also investigated the formation of a crowded environment during post-mitotic reassembly of the NPC. Quantitative analysis of the probe signals from metaphase to G1 phase indicated that some Nups are in a less crowded environment when dispersed in the cytoplasm at metaphase than when assembled in the NPC during interphase. However, some Nups exhibit a similar amount of crowding in both cases, suggesting that some Nups remain in subcomplexes, but others dissociate during mitosis. In addition, a lag period between the localization of Nups around the chromatin surface and the detection of crowding suggests that there is a dynamic rearrangement of Nups after assembly on the chromatin surface. Transfer-RNA (tRNA) is the key adaptor molecule in protein synthesis, but recent studies demonstrate behavior and interactions that suggest a far greater role in cellular function. This includes priming viral protein synthesis, retrograde nuclear trafficking, binding to cytochrome C, and post-translational arginylation of proteins. Defects in tRNA modifications are also linked to a number of human diseases with relatively unknown mechanisms. Here we describe the use of live-cell imaging techniques to monitor tRNA nuclear-cytoplasmic trafficking and localization in real time via microinjection of fluorescently-labeled tRNA (fl-tRNA) into adherent mammalian cells. These results represent an important step toward our eventual goal of developing assays for the full characterization of the subcellular dynamics of specific isoacceptor tRNAs. The Nuclear pore complex (NPC) is a protein complex existing in the nuclear envelope which functions as a selective channel between the cytoplasm and the nucleoplasm. The central channel of the pore, which is composed of Phe/Gly motif-rich subunits (FG-nucleoporins), is thought to form a hydrophobic barrier and regulate the passage of soluble proteins and nucleotides. In this study, we combi...

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