In Vivo Analysis of Protein Crowding in the Nuclear Pore Complex during Interphase and Mitosis

Abstract

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.