The nuclear pore complex (NPC) is a pivotal subcellular structure governing nucleocytoplasmic transport through a selectively permeable barrier. Comprising approximately 30 distinct proteins, it includes FG-Nups with phenylalanine-glycine (FG) motifs and non-FG Nups forming the pore's scaffold. The selectively permeable barrier formed by FG-Nups enables the passive diffusion of small molecules and facilitates the transport of larger ones recognized by nuclear transport receptors (NTRs). Their roles are critical in regulating mRNA and pre-ribosome nuclear export and the nuclear import of transcription factors, underscoring their significance in cellular processes. However, studying NPCs remains challenging due to their structural complexity, heterogeneity, dynamic interactions, and inaccessibility within live cells. In this dissertation, three core questions were investigated to elucidate the structure and function of the NPC. First, the nuclear export dynamics of pre-ribosomal subunits revealed significantly higher transport efficiency compared to other large cargos. Through inhibition of nuclear transport receptor (NTR), CRM1, by small-molecule inhibitor, leptomycin B, we found a dose-dependent inhibition of CRM1s played a crucial role in pre-ribosome export efficiency. We confirmed these results through a series of controlled environments with both import and export NTRs. Our results suggest that cooperative NTR mechanisms may enhance the nucleocytoplasmic transport of not only pre-ribosomal subunits but other protein complexes as well. Second, we investigated the dynamic properties of the NPC’s selectivity barrier by altering the concentration of O-linked β-N-acetylglucosamine (O-GlcNAc) sites on nuclear pore proteins. Using small-molecule inhibitors of O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) to decrease or increase NPC O-GlcNAcylation, respectively, we found a significant change in the overall 3D spatial density of NPC O-GlcNAc sites. Then, by applying the same OGT- and OGA-inhibited conditions, we found that NPC O-GlcNAcylation significantly impacted the nuclear export of mRNA, suggesting that NPC O-GlcNAcylation regulates mRNA’s passage through the NPC’s selective permeability barrier. Third, we examined the nuclear transport mechanism for intrinsically disordered proteins (IDPs). Our findings revealed that IDPs, unlike large folded proteins, can passively diffuse through NPCs independent of size, and their diffusion behaviors are differentiated by the content ratio of charged (Ch) and hydrophobic (Hy) amino acids. Thus, we proposed a Ch/Hy-ratio mechanism for IDP nucleocytoplasmic transport. In summary, comprehending the dynamic behavior of the NPC selectivity barrier and its involvement in mediating large transiting complexes and IDPs has provided valuable insights into the fundamental nucleocytoplasmic transport mechanism, emphasizing the NPC's crucial role in cellular health and function. / Biology