Significance: The nuclear pore complex (NPC) consisting of hundreds of proteins embeds into the nuclear envelope and functions as a selectively permeable barrier regulating the bidirectional trafficking of macromolecules between the nucleoplasm and cytoplasm in eukaryotic cells. However, the exact copy number and the specific function of each composite protein within the NPC remain vague. To interrogate this gap in knowledge, in this paper, we utilized a high-speed single-molecule microscopy in conjunction with highly specific auxin-inducible degron strategies to detail the stoichiometric ratio of NPC’s nuclear basket proteins and also reveal their distinct roles in mediating nuclear export of messenger RNAs. The demonstrated techniques might also serve as an effective model for characterizing other large macromolecule complexes.

The nuclear envelope (NE) surrounds the nucleus with a double membrane in eukaryotic cells. The double membranes are embedded with proteins that are synthesized on the endoplasmic reticulum and often destined specifically for either the outer nuclear membrane (ONM) or the inner nuclear membrane (INM). These nuclear envelope transmembrane proteins (NETs) play important roles in cellular function and participate in transcription, epigenetics, splicing, DNA replication, genome architecture, nuclear structure, nuclear stability, nuclear organization, and nuclear positioning. These vital functions are dependent upon both the correct localization and relative concentrations of NETs on the appropriate membrane of the NE. It is, therefore, important to understand the distribution and abundance of NETs on the NE. This review will evaluate the current tools and methodologies available to address this important topic.

The transient nature of RNA has rendered it one of the more difficult biological targets for imaging. This difficulty stems both from the physical properties of RNA as well as the temporal constraints associated therewith. These concerns are further complicated by the difficulty in imaging endogenous RNA within a cell that has been transfected with a target sequence. These concerns, combined with traditional concerns associated with super-resolution light microscopy has made the imaging of this critical target difficult. Recent advances have provided researchers the tools to image endogenous RNA in live cells at both the cellular and single-molecule level. Here, we review techniques used for labeling and imaging RNA with special emphases on various labeling methods and a virtual 3D super-resolution imaging technique.

Efficient gene expression depends on the tightly regulated export of messenger RNA (mRNA) through nuclear pore complexes (NPCs), which are densely modified by O-linked N-acetylglucosamine (O-GlcNAc). Although dysregulated O-GlcNAcylation has been linked to a variety of human diseases, the precise distribution of O-GlcNAc within the NPC and its effects on mRNA export remain poorly understood. Here, we combined single-point edge-excitation subdiffraction (SPEED) microscopy with stochastic optical reconstruction microscopy (STORM) to map the nanometer-scale distribution of an O-GlcNAc analog (GlcNAz) within NPCs and to quantify the export kinetics of mRNA–protein complexes (mRNPs) under both normal and perturbed O-GlcNAcylation conditions. Under basal conditions, GlcNAz is predominantly localized around the central channel of the NPC. However, both hypo- and hyper-O-GlcNAcylation cause GlcNAz to redistribute toward the nuclear and cytoplasmic peripheries. This shift is paralleled by changes in mRNP localization and altered distributions of key, highly O-GlcNAcylated, phenylalanine-glycine nucleoporins. These architectural rearrangements are accompanied by functional consequences: Elevated O-GlcNAcylation nearly doubles mRNA export efficiency (~61%), while reduced O-GlcNAcylation lowers it to ~16%, along with reduced NPC engagement. The transport receptor TAP exhibits analogous efficiency changes, reinforcing the role of O-GlcNAcylation as a key regulator of nucleocytoplasmic transport. Together, these results suggest that O-GlcNAcylation modulates NPC architecture and transport dynamics to fine-tune mRNA export, and indicate that targeted modulation of NPC O-GlcNAc levels may offer a promising strategy for addressing diseases associated with nuclear transport dysfunction.