The flow of genetic information is regulated by selective nucleocytoplasmic transport of messenger RNA:protein complexes (mRNPs) through the nuclear pore complexes (NPCs) of eukaryotic cells. However, the three-dimensional (3D) pathway taken by mRNPs as they transit through the NPC, and the kinetics and selectivity of transport, remain obscure. Here we employ single-molecule fluorescence microscopy with an unprecedented spatiotemporal accuracy of 8 nm and 2 ms to provide new insights into the mechanism of nuclear mRNP export in live human cells. We find that mRNPs exiting the nucleus are decelerated and selected at the centre of the NPC, and adopt a fast-slow-fast diffusion pattern during their brief, ~12 ms, interaction with the NPC. A 3D reconstruction of the export route indicates that mRNPs primarily interact with the periphery on the nucleoplasmic side and in the centre of the NPC, without entering the central axial conduit utilized for passive diffusion of small molecules, and eventually dissociate on the cytoplasmic side.

Adeno-associated virus (AAV) has been developed as a promising human gene therapy vector. Particularly, recombinant AAV vector (rAAV) achieves its transduction of host cells by crossing at least three physiological barriers including plasma membrane, endosomal membrane, and nuclear envelope (NE). So far, the AAV transduction mechanism has not been explored thoroughly at the single viral particle level. In this study, we employed high-speed super-resolution single-point edge-excitation sub-diffraction (SPEED) microscopy to map the events of single rAAV2 particles infecting live human cells with an unprecedented spatiotemporal resolution of 9–12 nm and 2–20 ms. Data reveal that rAAV2 particles are imported through nuclear pore complexes (NPCs) rather than nuclear membrane budding into the nucleus. Moreover, approximately 17% of the rAAV2 molecules starting from the cytoplasm successfully transverse the NPCs to reach the nucleoplasm, revealing that the NPCs act as a strict selective step for AAV delivery. This study lastly suggests a new pathway to improve AAV vectors for human gene therapy.

The nuclear pore complex (NPC) mediates the shuttle transport of macromolecules between the nucleus and cytoplasm in eukaryotic cells. The permeability barrier formed by intrinsically disordered phenylalanine-glycine-rich nucleoporins (FG-Nups) in the NPC functions as the critical selective control for nucleocytoplasmic transport. Signal-independent small molecules (< 40 kDa) passively diffuse through the pore, but passage of large cargo molecules is inhibited unless they are chaperoned by nuclear transport receptors (NTRs). NTRs are capable of interacting with FG-Nups and guide the cargos to cross the barrier by facilitated diffusion. The native conformation of the FG-Nups permeability barrier and the competition among multiple NTRs interacting with this barrier in the native NPCs are the 2 core questions still being highly debated in the field. Recently, we applied high-speed super-resolution fluorescence microscopy to map out the natural structure of the FG-Nups barrier and determined the competition among multiple NTRs as they interact with the barrier in the native NPCs. In this extra-view article, we will review the current understanding in the configuration and function of FG-Nups barrier and highlight the new evidence obtained recently to answer the core questions in nucleocytoplasmic transport.

The nuclear pore complex (NPC) is one of the largest supramolecular structures in eukaryotic cells. Its octagonal ring-scaffold perforates the nuclear envelope and features a unique molecular machinery that regulates nucleocytoplasmic transport. NPCs are composed of ~30 different nucleoporins (Nups), averaged at 8, 16 or 32 copies per NPC. This estimate has not been confirmed for individual NPCs in living cells due to the inherent difficulty of counting proteins inside single supramolecular complexes. Here we used single-molecule SPEED microscopy to directly count the copy-number of twenty-four different Nups within individual NPCs of live yeast, and found agreement as well as significant deviation from previous estimates. As expected, we counted 8 copies of four peripheral Nups and 16 copies of fourteen scaffold Nups. Unexpectedly, we counted a maximum of 16 copies of Nsp1 and Nic96, rather than 32 as previously estimated; and found only 10–15 copies of six other Nups, rather than 8 or 16 copies as expected. This in situ molecular-counting technology can test structure-function models of NPCs and other supramolecular structures in cells.

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.