Abstract
The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms.
Highlights
The nuclear pore complex (NPC), embedded in the nuclear envelope (NE), is a complex assembly of proteins that forms a gateway between the nucleus and the cytoplasm of eukaryotic cells
Microscopy allows the imaging of single molecules within a small pixel area of the CCD camera, resulting in a very fast detection speed. Supported by these new features, the SPEED methodology was further developed in two aspects: Firstly, 2D single-particle tracking (SPT) data within single NPCs were acquired by tracking single mRNA-protein complexes (mRNPs) through a singly illuminated NPC with a spatiotemporal resolution of 8 nm and 2 ms; secondly, the inherent 3D pathways of the mRNPs in the NPC were recovered via a 2D-to-3D deconvolution algorithm by utilizing the structural rotational symmetry of the NPC and the superposition of thousands of single-particle trajectories collected from multiple NPCs [6,9,30,110]
FG Nups located at the central scaffold region of the NPC would play the dominant role in gating which mRNP molecules should continue their transport into the cytoplasm
Summary
The nuclear pore complex (NPC), embedded in the nuclear envelope (NE), is a complex assembly of proteins that forms a gateway between the nucleus and the cytoplasm of eukaryotic cells. The human mRNP export process has been shown to be regulated by other protein complexes through differing mechanisms, including the transcription and export complexes (TREX and TREX-2) as well as factors involved in the release of cargo from the cytoplasmic surface of the NPC [46,47,48,49,50]. Locating the major selective barrier to nucleocytoplasmic export and a refinement of the measure of transport kinetics are important contributions to the understanding of the details of NPC’s role in mRNA export and other vital cellular processes, such as mislocalization of cytoplasmic proteins to the nucleus and deregulation of signaling pathways can have disastrous consequences (such as developmental defects or cancer) and directly or indirectly involve the interaction of the NPC with various proteins [63,64,65,66,67]. Is believed that precise localization of molecular interactions within the NPC’s central channel itself is directly relevant to cancer-drug targeting strategies [68,69,70,71]
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