Nuclear export of mRNA molecules studied by SPEED microscopy

Abstract

The nuclear exit of messenger RNA (mRNA) molecules through the nuclear pore complex (NPC) is an essential step in the translation process of all proteins. The current limitations of conventional fluorescence and electron microscopy have prevented elucidation of how mRNA exports through the NPCs of live cells. In the recent years, various single-molecule fluorescence (SMF) microscopy techniques have been developed to improve the temporal and spatial resolutions of live-cell imaging allowing a more comprehensive understanding of the dynamics of mRNA export through native NPCs. In this review, we firstly evaluate the necessity of single-molecule live-cell microscopy in the study of mRNA nuclear export. Then, we highlight the application of single-point edge-excitation sub-diffraction (SPEED) microscopy that combines high-speed SMF microscopy and a 2D-to-3D transformation algorithm in the studies of nuclear transport kinetics and route for mRNAs. Finally, we summarize the new features of mRNA nuclear export found with SPEED microscopy as well as the reliability and accuracy of SPEED microscopy in mapping the 3D spatial locations of transport routes adopted by proteins and mRNAs through the NPCs.

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high‐speed super‐resolution microscopy

“…So far, the method has been successfully applied to achieve 3D structural and functional information for 25–300 nm subcellular organelles that meet the rotational symmetry requirement such as NPC, primary cilium, microtubule and glass nanocapillary . To further introduce the method and respond to questions about the reproducibility in obtaining 3D information by 2D to 3D transformation, we have provided comprehensive analyses of this method by detailing the mathematical principle, the experimental data and the computational simulations used in our method . There are two critical factors in reproducing the accurate 3D super‐resolution information, the single molecule localization error and the number of single molecule locations.…”

Nucleocytoplasmic Transport: Regulatory Mechanisms and the Implications in Neurodegeneration

“…See Table 1 for more information about different nucleoporins. Nups contain phenylalanine-glycine (FG) motifs or repeats which create a permeability barrier against passive diffusion of larger cargo molecules (>60 KDa) [22,23]. FG repeats are intrinsically disordered domains [24], and they directly function in mediating the passage of the soluble transport receptors pass through the NPC [2,25,26].…”

Nuclear Import of Adeno-Associated Viruses Imaged by High-Speed Single-Molecule Microscopy

“…( A ) Diagramed illumination volumes of wide-field (light blue) and SPEED (blue) microscopy. ( B ) GFP-labeled NPCs embedded in the NE were highlighted inside (green) and outside (gray) the microscopy illumination volumes in the xy and yz planes [ 60 ]. ( C ) Selected area (yellow box) of GFP–NE florescence from wide-field illumination.…”