Single-Molecule Snapshots of Three-Dimensional Distribution of Transient Interactions in the Nuclear Pore Complex

Abstract

Translocation of macromolecules through the nuclear pore complex (NPC) is hindered by the phenylalanine-glycine (FG) repeats barrier unless they are chaperoned by transport receptors in eukaryotic cells. However, challenged by measuring a series of transient interactions between the transport receptor and the FG repeats, the precise mechanism of the facilitated translocation remains unclear. We applied single-point edge-excitation sub-diffraction (SPEED) microscopy to obtain a three-dimensional density map of the transient interactions with a spatiotemporal resolution of 9 nm and 400 μs. We observed novel features under real-time trafficking conditions that escape detection by conventional electron microscopy: the actual pathway of facilitated translocation through the NPC is not completely restricted by the NPC architecture; the primary interaction sites between Importin β1 (Imp β1, a major transport receptor) and the FG repeats locate symmetrically on the cytoplasmic and nucleoplasmic sides of the nuclear pore; Imp β1 only rarely occupies a central channel of approximate 10-20 nm diameter along the NPC axis, but the Imp β1-assisted cargo molecules expand their pathways into the central channel.