Abstract
Nuclear pore complexes (NPCs) embedded within the nuclear envelope mediate rapid, selective and bidirectional traffic between the cytoplasm and the nucleoplasm. Deciphering the mechanism and dynamics of this process is challenged by the need for high spatial and temporal resolution. We report here a multicolour imaging approach that enables direct three-dimensional visualization of cargo transport trajectories relative to a super-resolved octagonal double-ring structure of the NPC scaffold. The success of this approach is enabled by the high positional stability of NPCs within permeabilized cells, as verified by a combined experimental and simulation analysis. Hourglass-shaped translocation conduits for two cargo complexes representing different nuclear transport receptor pathways indicate rapid migration through the permeability barrier on or near the NPC scaffold. Binding sites for cargo complexes extend more than 100 nm from the pore openings, which is consistent with a wide distribution of the phenylalanine-glycine polypeptides that bind nuclear transport receptors.