Ribosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of â¼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s-1 in vivo.
Nuclear Pore/metabolism , Ribosome Subunits, Large, Eukaryotic/metabolism , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/ethics , Cell Nucleus/metabolism , Eukaryotic Initiation Factors/genetics , Eukaryotic Initiation Factors/metabolism , Fatty Acids, Unsaturated/pharmacology , HeLa Cells , Humans , Microscopy, Confocal , Nucleocytoplasmic Transport Proteins/genetics , Nucleocytoplasmic Transport Proteins/metabolism , Organelle Biogenesis , Pregnancy Proteins/genetics , Pregnancy Proteins/metabolism , Single Molecule Imaging
