Nuclear export of the pre-60S ribosomal subunit through single nuclear pores observed in real time.

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 ∼24 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.

Observing and tracking single small ribosomal subunits in vivo.

Ribosomes are formed of a small and a large subunit (SSU/LSU), both consisting of rRNA and a plethora of accessory proteins. While biochemical and genetic studies identified most of the involved proteins and deciphered the ribosomal synthesis steps, our knowledge of the molecular dynamics of the different ribosomal subunits and also of the kinetics of their intracellular trafficking is still limited. Adopting a labelling strategy initially used to study mRNA export we were able to fluorescently stain the SSU in vivo. We chose DIM2/PNO1 (Defective In DNA Methylation 2/Partner of NOb1) as labelling target and created a stable cell line carrying an inducible SNAP-DIM2 fusion protein. After bulk labelling with a green fluorescent dye combined with very sparse labelling with a red fluorescent dye the nucleoli and single SSU could be visualized simultaneously in the green and red channel, respectively. We used single molecule microscopy to track single SSU in the nucleolus and nucleoplasm. Resulting trajectory data were analyzed by jump-distance analysis and the variational Bayes single-particle tracking approach. Both methods allowed identifying the number of diffusive states and the corresponding diffusion coefficients. For both nucleoli and nucleoplasm we could identify mobile (D = 2.3-2.8 µm2/s), retarded (D = 0.18-0.31 µm2/s) and immobilized (D = 0.04-0.05 µm2/s) SSU fractions and, as expected, the size of the fractions differed in the two compartments. While the fast mobility fraction matches perfectly the expected nuclear mobility of the SSU (D = 2.45 µm2/s), we were surprised to find a substantial fraction (33%) of immobile SSU in the nucleoplasm, something not observed for inert control molecules.