Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Medical Subject Headings
Active Transport, Cell Nucleus; Antibodies; Binding Sites; Green Fluorescent Proteins; HeLa Cells; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; Immunohistochemistry; Kinetics; Membrane Glycoproteins; Microscopy, Fluorescence; Models, Molecular; Molecular Chaperones; Nuclear Envelope; Nuclear Localization Signals; Nuclear Pore; Nuclear Pore Complex Proteins; Nucleocytoplasmic Transport Proteins; Pregnancy Proteins; Protein Binding; Recombinant Proteins; Ribonucleoproteins; Thymus Hormones; Transfection; beta Karyopherins; ran GTP-Binding Protein
The mechanism by which macromolecules are selectively translocated through the nuclear pore complex (NPC) is still essentially unresolved. Single molecule methods can provide unique information on topographic properties and kinetic processes of asynchronous supramolecular assemblies with excellent spatial and time resolution. Here, single-molecule far-field fluorescence microscopy was applied to the NPC of permeabilized cells. The nucleoporin Nup358 could be localized at a distance of 70 nm from POM121-GFP along the NPC axis. Binding sites of NTF2, the transport receptor of RanGDP, were observed in cytoplasmic filaments and central framework, but not nucleoplasmic filaments of the NPC. The dwell times of NTF2 and transportin 1 at their NPC binding sites were 5.8 +/- 0.2 and 7.1 +/- 0.2 ms, respectively. Notably, the dwell times of these receptors were reduced upon binding to a specific transport substrate, suggesting that translocation is accelerated for loaded receptor molecules. Together with the known transport rates, our data suggest that nucleocytoplasmic transport occurs via multiple parallel pathways within single NPCs.
Kubitscheck, Ulrich; Grunwald, David; Hoekstra, Andreas; Rohleder, Daniel; Kues, Thorsten; Siebrasse, Jan Peter; and Peters, Reiner, "Nuclear transport of single molecules: dwell times at the nuclear pore complex" (2005). Biochemistry and Molecular Pharmacology Publications and Presentations. Paper 177.