Nuclear transport of single molecules: dwell times at the nuclear pore complex.
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
U
Institute of Medical Physics and Biophysics, Westfälische Wilhelms-Universität, D-48149 Münster, Germany. u.kubitscheck@uni-bonn.de
Grünwald
David
D
Hoekstra
Andreas
A
Rohleder
Daniel
D
Kues
Thorsten
T
Siebrasse
Jan Peter
JP
Peters
Reiner
R
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000906:Antibodies / Q000276:immunology; D001665:Binding Sites; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000032:analysis / Q000276:immunology; D009685:Nuclear Envelope / Q000648:ultrastructure; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism; D022022:Nuclear Pore / Q000737:chemistry / Q000502:physiology*; D028861:Nuclear Pore Complex Proteins / Q000032:analysis / Q000276:immunology; D029543:Nucleocytoplasmic Transport Proteins; D011257:Pregnancy Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry; D013951:Thymus Hormones / Q000737:chemistry; D014162:Transfection; D028961:beta Karyopherins / Q000737:chemistry / Q000378:metabolism; D020931:ran GTP-Binding Protein / Q000378:metabolism
D000906:Antibodies; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D008562:Membrane Glycoproteins; D018832:Molecular Chaperones; C038965:NUTF2 protein, human; D019913:Nuclear Localization Signals; D028861:Nuclear Pore Complex Proteins; D029543:Nucleocytoplasmic Transport Proteins; C094157:POM121 protein, human; C511307:POMZP3 protein, human; D011257:Pregnancy Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; C108121:TNPO1 protein, human; D013951:Thymus Hormones; D028961:beta Karyopherins; C094493:ran-binding protein 2; D049452:Green Fluorescent Proteins; D020931:ran GTP-Binding Protein