UMMS Affiliation

Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute

Date

1-17-2005

Document Type

Article

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

Abstract

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.

Comments

Citation: Kubitscheck U, Grünwald D, Hoekstra A, Rohleder D, Kues T, Siebrasse JP, Peters R. Nuclear transport of single molecules: dwell times at the nuclear pore complex. J Cell Biol. 2005 Jan 17;168(2):233-43. Link to article on publisher's site

Publisher PDF posted as allowed by the publisher's author rights policy at http://www.rupress.org/site/subscriptions/terms.xhtml.

At the time of publication, David Grünwald was not yet affiliated with the University of Massachusetts Medical School.

Related Resources

Link to Article in PubMed

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.