Title
Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Publication Date
2008-10-06
Document Type
Article
Subjects
Active Transport, Cell Nucleus; Binding Sites; Cell Nucleus; Cytoskeleton; Green Fluorescent Proteins; HeLa Cells; Humans; Kinetics; Membrane Glycoproteins; Microscopy, Fluorescence; Mutation; Nuclear Envelope; Nuclear Localization Signals; Nuclear Pore; Protein Binding; Protein Transport; Recombinant Fusion Proteins; Serum Albumin, Bovine; alpha Karyopherins; beta Karyopherins
Disciplines
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Cell Biology | Molecular Biology
Abstract
All molecular traffic between nucleus and cytoplasm occurs via the nuclear pore complex (NPC) within the nuclear envelope. In this study we analyzed the interactions of the nuclear transport receptors kapalpha2, kapbeta1, kapbeta1DeltaN44, and kapbeta2, and the model transport substrate, BSA-NLS, with NPCs to determine binding sites and kinetics using single-molecule microscopy in living cells. Recombinant transport receptors and BSA-NLS were fluorescently labeled by AlexaFluor 488, and microinjected into the cytoplasm of living HeLa cells expressing POM121-GFP as a nuclear pore marker. After bleaching the dominant GFP fluorescence the interactions of the microinjected molecules could be studied using video microscopy with a time resolution of 5 ms, achieving a colocalization precision of 30 nm. These measurements allowed defining the interaction sites with the NPCs with an unprecedented precision, and the comparison of the interaction kinetics with previous in vitro measurements revealed new insights into the translocation mechanism.
Rights and Permissions
Copyright 2008 Dange et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
DOI of Published Version
10.1083/jcb.200806173
Source
Dange T, Grünwald D, Grünwald A, Peters R, Kubitscheck U. Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study. J Cell Biol. 2008 Oct 6;183(1):77-86. doi: 10.1083/jcb.200806173. Link to article on publisher's site
Journal/Book/Conference Title
The Journal of cell biology
Related Resources
PubMed ID
18824568
Repository Citation
Dange, Thomas; Grunwald, David; Grunwald, Antje; Peters, Reiner; and Kubitscheck, Ulrich, "Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study" (2008). Biochemistry and Molecular Pharmacology Publications and Presentations. 172.
https://escholarship.umassmed.edu/bmp_pp/172
Comments
At the time of publication, David Grünwald was not yet affiliated with the University of Massachusetts Medical School.