Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
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
Biochemistry | Biochemistry, Biophysics, and Structural Biology | Cell Biology | Molecular Biology
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.
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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
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
The Journal of cell biology
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.