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Autonomy and robustness of translocation through the nuclear pore complex: a single-molecule study

¹ Institute for Physical and Theoretical Chemistry, Rheinische Friedrich-Wilhelms-University Bonn, D-53115 Bonn, Germany
² Institute of Medical Physics and Biophysics, Westfälische Wilhelms-Universität, D-48149 Münster, Germany

Correspondence to David Grünwald: dgruenwa{at}aecom.yu.edu

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 kap2, kapβ1, kapβ1N44, and kapβ2, 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.

T. Dange's present address is Albert Einstein College of Medicine, Department of Biochemistry, 1300 Morris Park Ave., Bronx, NY, 10461.

D. Grünwald's present address is Albert Einstein College of Medicine, Anatomy and Structural Biology, 1300 Morris Park Ave., Bronx, NY, 10461.

A. Grünwald's present address is Albert Einstein College of Medicine, Department of Medicine, Nephrology, 1300 Morris Park Ave., Bronx, NY, 10461.

Abbreviations used in this paper: FG, phenylalanine-glycine; FWHM, full width at half maximum; NE, nuclear envelope; NPC, nuclear pore complex; SNR, signal-to-noise ratio.

© 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/).

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