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J. Biol. Chem., Vol. 282, Issue 25, 18563-18572, June 22, 2007

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A Quantitative Kinetic Model for the in Vitro Assembly of Intermediate Filaments from Tetrameric Vimentin^*

Robert Kirmse¹², Stephanie Portet¹³, Norbert Mücke, Ueli Aebi^¶, Harald Herrmann^||, and Jörg Langowski⁴

From the Division of Biophysics of Macromolecules and the ^||Division of Molecular Genetics, German Cancer Research Center, Im Neuenheimer Feld 580, Heidelberg D-69120, Germany, the Department of Mathematics, University of Manitoba, Winnipeg MB, Canada R3T 2N2, and the ^¶M. E. Müller Institute for Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, Basel 4056, Switzerland

In vitro assembly of intermediate filament proteins is a very rapid process. It starts without significant delay by lateral association of tetramer complexes into unit-length filaments (ULFs) after raising the ionic strength from low salt to physiological conditions (100 mM KCl). We employed electron and scanning force microscopy complemented by mathematical modeling to investigate the kinetics of in vitro assembly of human recombinant vimentin. From the average length distributions of the resulting filaments measured at increasing assembly times we simulated filament assembly and estimated specific reaction rate parameters. We modeled eight different potential pathways for vimentin filament elongation. Comparing the numerical with the experimental data we conclude that a two-step mechanism involving rapid formation of ULFs followed by ULF and filament annealing is the most robust scenario for vimentin assembly. These findings agree with the first two steps of the previously proposed three-step assembly model (Herrmann, H., and Aebi, U. (1998) Curr. Opin. Struct. Biol. 8, 177-185). In particular, our modeling clearly demonstrates that end-to-end annealing of ULFs and filaments is obligatory for forming long filaments, whereas tetramer addition to filament ends does not contribute significantly to filament elongation.

Received for publication, February 5, 2007 , and in revised form, March 1, 2007.

^* This work was supported in part by the German Research Foundation (Deutsche Forschungsgemeinschaft, Grants HE 1853/4-2 and -/4-3 to H. H.) and by the Swiss National Science Foundation (Grant 3100AO-100448), a National Center of Competence in Research program grant on Nanoscale Science, the Swiss Society for Research on Muscular Diseases, the M. E. Müller Foundation of Switzerland, and the Canton Basel Stadt (to U. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Supported by a fellowship from the International Ph.D. program of the German Cancer Research Center.

³ Supported in part by a grant from the Natural Sciences and Engineering Research Council of Canada.

⁴ To whom correspondence should be addressed: Tel.: 49-6221-423-390; Fax: 49-6221-423-391; E-mail: jl{at}dkfz.de.

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