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Instabilité chimique dans les solutions microtubulaires : étude et recherche d'effecteurs

Abstract : Microtubules play a crucial role in cellular organisation and function. Microtubule assembly display puzzling non-linear features, like dynamic instability. Here, we intended first to determine the limiting factors of microtubule assembly, and second to identify new effectors of this process. The kinetics of the reaction were studied in simple experimental conditions, allowing simultaneous and precise measurement of all the parameters of the reaction. Our results indicate that the microtubule nucleation occurs in two steps : rapid and irreversible formation of tubulin oligomers then aggregation of these oligomers into a microtubule. The elongation rate of the microtubule appears to be independent of the GTP-tubulin concentration : this elongation rate is probably limited by structural properties of the microtubules. Finally, microtubule disassembly would occur through catastrophes, and would be triggered by tubulin oligomers. The tubulin oligomers controlling disassembly could possibly be the same as those controlling microtubule nucleation. We then produced tubulin oligomers triggering microtubule nucleation by a moderate covalent crosslinking of microtubules. These oligomers are constituted by lateral interactions of 10 tubulin dimmers, four of which aggregate to form a microtubule. Microtubules assembled in our conditions in the presence of nucleating tubulin oligomers do not spontaneously disassemble : these conditions are suitable to look for catastrophe factors. Finally, we developed an affinity chromatographic procedure to purify tubulin-binding proteins in a native state. Thus, these tubulin partners are amenable to functional tests. Peptide mapping and mass spectrometry allowed us to identify the purified proteins. Some are known to control microtubule assembly, but others are possibly new effectors of microtubule dynamics.
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https://tel.archives-ouvertes.fr/tel-00078986
Contributor : Nicolas Caudron <>
Submitted on : Thursday, June 8, 2006 - 2:21:09 PM
Last modification on : Friday, November 6, 2020 - 3:44:49 AM
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Nicolas Caudron. Instabilité chimique dans les solutions microtubulaires : étude et recherche d'effecteurs. Biochimie [q-bio.BM]. Université Joseph-Fourier - Grenoble I, 2001. Français. ⟨tel-00078986⟩

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