Microtubule Seeded-assembly in the Presence of Poorly Nucleating Nucleotide Analogues.
| Autor: | Ku S; Univ Rennes, CNRS, IGDR (Institut de genetique et developpement de Rennes) - UMR 6290, F-35000 Rennes, France., Heichette C; Univ Rennes, CNRS, IGDR (Institut de genetique et developpement de Rennes) - UMR 6290, F-35000 Rennes, France., Duchesne L; Univ Rennes, CNRS, IGDR (Institut de genetique et developpement de Rennes) - UMR 6290, F-35000 Rennes, France., Chrétien D; Univ Rennes, CNRS, IGDR (Institut de genetique et developpement de Rennes) - UMR 6290, F-35000 Rennes, France. |
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| Jazyk: | angličtina |
| Zdroj: | Bio-protocol [Bio Protoc] 2020 Aug 20; Vol. 10 (16), pp. e3714. Date of Electronic Publication: 2020 Aug 20 (Print Publication: 2020). |
| DOI: | 10.21769/BioProtoc.3714 |
| Abstrakt: | Microtubule dynamic instability is driven by the hydrolysis of the GTP bound to the β-subunit of the α-β tubulin heterodimer. Nucleotide analogues are commonly used to mimic the different steps of the tubulin GTPase cycle, but most of them are poor microtubule nucleators. Usually, microtubule assembly is seeded by guanylyl-(α, β)-methylene-diphosphonate (GMPCPP) or glycerol that can be limiting factors in monitoring the effect of other nucleotide analogs on their polymerization. Here, we describe a protocol that allows the assembly of microtubules in the presence of nucleotide analogues without the need of heterogeneous seeds and at a low final glycerol concentration. Microtubules are first assembled in the presence of the analogue of interest and glycerol to promote assembly. These microtubules are then sonicated to produce seeds that will be used to assemble microtubules in the absence of glycerol. This strategy produces homogeneous nucleotide-bound microtubules that can be further analyzed by biochemical or structural methods such as cryo-electron microscopy. Competing Interests: Competing interestsThe authors declare no competing interests. (Copyright © The Authors; exclusive licensee Bio-protocol LLC.) |
| Databáze: | MEDLINE |
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