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Article Dans Une Revue Bio-Protocol Année : 2023

Characterization of Microtubule Lattice Heterogeneity by Segmented Subtomogram Averaging

Résumé

Microtubule structure is commonly investigated using single-particle analysis (SPA) or subtomogram averaging (STA), whose main objectives are to gather high-resolution information on the alpha beta-tubulin heterodimer and on its interactions with neighboring molecules within the microtubule lattice. The maps derived from SPA approaches usually delineate a continuous organization of the alpha beta-tubulin heterodimer that alternate regularly head-to-tail along protofilaments, and that share homotypic lateral interactions between monomers (alpha-alpha, beta-beta), except at one unique region called the seam, made of heterotypic ones (alpha-beta, beta-alpha). However, this textbook description of the microtubule lattice has been challenged over the years by several studies that revealed the presence of multi-seams in microtubules assembled in vitro from purified tubulin. To analyze in deeper detail their intrinsic structural heterogeneity, we have developed a segmented subtomogram averaging (SSTA) strategy on microtubules decorated with kinesin motor-domains that bind every alpha beta-tubulin heterodimer. Individual protofilaments and microtubule centers are modeled, and sub-volumes are extracted at every kinesin motor domain position to obtain full subtomogram averages of the microtubules. The model is divided into shorter segments, and subtomogram averages of each segment are calculated using the main parameters of the full-length microtubule settings as a template. This approach reveals changes in the number and location of seams within individual microtubules assembled in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts. Key features center dot This protocol builds upon the method developed by J.M. Heumann to perform subtomogram averages of microtubules and extends it to divide them into shorter segments. center dot Microtubules are decorated with kinesin motor-domains to determine the underlying organization of its constituent alpha beta-tubulin heterodimers. center dot The SSTA approach allows analysis of the structural heterogeneity of individual microtubules and reveals multi-seams and changes in their number and location within their shaft.
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Dates et versions

hal-04197848 , version 1 (11-10-2023)

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Clément Bousquet, John M Heumann, Denis Chrétien, Charlotte Guyomar. Characterization of Microtubule Lattice Heterogeneity by Segmented Subtomogram Averaging. Bio-Protocol, 2023, 13 (14), ⟨10.21769/BioProtoc.4723⟩. ⟨hal-04197848⟩
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