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Microtubules are polymers assembled from αβ-tubulin heterodimers. They typically display lateral α-α and β-β homotypic interactions, except at one region, called the seam, where heterotypic α-β and β-α interactions occur. Here, we decorated microtubules assembled in vitro or in cytoplasmic Xenopus egg extracts with kinesin-motor domains, and analyzed their lattice organization using dual axis cryo-electron tomography followed by segmented sub-tomogram averaging. In both conditions, microtubules incorporated variable protofilament and/or tubulin subunit helix start numbers. While microtubules assembled in vitro displayed variable numbers of seams, those assembled in extracts displayed preferentially one seam. The seam location varied within individual microtubules implying the presence of lattice holes. Thus, the formation of discontinuous microtubule lattices is an intrinsic property of tubulin assembly, a process that is controlled in cells. One-Sentence Summary Microtubules assembled from purified tubulin form discontinuous lattices, an intrinsic property strictly controlled in cytoplasmic Xenopus egg extracts.

Microtubules are polymers assembled from αβ-tubulin heterodimers. They typically display lateral α-α and β-β homotypic interactions, except at one region, called the seam, where heterotypic α-β and β-α interactions occur. Here, we decorated microtubules assembled in vitro or in cytoplasmic Xenopus egg extracts with kinesin-motor domains, and analyzed their lattice organization using dual axis cryo-electron tomography followed by segmented sub-tomogram averaging. In both conditions, microtubules incorporated variable protofilament and/or tubulin subunit helix start numbers. While microtubules assembled in vitro displayed variable numbers of seams, those assembled in extracts displayed preferentially one seam. The seam location varied within individual microtubules implying the presence of lattice holes. Thus, the formation of discontinuous microtubule lattices is an intrinsic property of tubulin assembly, a process that is controlled in cells. One-Sentence Summary Microtubules assembled from purified tubulin form discontinuous lattices, an intrinsic property strictly controlled in cytoplasmic Xenopus egg extracts.

35 p.-12 fig.-2 tab.-3 animat.-1 schem.This article is dedicated to the memory of Dr Linda Amos, a dear friend and pioneer in the study of microtubules and the mechanism of action of paclitaxel (Amos and Löwe, 1999), who passed away while we were assembling this manuscript., Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules’ diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a βM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding., Ministerio de Ciencia e Innovación (PID2019-104545RB-I00 (AEI/10.13039/501100011033)) J Fernando Díaz Consejo Superior de Investigaciones Científicas (PIE 201920E111) J Fernando Díaz Fundación Tatiana Pérez de Guzmán el Bueno (Proyecto de Investigación en Neurociencia 2020) J Fernando Díaz European Union NextGenerationEU (H2020-MSCA-ITN-2019 860070 TUBINTRAIN) Andrea E Prota J Fernando Díaz Swiss National Science Foundation (310030_192566) Michel O Steinmetz JSPS KAKENHI (16K07328/17H03668) Shinji Kamimura National Natural Science Foundation of China (30930108) Wei-Shuo Fang Chinese Academy of Medical Sciences (2016-I2M-1-010) Wei-Shuo Fang Ministerio de Ciencia e Innovación (PID2019-104070RB-C22 (AEI/10.13039/501100011033)) Federico Gago We thank Ganadería Fernando Díaz for calf brains supply and staff of beamlines X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen PSI, Switzerland) and BL11-NDC-SWEET (ALBA, Cerdanyola del Vallès, Spain) for their support. We also thank Mr Pedro Gascón Blanco for his private donation to the project to support a month of a student salary.