Tubulin_GTP

Microtubules are essential components of the cytoskeleton involved in fundamental processes such as intracellular trafficking or cell division. A large part of these functions is underpinned by their dynamic properties, which allow microtubules to switch rapidly between polymerization and depolymerization phases upon cell needs. While microtubule dynamics is finely tuned in cells by accessory proteins, the ability to alternate between growing and shrinkage states is intrinsic to microtubules assembled from purified tubulin. The hydrolysis of the GTP bound to the exchangeable site of tubulin sustains this particular dynamic behavior, called 'dynamic instability'. According to the most widely spread model in the literature, a delay between polymerization and GTP-hydrolysis generates a 'GTP-cap' at microtubule growing ends. Stochastic loss of this GTP-cap would result in fast disassembly of the microtubule, while re-capping would allow the reverse event. Analysis of the GTP-cap has stimulated a large number of studies in the past. However, the absence of specific probes for the nucleotide state of tubulin in microtubules did not allow a detailed characterization of this structure at the molecular level. This project involves a collaboration between the Institute of Genetics and Development of Rennes (D. Chrétien, partner 1) and the Paul Scherrer Institute (M. Steinmetz, Villigen, Switzerland, partner 2). During a previous collaboration, we characterized the architecture of the GTP-cap by cryo-electron tomography (cryo-ET) using the plus-end binding protein 1 (EB1) conjugated to gold nanoparticles. EB1, which binds specifically to microtubule growing ends, displays a high affinity for microtubules assembled in the presence of some GTP-analogues, and thus is currently considered as a specific marker of the GTP-cap region. Our results suggest that the GTP-cap structure includes outwardly curved sheets present at microtubule growing ends, and extends onto closed parts of the microtubule lattice. In the present project, our objective is to extend this study by correlating tubulin conformational changes with its GTPase cycle. In this respect, our methodological approach will aim at characterizing at the highest possible resolution dynamic microtubules assembled in the presence of GTP and GDP-Pi analogues. This work will involve solving the high-resolution structure of tubulin in the presence of the analogues by X-ray crystallography (partner 2), analysis by cryo-ET of dynamic microtubules in the presence of the analogues (partner 2), and docking of the high-resolution structures of tubulin into the molecular envelopes determined by cryo-ET (partners 1 & 2). Malfunctions of microtubule dynamics are associated with a range of pathologies such as neuronal disorders or cell cycle anomalies. In this context, a better understanding of the molecular basis of microtubule dynamics is essential to develop new strategies against these pathologies. Our project will also bring to the scientific community new methodological tools to analyze the structure and dynamics of complex macromolecular assemblages. Methodological approaches will be disseminated though practical and theoretical workshops. Results will be published in high-impact journals and presented at international conferences. A particular effort will be devoted to presenting our work to the non-scientific public.