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Abstract

The cytoskeleton is an essential component of cells composed of three types of polymer networks- actin filaments, intermediate filaments and microtubules. Together they ensure compartmentation, spatial and temporal regulation of cargo flow and regulate the cell shape. Microtubules, the largest polymers of the cytoskeleton, are hollow cylinders composed of alpha/beta-tubulin dimers arranged in protofilaments. The orientation of alpha/beta-tubulin dimers in protofilaments is giving rise to the polarity of microtubules with the beta-tubulin-facing plus end and the alpha/-tubulin-facing minus end. The microtubule plus end is dynamic while the minus end is the origin of microtubule nucleation. In cells, microtubules are formed de novo in a process termed microtubule nucleation that is mediated by gamma-TuCs composed of gamma-tubulins and gamma-complex proteins (GCPs). These complexes serve as structural templates for microtubule nucleation, accelerating the process by decreasing an initial kinetic barrier. In fungi, the principal microtubule template is the gamma-TuSC, which is assembled from Spc97 and Spc98 and two copies of gamma-tubulin, and oligomerizes at the SPB into a ring-like structure templating microtubule nucleation. The vertebrate principal microtubule template, the gamma-TuRC, is compositionally more complex. Besides containing homologs of fungal Spc97 and Spc98 termed GCP2 and GCP3, respectively, three additional GCP variants-GCP4, GCP5 and GCP6 - are present as well. Structural studies of the gamma-TuRC have been not successful, preventing description of its molecular architecture and geometry and cryo-EM of the gamma-TuSC reached only a medium resolution not allowing to elucidate details as an interface on a residue level or small conformational differences. In this study, I present detailed structures of both types of gamma-TuCs complemented with biological approaches performed by my collaborators. Using cryo-EM and negative stain EM analysis, I gained insights into the structure, conformation and geometry of these complexes. Combining cryo-EM and negative stain EM analysis, I described the structure of the Candida albicans gamma-TuSC heterotetramer as a representative of the fungal microtubule template. The high-resolution reconstruction allowed me to describe in detail the intermolecular interface of Candida albicans gamma-TuSC subunits and to identify an extended interface specific for the fungal class of Saccharomycetes, which underlies conformational stability of the gamma-TuSC. This interface is absent in the vertebrate gamma-TuRC, where it is replaced by electrostatic interactions between neighbouring gamma-tubulins. A comparison of representative genomes across evolution allowed me to dissect the evolutionary relationship of microtubule nucleators, revealing that the gamma-TuSC evolved by simplification of the more complex -TuRC system. Applying cryo-EM, I successfully resolved the vertebrate gamma-TuRC at sufficient resolution to describe its shape and unambiguously assign after more than 20 years its molecular components in a specific order and position. Furthermore, I resolved an unexpected region in the lumen of the gamma-TuRC composed of a helical scaffold binding one copy of actin. My model of the gamma-TuRC allowed me to measure its helical parameters and compare it with microtubule parameters, identifying deviations that overall suggested a conformational activation mechanism for the gamma-TuRC. Cryo-EM of the gamma-TuRC revealed its uniform structure assembled from more than 30 individual proteins, which I aimed to understand by addressing the gamma-TuRC assembly mechanism. Reconstructing gamma-TuRC assembly intermediates by cryo-EM, I proposed an assembly mechanism starting from a 6-spoked assembly intermediate containing GCP4,5,6, which is expanded by preformed gamma-TuSC units. The expansion process is coupled to conformational rearrangements. Furthermore, preventing incorporation of actin into the gamma-TuRC, I demonstrated that actin is a dispensable component for gamma-TuRC structure and assembly. In conclusion, this study represents a major breakthrough in the understanding of gamma-TuC structures and their role in microtubule nucleation. It creates a basis for follow-up studies, e.g. structural studies of gamma-TuC with their binding partners from the yeast SPB of yeasts or the vertebrate centrosome, aiming to elucidating their effect on microtubule nucleation.