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Abstract

Microtubules (MTs) are hollow tubular structures composed of protofilaments of α/β-tubulin dimers that play a crucial role in vital cellular processes, such as chromosome separation, intracellular trafficking and organelle organisation. These processes critically rely on the de novo generation of MTs, termed MT nucleation, in the right number, at the correct time and place. Therefore, MTs are not nucleated randomly, but by γ-tubulin-containing complexes (γ-TuCs), which function as a template MT nucleation through 14 γ-tubulin subunits in a helical arrangement, similar to the α/β-tubulin subunits in the MT. In vertebrates, the γ-TuC consists of 14 spokes that are pre-assembled from over 40 proteins in the cytoplasm and is called the γ-tubulin ring complex (γ-TuRC). The γ-TuRC is then recruited to a MT-organising center (MTOC)such as the centrosome, where it is activated. In ascomycete yeast model organisms, on the other hand, the compositionally simpler 2-spoked γ-TuSC (γ-tubulin small complex) is only assembled into a 14-spoked γ-TuRC and activated upon their recruitment to the ascomycete yeast MTOC, the nuclear envelope-embedded spindle-pole body (SPB). Recent cryo-EM reconstructions have revealed the architecture of the purified vertebrate γ-TuRC as well as the ascomycete yeast γ-TuSC. However, many aspects of the spatiotemporal regulation of MT nucleation still remained unclear. In this thesis, I aimed to advance the understanding of the MTOC recruitment and activation of γ-TuCs at the structural level using cryo-EM.

The structural basis for how vertebrate γ-TuRCs are recruited to MTOCs, including the centrosome, has thus far remained unknown. In the first part of my work, I used cryo- EM to show how NEDD1, an essential adaptor protein mediating γ-TuRC localisation to various MTOCs, binds to the γ-TuRC as part of a native complex purified from Xenopus laevis egg extracts (purification by Qi Gao). NEDD1 forms a tetrameric grapnel-shaped complex together with four of the N-GCP3/MZT1 modules that are part of the γ-TuRC. The NEDD1 grapnel docks the bottom of the γ-TuRC in a defined orientation, revealing how NEDD1 allows to pre-orient the γ-TuRC for directed MT nucleation. This study was published in Nature Communications, with me as a shared first author.

After recruitment to the MTOC, γ-TuRCs are activated for MT nucleation. Available cryo-EM reconstructions of the purified vertebrate γ-TuRC surprisingly revealed an asymmetric γ-tubulin arrangement, called the open conformation. This conformation is incompatible with the symmetry of a 13 protofilament MT, raising the question whether and how the γ-TuRC adopts its conformation to accommodate MT nucleation. In the second part of my work, in collaboration with Dr. Anna Böhler and Qi Gao, I obtained cryo-EM reconstructions of the γ-TuRC capping a MT nucleated through a native spindle assembly pathway in cytoplasmic extracts from X. laevis. I could show that the MT-capping γ-TuRC partially closes towards, but does not reach full MT-like symmetry. To adapt to the remaining γ-TuRC asymmetry, the MT assumes a partially asymmetric protofilament arrangement featuring inter-protofilament gaps and exhibits local misalignment to the γ-TuRC. Together with Qi Gao, I showed that the asymmetric arrangement of the γ-TuRC-capped MT end can be specifically recognised by MT-binding protein CAMSAP2. This study was published in EMBO Journal, with me as a shared first author.

The activation and recruitment of γ-TuRCs follows a different mechanism in ascomycete yeasts compared to vertebrates. Previous work had shed light on the assembly of γ-TuRCs at the nuclear side of the ascomycete yeast SPB. Yet, the architecture of the cytoplasmic MT nucleation unit, consisting of the γ-TuSC with CM1- containing γ-TuC receptor Spc72 and nucleation-promoting MT polymerase Stu2, was poorly understood. In the third part of my work, performed in collaboration with Dr. Anjun Zheng, I used cryo-EM to reveal how Candida albicans Spc72 assembles the γ-TuRC through its dimeric CM1 motif. In contrast to other CM1-containing proteins, Spc72 stoichiometrically converts the γ-TuRC into a conformationally fully closed MT template, primed for efficient MT nucleation. In addition, I used AlphaFold2 to predict how Stu2 binds to two Spc72 coiled-coil modules through its conserved C-terminal α-helix, which was confirmed by biochemical data from Dr. Anjun Zheng and Dr. Martin Würtz (Schiebel lab). This study was published in Nature Communications, with me as a shared first author.

In conclusion, my work provides structural insights into the MTOC recruitment and conformational regulation of the γ-TuRC, decisively advancing the understanding of the mechanism and spatiotemporal regulation of MT nucleation.