TY  - GEN
ID  - heidok34632
AV  - public
UR  - https://archiv.ub.uni-heidelberg.de/volltextserver/34632/
Y1  - 2024///
N2  - Microtubules, dynamic cytoskeletal filaments with a cylindrical shape made up of ??-tubulin dimers, are essential for the process of mitosis and meiosis, where their precise control in the spatial and temporal dimension is critical. ?-tubulin complexes, universally present in eukaryotes, are the most important factors for microtubule nucleation, the de novo formation of microtubules from ??-tubulin dimers. Recent cryo-electron microscopy (cryo-EM) studies of ?-tubulin complexes marked a breakthrough in the microtubule nucleation field. In these studies, the structure of the vertebrate ?-tubulin ring complex (?-TuRC) was determined, uncovering that 14 gamma-tubulin complex proteins (GCP) bound to 14 ?-tubulin proteins (14 spokes) together with the microproteins MZT1/2 and one actin molecule assemble into a defined asymmetrical left-handed spiral that forms a structural template for a 13-protofilament microtubule. However, the absence of a finely tunable system for a bottom-up dissection of the individual components? functions hinders a comprehensive understanding of ?-TuRC ?s action in microtubule organizing centers (MTOCs), like the centrosome.
Building upon the architectural consensus of the ?-TuRC subunits determined by cryo-EM studies, my PhD work focused on the development of a recombinant expression system in insect cells for the reconstitution of human ?-TuRC and related complexes. This recombinant system yielded protein complexes with structural and functional properties highly similar to the native ?-TuRC and enabled targeted analysis of individual ?-TuRC components, such as the function of the actin molecule that was surprisingly found in the lumen of the vertebrate ?-TuRC. In collaboration with Erik Zupa, I could demonstrate by cryo-EM analysis that the absence of actin in mutant ?-TuRC does not compromise the assembly and structural integrity of the complex, but its integration in the lumenal bridge is important for controlling ?-TuRC conformation and its function inside cells. Additionally, I discovered novel MZT1 docking sites and a modular assembly pathway of the human ?-TuRC, evolving from a 4-spoke GCP4- GCP5-GCP4-GCP6 intermediate to the 14-spoke asymmetric ring by successive addition of ?-tubulin small complexes (GCP2-GCP3).
Furthermore, I studied the augmin complex, a hetero-octamer of HAUS (homologous to augmin subunits) proteins, which is a crucial ?-TuRC recruiting factor and enables microtubule nucleation from pre-existing microtubules. Augmin plays a conserved role across species, from plants to humans, orchestrating microtubule amplification via the microtubule branching pathway, which is especially relevant to build the mitotic spindle. Despite its central role, a lack of structural information limits our understanding of augmin's functional sites. Employing an integrative approach, I determined the molecular architecture of the augmin complex in collaboration with Erik Zupa, revealing the collective contribution of the N-termini of HAUS2, 6, 7, and 8 to the formation of a composite microtubule binding unit.
In summary, this work represents a significant advance in the characterization of two key components of the microtubule branching pathway. Moreover, it lays the foundation for further targeted investigations of ?-TuRC and augmin, in particular their cooperation on microtubules, a fundamental aspect of cell division.
CY  - Heidelberg
TI  - Integrative structural biology of recombinant ?-tubulin-related complexes
A1  - Würtz, Martin
ER  -