eprintid: 34632
rev_number: 15
eprint_status: archive
userid: 8036
dir: disk0/00/03/46/32
datestamp: 2024-03-28 08:03:25
lastmod: 2024-03-28 09:15:02
status_changed: 2024-03-28 08:03:25
type: doctoralThesis
metadata_visibility: show
creators_name: Würtz, Martin
title: Integrative structural biology of recombinant γ-tubulin-related complexes
subjects: ddc-570
divisions: i-140001
adv_faculty: af-14
cterms_swd: Zellbiologie
cterms_swd: Zytoskelett
cterms_swd: Biologische Chemie
abstract: Microtubules, dynamic cytoskeletal filaments with a cylindrical shape made up of αβ-tubulin dimers, are essential for the process of mitosis and meiosis, where their precise control in the spatial and temporal dimension is critical. γ-tubulin complexes, universally present in eukaryotes, are the most important factors for microtubule nucleation, the de novo formation of microtubules from αβ-tubulin dimers. Recent cryo-electron microscopy (cryo-EM) studies of γ-tubulin complexes marked a breakthrough in the microtubule nucleation field. In these studies, the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) was determined, uncovering that 14 gamma-tubulin complex proteins (GCP) bound to 14 γ-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 γ-TuRC ́s action in microtubule organizing centers (MTOCs), like the centrosome.
Building upon the architectural consensus of the γ-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 γ-TuRC and related complexes. This recombinant system yielded protein complexes with structural and functional properties highly similar to the native γ-TuRC and enabled targeted analysis of individual γ-TuRC components, such as the function of the actin molecule that was surprisingly found in the lumen of the vertebrate γ-TuRC. In collaboration with Erik Zupa, I could demonstrate by cryo-EM analysis that the absence of actin in mutant γ-TuRC does not compromise the assembly and structural integrity of the complex, but its integration in the lumenal bridge is important for controlling γ-TuRC conformation and its function inside cells. Additionally, I discovered novel MZT1 docking sites and a modular assembly pathway of the human γ-TuRC, evolving from a 4-spoke GCP4- GCP5-GCP4-GCP6 intermediate to the 14-spoke asymmetric ring by successive addition of γ-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 γ-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 γ-TuRC and augmin, in particular their cooperation on microtubules, a fundamental aspect of cell division.
date: 2024
id_scheme: DOI
id_number: 10.11588/heidok.00034632
ppn_swb: 1884571727
own_urn: urn:nbn:de:bsz:16-heidok-346329
date_accepted: 2024-03-19
advisor: HASH(0x5608dc6d5fc8)
language: eng
bibsort: WURTZMARTIINTEGRATIV
full_text_status: public
place_of_pub: Heidelberg
citation:   Würtz, Martin  (2024) Integrative structural biology of recombinant γ-tubulin-related complexes.  [Dissertation]     
document_url: https://archiv.ub.uni-heidelberg.de/volltextserver/34632/1/PhD_Thesis_Martin_W%C3%BCrtz2024Final.pdf