Microtubule Nucleating complexes: A structural view

Microtubule Organizing Centers (MTOCs) organize the spatial and temporal patterns of microtubules by controlling their nucleation. Microtubule nucleation is mediated by γ-Tubulin. From yeast to humans, γ-Tubulin is found in a ~300kDa protein complex called γ-Tubulin Small Complex or γTuSC. γTuSC assembles into a ~2.2MDa complex called γ-Tubulin Ring Complex (γTuRC) that nucleates Microtubules by providing a template from which protofilaments can nucleate. The structure of the S. cereviseae γTuRC has been determined at a subnanometer resolution, indicating the organization of the γTuSC within the ring. While the overall structure of the γTuRC is conserved, in higher-eukaryotes γTuRC contains 5 more proteins (GCPs) than its yeast counterpart. Studies at the cell level indicate that these proteins are used as localization markers for γTuRCs within the cell. Structural work in one of the human non-γTuSC proteins has revealed a striking similarity to the yeast proteins indicating that the GCPs localize within the ring. Despite this indications, the organization of the GCPs within these complexes remains unclear. The following manuscript details the work done in understanding the organization of the proteins that make up the S. cereviseae γTuSC and the first Cryo-Electron Microscopy structure of the D. melanogaster γTuRC. The S. cereviseae work describes the orientation of the proteins within the complex which helped in understanding the sequence to structure role. The structural work of the D. melanogaster γTuRC strengthens the theory of the localization of the extra GCPs and an initial indication of the stoichiometry of these components. As part of the structural work on γTuRC, an acquisition paradigm was designed to speed up cryo-EM tomography data collection as well as significantly improving the data quality and resolution