Consolati, Tanja;
(2019)
Regulation of γTuRC-mediated microtubule nucleation studied in vitro.
Doctoral thesis (Ph.D), UCL (University College London).
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Abstract
Microtubules are cytoskeletal filaments central to a wide range of cellular processes: they serve as tracks for intracellular transport, provide mechanical support, serve as building blocks for flagella and cilia and drive the separation of the genomic material for cell division. The organization of the microtubule cytoskeleton into different architectures is crucial for its function. Cells regulate the formation of these microtubule arrays by tight temporal and spatial control over the nucleation of new microtubules. // The main microtubule nucleator in vivo is the γ-tubulin ring complex (γTuRC), a multi-subunit complex conserved in all eukaryotic cells. Currently, the molecular mechanism of γTuRC-mediated microtubule nucleation is poorly understood. This is in part due to the difficulty to purify γTuRCs with good yields, in particular from higher eukaryotes, and the lack of an assay able to visualize and quantify microtubule nucleation kinetics in real time. // In cells, the γTuRC interacts with a multitude of proteins important for γTuRC localization and/or activation. In addition, it has emerged that γTuRC nucleation efficiency can be modulated by microtubule binding proteins via their effects on microtubule dynamics. What is missing is a framework to understand how these different factors regulate the γTuRC activity and how they can mechanistically work together to control γTuRC-mediated microtubule nucleation in cells. // To address this question, I established a new method for the purification of human γTuRCs from a stable cell line and developed a new real-time dynamic TIRF microscopy-based microtubule nucleation assay. The activity of purified γTuRCs was studied in the presence and in the absence of potential regulators and microtubule binding proteins in order to better understand the mechanism of microtubule nucleation and its regulation.
| Type: | Thesis (Doctoral) |
|---|---|
| Qualification: | Ph.D |
| Title: | Regulation of γTuRC-mediated microtubule nucleation studied in vitro |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| Additional information: | Copyright © The Author 2019. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. |
| Keywords: | microtubule nucleation, γ-tubulin ring complex, γTuRC |
| UCL classification: | UCL UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/10082151 |
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