The microtubule cytoskeleton plays important roles in various cellular functions.

Microtubules are nucleated by the γ-tubulin ring complex (γ-TuRC) in the microtubule organizing center (MTOC). Even the structure of γ-TuRC has been reported, the regulation of γ-TuRC and γ-TuRC-mediated microtubule nucleation is still unclear. In this study, the regulation of microtubules is investigated in 2 aspects: 1) by studying the ubiquitin-proteasome degradation pathway of γ-tubulin, and 2) by discovering novel microtubule regulators using high-throughput CRISPR screening. In the first part, I found potential ubiquitination sites of γ-tubulin degradation, confirmed cullin-RING ligase (CRL) is responsible for the pathway, and identified ubiquitin linkage K48 as the only linkage for γ-tubulin degrad...[ Read more ]

The microtubule cytoskeleton plays important roles in various cellular functions.

Microtubules are nucleated by the γ-tubulin ring complex (γ-TuRC) in the microtubule organizing center (MTOC). Even the structure of γ-TuRC has been reported, the regulation of γ-TuRC and γ-TuRC-mediated microtubule nucleation is still unclear. In this study, the regulation of microtubules is investigated in 2 aspects: 1) by studying the ubiquitin-proteasome degradation pathway of γ-tubulin, and 2) by discovering novel microtubule regulators using high-throughput CRISPR screening. In the first part, I found potential ubiquitination sites of γ-tubulin degradation, confirmed cullin-RING ligase (CRL) is responsible for the pathway, and identified ubiquitin linkage K48 as the only linkage for γ-tubulin degradation. In the second part, I used fluorescence-activated cell sorting (FACS) for high-throughput CRISPR screening of microtubule regulators based on fluorescence intensities. I established a novel microtubule regrowth assay, a common approach for studying microtubule nucleation, especially for FACS analysis. This study provides insights into a novel approach to microtubule regulation by degradation and finds out novel players of microtubule nucleation.

Keywords: microtubule, γ-tubulin, γ-tubulin ring complex, ubiquitination, cullin-RING ligase, ubiquitin-proteasome degradation, CRISPR, genetic screening