Microtubules are crucial for maintaining cell shape, mediating chromosome alignment during mitosis and providing tracks for subcellular molecular and organelle transportation. These microtubule-dependent processes require proper regulation of microtubules nucleation. As the principle microtubule nucleator, the γ-tubulin ring complex (γ-TuRC) basically consists of γ-tubulin and gama-tubulin complex proteins (GCPs, GCP2-6). And several γ-TuRC-associated proteins regulate the γTuRC functions, frequently through mediating the attachment of the complex to microtubule-organizing centers (MTOCs), at least the centrosomes and the Golgi. However, a more detailed control system of γTuRC-mediated microtubule nucleation remains elusive.

Among the several γTuRC associated proteins, CDK5RAP2 stimula...[ Read more ]

Microtubules are crucial for maintaining cell shape, mediating chromosome alignment during mitosis and providing tracks for subcellular molecular and organelle transportation. These microtubule-dependent processes require proper regulation of microtubules nucleation. As the principle microtubule nucleator, the γ-tubulin ring complex (γ-TuRC) basically consists of γ-tubulin and gama-tubulin complex proteins (GCPs, GCP2-6). And several γ-TuRC-associated proteins regulate the γTuRC functions, frequently through mediating the attachment of the complex to microtubule-organizing centers (MTOCs), at least the centrosomes and the Golgi. However, a more detailed control system of γTuRC-mediated microtubule nucleation remains elusive.

Among the several γTuRC associated proteins, CDK5RAP2 stimulates γ-TuRC-mediated microtubule nucleation and NEDD1 is indispensable for chromosome-mediate microtubule nucleation. My previous lab mate Mr. Yu Hu used CDK5RAP2 and NEDD1 to isolate the γ-TuRC and identified several γ-TuRC-associated candidates by mass spectrometry. Among these proteins, NDR1, FDH and Eg5 attracted our interests. NDR1 is involved in centrosome duplication, chromosome alignment, cell cycle regulation and tumor suppression. NDR2, which has similar amino acid sequence to NDR1, is also considered as a potential candidate. FDH is an aldehyde dehydrogenase, and the only known function is its involvement in formate oxidation. Eg5 is a motor protein important for the polarity of the spindle. Considering their potential roles in regulating γTuRC function, I characterized their binding properties of γTuRC and uncover the functions of these bindings. Immunoprecipitation studies revealed that only NDR1 and NDR2 binded with γTuRC. Knockdown of NDR1 or NDR2 inhibited the centrosomal microtubule nucleation. However, knockdown of NDR1 or NDR2 did not affect the centrsomal γTuRC components, which indicated that NDR kinases did not regulates γ-TuRC-mediated nucleation by targeting the γ-TuRC to the centrosome. Interestingly, overexpression of NDR1 or NDR2 decreased the centrosomal the level of γ-tubulin, which indicated the γTuRC-binding properties of NDR kinases. In conclusion, my thesis study shows the discovery of new γTuRC-associated proteins, NDR1 and NDR2, regulating the γTuRC-mediated microtubule nucleation. Since both MST1-MOB1-NDR axis and γTuRCs are involved in centrosome duplication. It is possible that the MST1-MOB1-NDR axis may also activate the γTuRCs on the cartwheel base of the procentriole nucleation site to nucleate microtubule triplets of the centriole. So in addition to discovery of a new γTuRC-associated protein regulates the γTuRC-mediated microtubule nucleation, my thesis study also provide an insight into the mechanism of γTuRCs initiate centriolar microtubule triplets.