Regulation of the Microtubule-Nucleating Activity of the gamma-Tubulin Ring Complex

Polymerization dynamics enable microtubules to carry out spatial arrangements in response to cellular necessities. Like many other protein polymerization processes, the nucleation steps of microtubule polymerization reactions are slower than the later elongation processes. This allows biological systems to spatially control the reactions. gamma-Tubulin complex (gamma-TuC) plays a critical role in microtubule nucleation occurring at least at centrosomes, chromatins, and spindle microtubules. However, the control mechanism of gamma-TuC-mediated microtubule nucleation remains elusive.;CDK5RAP2 is a human microcephaly protein that binds to the gamma-TuC and is involved in the centrosomal attachment of gamma-tubulin. The gamma-TuC-binding domain found in CDK5RAP2 is conserved in Drosophila centrosomin, and Schizosaccharomyces pombe Mto1p and Pcp1p, which are gamma-TuC-tethering proteins in the respective organisms. In the first project, I show that this domain within CDK5RAP2 associates with the gamma-tubulin ring complex (gamma-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the gamma-TuRC-mediated nucleation activator (gamma-TuNA). The gamma-TuNA but not its gamma-TuRC-binding deficient mutant stimulates microtubule nucleation by purified gamma-TuRC in vitro and induces extensive, gamma-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. gamma-TuRC bound to the gamma-TuNA contains NME7, FAM128A/B, and actin in addition to gamma-tubulin and GCP2-6. RNAi-mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although gamma-TuRC assembly is unaffected. Collectively, these results suggest that the gamma-TuNA found in CDK5RAP2 has regulatory functions in gamma-TuRC-mediated microtubule nucleation.;I also investigated the novel gamma-TuRC-associated protein NME7 and have found that NME7 is a regulatory component of gamma-TuRC and plays an important role in gamma-TuRC-mediated microtubule nucleation and ciliogenesis. Immunofluorescence studies show that NME7 localizes to centrosomes throughout all stages of cell cycle, in addition to the basal body and axoneme of the early formed cilium. Furthermore, the NME7 centrosomal localization depends on its incorporation into the gamma-TuRC. Although NME7 is not required for gamma-TuRC assembly or the assembly of the gamma-TuRC into centrosomes, RNAi-mediated depletion of NME7 perturbed the centrosomal microtubule nucleation and ciliogenesis. gamma-Tubulin is a GDP/GTP binding protein, however, whether GDP/GTP regulates gamma-TuRC-mediated microtubule nucleation remains unknown. In the in vitro assays, the microtubule nucleating-activity of gamma-TuRC isolated by gamma-TuNA was severely compromised by GDP. In the same settings, NME7 stimulated the microtubule nucleating-activity of GDP-preloaded gamma-TuRC and promoted GTP binding to gamma-tubulin in gamma-TuRC regardless of its autophosphorylating activity. Taken together, these results authenticate that NME7 stimulates the gamma-TuRC-mediated microtubule nucleation by facilitating the GTP-bound form of gamma-TuRC.;In summary, gamma-TuRC-mediated microtubule nucleation is under multiple controls by gamma-TuNA found in CDK5RAP2 and NME7. Multimeric gamma-tubulins assembly to gamma-TuRC is the prerequisite for the association of gamma-TuNA and NME7. The gamma-TuNA associates with the gamma-TuRC to stimulate gamma-TuRC-mediated microtubule nucleation. In addition, NME7 promotes the GTP-bound form of gamma-TuRC and facilitates gamma-TuRC-mediated microtubule nucleation process.