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. γ-Tubulin complex (γ-TuC) plays a critical role in microtubule nucleation occurring at least at centrosomes, chromatins, and spindle microtubules. However, the control mechanism of γ-TuC-mediated microtubule nucleation remains elusive....[ Read more ]

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. γ-Tubulin complex (γ-TuC) plays a critical role in microtubule nucleation occurring at least at centrosomes, chromatins, and spindle microtubules. However, the control mechanism of γ-TuC-mediated microtubule nucleation remains elusive.

CDK5RAP2 is a human microcephaly protein that binds to the γ-TuC and is involved in the centrosomal attachment of γ-tubulin. The γ-TuC-binding domain found in CDK5RAP2 is conserved in Drosophila centrosomin, and Schizosaccharomyces pombe Mto1p and Pcp1p, which are γ-TuC-tethering proteins in the respective organisms. In the first project, I show that this domain within CDK5RAP2 associates with the γ-tubulin ring complex (γ-TuRC) to stimulate its microtubule-nucleating activity and is therefore referred to as the γ-TuRC-mediated nucleation activator (γ-TuNA). The γ-TuNA but not its γ-TuRC-binding deficient mutant stimulates microtubule nucleation by purified γ-TuRC in vitro and induces extensive, γ-TuRC-dependent nucleation of microtubules in a microtubule regrowth assay. γ-TuRC bound to the γ-TuNA contains NME7, FAM128A/B, and actin in addition to γ-tubulin and GCP2-6. RNAi-mediated depletion of CDK5RAP2 impairs both centrosomal and acentrosomal microtubule nucleation, although γ-TuRC assembly is unaffected. Collectively, these results suggest that the γ-TuNA found in CDK5RAP2 has regulatory functions in γ-TuRC-mediated microtubule nucleation.

I also investigated the novel γ-TuRC-associated protein NME7 and have found that NME7 is a regulatory component of γ-TuRC and plays an important role in γ-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 γ-TuRC. Although NME7 is not required for γ-TuRC assembly or the assembly of the γ-TuRC into centrosomes, RNAi-mediated depletion of NME7 perturbed the centrosomal microtubule nucleation and ciliogenesis. γ-Tubulin is a GDP/GTP binding protein, however, whether GDP/GTP regulates γ-TuRC-mediated microtubule nucleation remains unknown. In the in vitro assays, the microtubule nucleating-activity of γ-TuRC isolated by γ-TuNA was severely compromised by GDP. In the same settings, NME7 stimulated the microtubule nucleating-activity of GDP-preloaded γ-TuRC and promoted GTP binding to γ-tubulin in γ-TuRC regardless of its autophosphorylating activity. Taken together, these results authenticate that NME7 stimulates the γ-TuRC-mediated microtubule nucleation by facilitating the GTP-bound form of γ-TuRC.

In summary, γ-TuRC-mediated microtubule nucleation is under multiple controls by γ-TuNA found in CDK5RAP2 and NME7. Multimeric γ-tubulins assembly to γ-TuRC is the prerequisite for the association of γ-TuNA and NME7. The γ-TuNA associates with the γ-TuRC to stimulate γ-TuRC-mediated microtubule nucleation. In addition, NME7 promotes the GTP-bound form of γ-TuRC and facilitates γ-TuRC-mediated microtubule nucleation process.