The microtubule cytoskeleton is involved in many essential cellular processes, such as intracellular transport, cell division, cell locomotion and morphogenesis. In animal cells, the centrosomes control the temporal and spatial distribution of microtubules, and thus are known as the primary microtubule-organizing centers. Centrosomes are composed of a pair of centrioles (i.e. mother and daughter centrioles) surrounded by a pericentriolar material in which microtubule nucleation occurs and is mediated by the γ-tubulin ring complex (γ-TuRC). Recent works have greatly improved our understanding on the microtubule nucleation mediated by γ-TuRC, and implicated that this process involves the integration of multiple mechanisms. However, the molecular composition and regulation of the n...[ Read more ]

The microtubule cytoskeleton is involved in many essential cellular processes, such as intracellular transport, cell division, cell locomotion and morphogenesis. In animal cells, the centrosomes control the temporal and spatial distribution of microtubules, and thus are known as the primary microtubule-organizing centers. Centrosomes are composed of a pair of centrioles (i.e. mother and daughter centrioles) surrounded by a pericentriolar material in which microtubule nucleation occurs and is mediated by the γ-tubulin ring complex (γ-TuRC). Recent works have greatly improved our understanding on the microtubule nucleation mediated by γ-TuRC, and implicated that this process involves the integration of multiple mechanisms. However, the molecular composition and regulation of the nucleating activity of the γ-TuRC is still unclear. In quiescent cells, the mother centrioles become the basal bodies and organize the formation of cilia. Protruding from the cell surface, cilia are microtubule-based organelles and are present in most cell types. The functions of cilia include clearing environment by rhythmical motion, generating fluid flow by coordinated beating, sensing the stimuli through various receptors and mediating signal transduction. Formation of functional cilia is critical for animal development. Disruption of cilia function results in ciliopathies, cilia-related disorders. Nonetheless, the molecular mechanisms underlying ciliogenesis and ciliopathies are still largely unknown.

In my research, I characterize NME7, an uncharacterized member of the NME (proteins expressed in non-metastatic cells) family, and show that NME7 is both involved in the microtubule organization and cilia function. Several characterized NME family members possess nucleoside-diphosphate kinase (NDPK) and protein histidine kinase activity. NME7 contains a DM10 domain and two putative kinase domains, kinase domains A and B. Here I demonstrate that the kinase domain A of NME7 is required for autophosphorylation, although I did not detect the NDP kinase activity of NME7. NME7 also localizes to the centrosome and NME7 is not essential for centrosome duplication and cell cycle progression. I also show that NME7 is a component of the γ-TuRC and the kinase domain B is crucial for the γ-TuRC binding. However, NME7 is not required for the γ-TuRC assembly or centrosome targeting. The centrosome localization of NME7 depends on its incorporation into the γ-TuRC. Depletion of NME7 impairs γ-TuRC-mediated microtubule nucleation. Moreover, NME7 facilitates the γ-TuRC-mediated microtubule nucleation in vitro through its kinase action.

The NME7^-/- mice has primary ciliary dyskinesia (PCD) symptoms, implying that NME7 may be involved in cilia biogenesis or function. However, in these mice, histologically normal cilia are present on ependymal cells and respiratory epithelia. Therefore, the role of NME7 in cilia function is unclear. In my study, I provide evidence for a role of NME7 in cilia function in cultured cells and in zebrafish. I show that NME7 localizes to the basal body and the axoneme of the immature cilia, and the axoneme targeting of NME7 requires DM10 domain and γ-TuRC binding. I also examined the role of NME7 in zebrafish development. Cilia formation was compromised in nme7 morphants at the early developmental stage. And the defects in cilia resulted in hydrocephalus and disrupted left-right axis establishment, suggesting that NME7 is required for cilia formation and function during animal development.

To summarize, my study reveals that NME7 is both involved in γ-TuRC-mediated microtubule nucleation and cilia function. My study uncovers the kinase function of NME7 and suggests a new mechanism that controls the microtubule nucleation mediated by the γ-TuRC. My study also provides insight into the pathogenic mechanisms underlying ciliopathies.