MABT29 Sigma-AldrichAnti-Ninein Antibody, clone 79-160-7

OBJECTIVE: Angiogenesis is an integral part of many physiological processes but may also aggravate pathological conditions such as cancer. Development of effective angiogenesis inhibitors requires a thorough understanding of the molecular mechanisms regulating vessel formation. The aim of this project was to identify proteins that regulate tubular morphogenesis of endothelial cells. METHODS AND RESULTS: Phosphotyrosine-dependent affinity-purification and mass spectrometry showed tyrosine phosphorylation of ninein during tubular morphogenesis of endothelial cells. Ninein was recently identified as a centrosomal microtubule-anchoring protein. Our results show that ninein is localized in the cytoplasm in endothelial cells, and that it is highly expressed in the vasculature in normal and pathological human tissues. Using embryoid bodies as a model of vascular development, we found that ninein is abundantly expressed in the cytoplasm of endothelial cells during sprouting angiogenesis, in particular in the sprouting tip-cell. In accordance, siRNA-dependent silencing of ninein in endothelial cells inhibited tubular morphogenesis. CONCLUSIONS: In this study, we show that ninein is expressed in developing vessels and in endothelial tip cells, and that ninein is critical for formation of the vascular tube. These data strongly implicate ninein as an important new regulator of angiogenesis.

Cell-to-cell contact and polarisation of epithelial cells involve a major reorganisation of the microtubules and centrosomal components. The radial microtubule organisation is lost and an apico-basal array develops that is no longer anchored at the centrosome. This involves not only the relocation of microtubules but also of centrosomal anchoring proteins to apical non-centrosomal sites. The relocation of microtubule minus-end-anchoring proteins such as ninein to the apical sites is likely to be essential for the assembly and stabilisation of the apico-basal arrays in polarised epithelial cells. In this study, we establish that ninein is highly dynamic and that, in epithelial cells, it is present not only at the centrosome but also in the cytoplasm as distinct speckles. Live-cell imaging reveals that GFP-ninein speckles are released from the centrosome and move in a microtubule-dependent manner within the cytoplasm and thus establishes that epithelial cells possess the mechanical means for relocation of ninein to non-centrosomal anchoring sites. We also provide evidence for the deployment of ninein speckles to apical anchoring sites during epithelial differentiation in both an in situ tissue and an in vitro culture system. In addition, the findings suggest that the non-centrosomal microtubule anchoring sites associate with adherens junctions in polarised epithelial cells.

Primary cilia (PC) function as microtubule-based sensory antennae projecting from the surface of many eukaryotic cells. They play important roles in mechano- and chemosensory perception and their dysfunction is implicated in developmental disorders and severe diseases. The basal body that functions in PC assembly is derived from the mature centriole, a component of the centrosome. Through a small interfering RNA screen we found several centrosomal proteins (Ceps) to be involved in PC formation. One newly identified protein, Cep164, was indispensable for PC formation and hence characterized in detail. By immunogold electron microscopy, Cep164 could be localized to the distal appendages of mature centrioles. In contrast to ninein and Cep170, two components of subdistal appendages, Cep164 persisted at centrioles throughout mitosis. Moreover, the localizations of Cep164 and ninein/Cep170 were mutually independent during interphase. These data implicate distal appendages in PC formation and identify Cep164 as an excellent marker for these structures.

The centrosome organizes microtubules by controlling nucleation and anchoring processes. In mammalian cells, subdistal appendages of the mother centriole are major microtubule-anchoring structures of the centrosome. It is not known how newly nucleated microtubules are anchored to these appendages. We show here that ninein, a component of subdistal appendages, localizes to the centriole via its C-terminus and interacts with gamma-tubulin-containing complexes via its N-terminus. Expression of a construct encoding the ninein C-terminus displaced endogenous ninein and the gamma-tubulin ring complex (gamma-TuRC) from the centrosome, leading to microtubule nucleation and anchoring defects. By contrast, expression of a fusion consisting of the N- and C-terminal domains (lacking the central coiled-coil region) displaced endogenous ninein without perturbing gamma-TuRC localization. Accordingly, only anchoring defects were observed in this case. Therefore, expression of this fusion appeared to uncouple microtubule nucleation and anchorage activities at the centrosome. Our results suggest that ninein has a role not only in microtubule anchoring but also in promoting microtubule nucleation by docking the gamma-TuRC at the centrosome. In addition, we show that the gamma-TuRC might not be sufficient to anchor microtubules at the centrosome in the absence of ninein. We therefore propose that ninein constitutes a molecular link between microtubule-nucleation and -anchoring activities at the centrosome.