Microtubule nucleation in the cytoplasm of developing cortical neurons and its regulation by brain-derived...

The centrosomes of neurons do not nucleate microtubules (MTs). It is not well known where MTs are nucleated in neurons. We performed MT regrowth experiments in primary cultured cortical neurons from mice. After cold-nocodazole depolymerization of MTs in neurons at 3 days in vitro (DIV), we observed numerous short MTs that regenerated in the cytoplasm; they were roughly equal in length and randomly oriented. Gamma-tubulin and MZT2 were detected at one end of these MTs, indicating that the short MTs were nucleated by γ-tubulin-ring complexes; the Golgi apparatus was not the origin site of their nucleation. The ratio of MT-regenerating cells, the density of regenerated MTs, and their lengths decreased at 7 DIV. Pretreatment of neurons with protein-kinase inhibitors, K-252a, staurosporine, or H7 diminished the length of the regenerated MTs, while pretreatment of neurons with brain-derived neurotrophic factor increased the length of regenerated MTs. These results support the idea that short MTs with mixed orientations are transiently generated in the cytoplasm of differentiating neurons, and that they are possibly transported into dendrites to provide seeds for MTs with mixed polarity.