γ-Tubulin Distribution in the Neuron: Implications for the Origins of Neuritic Microtubules

Axons and dendrites contain dense microtubule (MT) arrays that are not attached to a traditional MT nucleating structure such as the centrosome. Nevertheless, the MTs within these neurites are highly organized with respect to their polarity, and consist of a regular 13-protofilament lattice, the two known characteristics of MTs nucleated at the centrosome. These observations suggest either that axonal and dendritic MTs arise at the centrosome, or that they are nucleated locally, following a redistribution of MT nucleating material from the centrosome during neuronal development. To begin distinguishing between these possibilities, we have determined the distribution of γ-tubulin within cultured sympathetic neurons. γ-tubulin, a newly discovered protein which is specifically localized to the pericentriolar region of nonneuronal cells (Zheng, Y., M. K. Jung, and B. R. Oakley. 1991. Cell. 65:817-823; Stearns, T., L. Evans, and M. Kirschner. 1991. Cell. 65:825-836), has been shown to play a critical role in MT nucleation in vivo (Joshi, H. C., M. J. Palacios, L. McNamara, and D. W. Cleveland. 1992. Nature (Lond.). 356:80-83). Because the γ-tubulin content of individual cells is extremely low, we relied principally on the high degree of resolution and sensitivity afforded by immunoelectron microscopy. Our studies reveal that, like the situation in nonneuronal cells, γ-tubulin is restricted to the pericentriolar region of the neuron. Furthermore, serial reconstruction analyses indicate that the minus ends of MTs in both axons and dendrites are free of γ-tubulin immunoreactivity. The absence of γ-tubulin from the axon was confirmed by immunoblot analyses of pure axonal fractions obtained from explant cultures. The observation that γ-tubulin is restricted to the pericentriolar region of the neuron provides compelling support for the notion that MTs destined for axons and dendrites are nucleated at the centrosome, and subsequently released for translocation into these neurites.