Differential expression of human γ-tubulin isotypes during neuronal development and oxidative stress points to a γ-tubulin-2 prosurvival function.

gamma-Tubulins are highly conserved members of the tubulin superfamily essential for microtubule nucleation. Humans possess 2 gamma-tubulin genes. It is thought that gamma-tubulin-1 represents aubiquitous isotype, whereas gamma-tubulin-2 is found predominantly in the brain, where it may be endowed with divergent functions beyond microtubule nucleation. The molecular basis of the purported functional differences between gamma-tubulins is unknown. We report discrimination of human gamma-tubulins according to their electrophoretic and immunochemical properties. In vitro mutagenesis revealed that the differences in electrophoretic mobility originate in the C-terminal regions of the gamma-tubulins. Using epitope mapping, we discovered mouse monoclonal antibodies that can discriminate between human gamma-tubulin isotypes. Real time quantitative RT-PCR and 2-dimensional-PAGE showed that gamma-tubulin-1 is the dominant isotype in fetal neurons. Although gamma-tubulin-2 accumulates in the adult brain, gamma-tubulin-1 remains the major isotype in various brain regions. Localization of gamma-tubulin-1 in mature neurons was confirmed by immunohistochemistry and immunofluorescence microscopy on clinical samples and tissue microarrays. Differentiation of SH-SY5Y human neuroblastoma cells by all-trans retinoic acid, or oxidative stress induced by mitochondrial inhibitors, resulted in upregulation of gamma-tubulin-2, whereas the expression of gamma-tubulin-1 was unchanged. Fractionation experiments and immunoelectron microscopy revealed an association of gamma-tubulins with mitochondrial membranes. These data indicate that in the face of predominant nu-tubulin-1 expression, the accumulation of gamma-tubulin-2 in mature neurons and neuroblastoma cells during oxidative stress may denote a prosurvival role of gamma-tubulin-2 in neurons.-Draberova, E., Sulimenko, V., Vinopal, S., Sulimenko, T., Sladkova, V., D'Agostino, L., Sobol, M., Hozak, P., Kren, L., Katsetos, C. D., Draber, P. Differential expression of human gamma-tubulin isotypes during neuronal development and oxidative stress points to gamma-tubulin-2 prosurvival function.