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Differential expression of human γ‐tubulin isotypes during neuronal development and oxidative stress points to a γ‐tubulin‐2 prosurvival function
Author(s) -
Dráberová Eduarda,
Sulimenko Vadym,
Vinopal Stanislav,
Sulimenko Tetyana,
Sládková Vladimíra,
D'Agostino Luca,
Sobol Margaryta,
Hozák Pavel,
Křen Leoš,
Katsetos Christos D.,
Dráber Pavel
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.201600846rr
Subject(s) - tubulin , biology , microtubule , isotype , microbiology and biotechnology , epitope , neurite , monoclonal antibody , biochemistry , antibody , genetics , in vitro
ABSTRACT γ‐Tubulins are highly conserved members of the tubulin superfamily essential for microtubule nucleation. Humans possess 2 γ‐tubulin genes. It is thought that γ‐tubulin‐1 represents a ubiquitous isotype, whereas γ‐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 γ‐tubulins is unknown. We report discrimination of human γ‐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 γ‐tubulins. Using epitope mapping, we discovered mouse monoclonal antibodies that can discriminate between human γ‐tubulin isotypes. Real time quantitative RT‐PCR and 2‐dimensional‐PAGE showed that γ‐tubulin‐1 is the dominant isotype in fetal neurons. Although γ‐tubulin‐2 accumulates in the adult brain, γ‐tubulin‐1 remains the major isotype in various brain regions. Localization of γ‐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 γ‐tubulin‐2, whereas the expression of γ‐tubulin‐1 was unchanged. Fractionation experiments and immunoelectron microscopy revealed an association of γ‐tubulins with mitochondrial membranes. These data indicate that in the face of predominant γ‐tubulin‐1 expression, the accumulation of γ‐tubulin‐2 in mature neurons and neuroblastoma cells during oxidative stress may denote a prosurvival role of γ‐tubulin‐2 in neurons.—Dráberová, E., Sulimenko, V., Vinopal, S., Sulimenko, T., Sládková, V., D'Agostino, L., Sobol, M., Hozák, P., Křen, L., Katsetos, C. D., Dráber, P. Differential expression of human γ‐tubulin isotypes during neuronal development and oxidative stress points to γ‐tubulin‐2 prosurvival function. FASEB J. 31, 1828–1846 (2017). www.fasebj.org