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

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. 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- 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.

The GTPase domain of gamma-tubulin is required for normal mitochondrial function and spatial organization

“…Similar to the nuclear compartment, mitochondria contains both DNA and is surrounded by a double membrane 12 . Recent work demonstrates an association of γ-tubulin with mitochondrial membranes 13 . In addition, the γ-tubulin’s DNA binding motif is encoded in the γ-tubulin C terminus 14 .…”

“…To test this, we mutated Cyst 13 to an Ala (GFP-A 13 γ-tubulin resist ), as this mutation impairs GTP binding to the GTPase domain 11, 28 and stably co-expresses the mutated recombinant protein in γTubulin sh-U2OS cells ( γTubulin sh-U2OS-A 13 γ-tubulin resist ; Supplementary Fig. 5b).…”

“…9Increased mitochondria protein transport and low cellular mitochondria content affect the γ-tubulin meshwork. a – c Confocal fluorescent microscopy of fixed and live U2OS cells that stably expressed γTubulin shRNA and co-expressed the sh-resistant GFP-γ-tubulin resist ( a ), the Cyst 13 to Ala 13 GFP-A 13 γ-tubulin resist ( b ) or GFP ( c ) and transiently expressed pmTurquoise2-mito (mito), as indicated. Endogenous MTCO2 was stained with an anti-MTCO2 antibody ( N  = 3).…”

“…The γ-string meshwork interacts with both the mtDNA, and with the inner mitochondrial membrane protein MTCO2, and also affects the replication of mtDNA and expression of mitochondria-related genes. Manipulation of the cellular protein levels of γ-tubulin, or of γ-tubulin GTP binding by either CDA/DMF treatment, or by mutating Cys 13 on its GTPase domain, disrupts the structure of the γ-tubulin meshwork 11 and affects mitochondrial respiratory capacity and mass. As the γ-tubulin protein levels drop below 40 % in γTubulin sgRNA expressing cells, the mitochondrial membrane potential is impaired and finally the mitochondrial integrity is lost, subsequently realising cytochrome c. In contrast, increased transport to the mitochondria, the expression of the C-terminal DNA-binding domain of γ-tubulin or disruption of γ-tubulin’s NLS, relocates γ-tubulin to the mitochondrial meshwork.…”

“…In human neuroblastoma cell lines, both neuronal development and mitochondrial-induced oxidative stress results in upregulation of TUBG2 , which is considered to be a pro-survival signal 13 . Here we show that γTubulin sgRNA mediated depletion of γ-tubulin protein kills cell lines, whereas cell lines stably expressing γTubulin shRNA survive with approximately 50% of the γ-tubulin pool, demonstrating an important role of γ-tubulin in cellular homeostasis and cell survival.…”