Activation of mast cells by aggregation of the high-affinity IgE receptors (FcεRI) initiates signaling events leading to the release of inflammatory and allergic mediators stored in cytoplasmic granules. A key role in this process play changes in concentrations of intracellular Ca²⁺ controlled by store-operated Ca²⁺ entry (SOCE). Although microtubules are also involved in the process leading to degranulation, the molecular mechanisms that control microtubule rearrangement during activation are largely unknown. In this study, we report that activation of bone marrow-derived mast cells (BMMCs) induced by FcεRI aggregation or treatment with pervanadate or thapsigargin results in generation of protrusions containing microtubules (microtubule protrusions). Formation of these protrusions depended on the influx of extracellular Ca²⁺. Changes in cytosolic Ca²⁺concentration also affected microtubule plus-end dynamics detected by microtubule plus-end tracking protein EB1. Experiments with knockdown or reexpression of STIM1, the key regulator of SOCE, confirmed the important role of STIM1 in the formation of microtubule protrusions. Although STIM1 in activated cells formed puncta associated with microtubules in protrusions, relocation of STIM1 to a close proximity of cell membrane was independent of growing microtubules. In accordance with the inhibition of Ag-induced Ca²⁺ response and decreased formation of microtubule protrusions in BMMCs with reduced STIM1, the cells also exhibited impaired chemotactic response to Ag. We propose that rearrangement of microtubules in activated mast cells depends on STIM1-induced SOCE, and that Ca²⁺ plays an important role in the formation of microtubule protrusions in BMMCs.

γ-Tubulin is the key protein for microtubule nucleation. Duplication of the γ-tubulin gene occurred several times during evolution, and in mammals γ-tubulin genes encode proteins which share ∼97% sequence identity. Previous analysis of Tubg1 and Tubg2 knock-out mice has suggested that γ-tubulins are not functionally equivalent. Tubg1 knock-out mice died at the blastocyst stage, whereas Tubg2 knock-out mice developed normally and were fertile. It was proposed that γ-tubulin 1 represents ubiquitous γ-tubulin, while γ-tubulin 2 may have some specific functions and cannot substitute for γ-tubulin 1 deficiency in blastocysts. The molecular basis of the suggested functional difference between γ-tubulins remains unknown. Here we show that exogenous γ-tubulin 2 is targeted to centrosomes and interacts with γ-tubulin complex proteins 2 and 4. Depletion of γ-tubulin 1 by RNAi in U2OS cells causes impaired microtubule nucleation and metaphase arrest. Wild-type phenotype in γ-tubulin 1-depleted cells is restored by expression of exogenous mouse or human γ-tubulin 2. Further, we show at both mRNA and protein levels using RT-qPCR and 2D-PAGE, respectively, that in contrast to Tubg1, the Tubg2 expression is dramatically reduced in mouse blastocysts. This indicates that γ-tubulin 2 cannot rescue γ-tubulin 1 deficiency in knock-out blastocysts, owing to its very low amount. The combined data suggest that γ-tubulin 2 is able to nucleate microtubules and substitute for γ-tubulin 1. We propose that mammalian γ-tubulins are functionally redundant with respect to the nucleation activity.

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