title: γ-Tubulin 2 nucleates microtubules and is downregulated in mouse early embryogenesis.
abstract: 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.
authors.affiliation: Department of Biology of Cytoskeleton, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Stanislav
authors.initials: S
authors.lastname: Vinopal
authors.forename: Markéta
authors.initials: M
authors.lastname: Cernohorská
authors.forename: Vadym
authors.initials: V
authors.lastname: Sulimenko
authors.forename: Tetyana
authors.initials: T
authors.lastname: Sulimenko
authors.forename: Věra
authors.initials: V
authors.lastname: Vosecká
authors.forename: Matyáš
authors.initials: M
authors.lastname: Flemr
authors.forename: Eduarda
authors.initials: E
authors.lastname: Dráberová
authors.forename: Pavel
authors.initials: P
authors.lastname: Dráber
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D015536:Down-Regulation; D010064:Embryo Implantation; D047108:Embryonic Development / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000235:genetics; D020033:Protein Isoforms / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D013997:Time Factors; D014404:Tubulin / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism*
chemical_list: D020033:Protein Isoforms; D014404:Tubulin