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.

Department of Biology of Cytoskeleton Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

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PLoS One. 2012;7(6): doi/10.1371/annotation/5dd084b1-20e6-4e1f-88e0-dfe05289da08 PubMed

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Oakley BR, Oakley CE, Yoon Y, Jung M. γ-Tubulin is a component of the spindle pole body that is essential for microtubule function in Aspergillus nidulans. Cell. 1990;61:1289–1301. PubMed

Stearns T, Evans L, Kirschner M. γ-Tubulin is highly conserved component of the centrosome. Cell. 1991;65:825–836. PubMed

Joshi HC, Palacios MJ, McNamara L, Cleveland DW. γ-Tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation. Nature. 1992;356:80–83. PubMed

Wiese C, Zheng Y. Microtubule nucleation: gamma-tubulin and beyond. J Cell Sci. 2006;119:4143–4153. PubMed

Raynaud-Messina B, Merdes A. γ-Tubulin complexes and microtubule organization. Curr Opin Cell Biol. 2007;19:24–30. PubMed

Moritz M, Braunfeld MB, Guenebaut V, Heuser J, Agard DA. Structure of the γ-tubulin ring complex: a template for microtubule nucleation. Nat Cell Biol. 2000;2:365–370. PubMed

Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA. Microtubule nucleating γ-TuSC assembles structures with 13-fold microtubule-like symmetry. Nature. 2010;466:879–882. PubMed PMC

Lüders J, Stearns T. Microtubule-organizing centres: a re-evaluation. Nat Rev Mol Cell Biol. 2007;8:161–167. PubMed

Hořejší B, Vinopal S, Sládková V, Dráberová E, Sulimenko V, et al. Nuclear γ-tubulin associates with nucleoli and interacts with tumor suppressor protein C53. J Cell Physiol. 2011;227:367–382. PubMed

Moudjou M, Bordes N, Paintrand M, Bornens M. γ-Tubulin in mammalian cells: the centrosomal and the cytosolic forms. J Cell Sci. 1996;109:875–887. PubMed

Haren L, Remy MH, Bazin I, Callebaut I, Wright M, et al. NEDD1-dependent recruitment of the γ-tubulin ring complex to the centrosome is necessary for centriole duplication and spindle assembly. J Cell Biol. 2006;172:505–515. PubMed PMC

Dammermann A, Maddox PS, Desai A, Oegema K. SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the gamma-tubulin-mediated addition of centriolar microtubules. J Cell Biol. 2008;180:771–785. PubMed PMC

Zimmerman S, Chang F. Effects of γ-tubulin complex proteins on microtubule nucleation and catastrophe in fission yeast. Mol Biol Cell. 2005;16:2719–2733. PubMed PMC

Cuschieri L, Miller R, Vogel J. Gamma-tubulin is required for proper recruitment and assembly of Kar9-Bim1 complexes in budding yeast. Mol Biol Cell. 2006;17:4420–4434. PubMed PMC

Bouissou A, Verollet C, Sousa A, Sampaio P, Wright M, et al. γ-Tubulin ring complexes regulate microtubule plus end dynamics. J Cell Biol. 2009;187:327–334. PubMed PMC

Nayak T, Edgerton-Morgan H, Horio T, Xiong Y, De Souza CP, et al. Gamma-tubulin regulates the anaphase-promoting complex/cyclosome during interphase. J Cell Biol. 2010;190:317–330. PubMed PMC

Rodriguez AS, Batac J, Killilea AN, Filopei J, Simeonov DR, et al. Protein complexes at the microtubule organizing center regulate bipolar spindle assembly. Cell Cycle. 2008;7:1246–1253. PubMed

Liu B, Joshi HC, Wilson TJ, Silflow CD, Palevitz BA, et al. γ-Tubulin in Arabidopsis: gene sequence, immunoblot, and immunofluorescence studies. Plant Cell. 1994;6:303–314. PubMed PMC

Ruiz F, Beisson J, Rossier J, Dupuis-Williams P. Basal body duplication in Paramecium requires gamma-tubulin. Current Biology. 1999;9:43–46. PubMed

Tan M, Heckmann K. The two gamma-tubulin-encoding genes of the ciliate Euplotes crassus differ in their sequences, codon usage, transcription initiation sites and poly(A) addition sites. Gene. 1998;210:53–60. PubMed

Wilson PG, Zheng Y, Oakley CE, Oakley BR, Borisy GG, et al. Differential expression of two γ-tubulin isoforms during gametogenesis and development in Drosophila. Dev Biol. 1997;184:207–221. PubMed

Wise DO, Krahe R, Oakley BR. The γ-tubulin gene family in humans. Genomics. 2000;67:164–170. PubMed

Yuba-Kubo A, Kubo A, Hata M, Tsukita S. Gene knockout analysis of two γ-tubulin isoforms in mice. Develop Biol. 2005;282:361–373. PubMed

Carson AR, Scherer SW. Identifying concerted evolution and gene conversion in mammalian gene pairs lasting over 100 million years. BMC Evol Biol. 2009;9:156. PubMed PMC

Khodjakov A, Rieder CL. The sudden recruitment of γ-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules. J Cell Biol. 1999;146:585–596. PubMed PMC

Piehl M, Tulu US, Wadsworth P, Cassimeris L. Centrosome maturation: Measurement of microtubule nucleation throughout the cell cycle by using GFP-tagged EB1. Proc Natl Acad Sci USA. 2004;101:1584–1588. PubMed PMC

Piko L, Clegg KB. Quantitative changes in total RNA, total poly(A), and ribosomes in early mouse embryos. Dev Biol. 1982;89:362–378. PubMed

Schultz RM, Wassarman PM. Biochemical studies of mammalian oogenesis: Protein synthesis during oocyte growth and meiotic maturation in the mouse. J Cell Sci. 1977;24:167–194. PubMed

Sellens MH, Stein S, Sherman MI. Protein and free amino acid content in preimplantation mouse embryos and in blastocysts under various culture conditions. J Reprod Fertil. 1981;61:307–315. PubMed

Guillet V, Knibiehler M, Gregory-Pauron L, Remy MH, Chemin C, et al. Crystal structure of gamma-tubulin complex protein GCP4 provides insight into microtubule nucleation. Nat Struct Mol Biol. 2011;18:915–919. PubMed PMC

Lüders J, Patel UK, Stearns T. GCP-WD is a γ-tubulin targeting factor required for centrosomal and chromatin mediated microtubule nucleation. Nature Cell Biol. 2006;8:137–147. PubMed

Uehara R, Nozawa RS, Tomioka A, Petry S, Vale RD, et al. The augmin complex plays a critical role in spindle microtubule generation for mitotic progression and cytokinesis in human cells. Proc Natl Acad Sci U S A. 2009;106:6998–7003. PubMed PMC

Uehara R, Goshima G. Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase. J Cell Biol. 2010;191:259–267. PubMed PMC

Hutchins JR, Toyoda Y, Hegemann B, Poser I, Heriche JK, et al. Systematic analysis of human protein complexes identifies chromosome segregation proteins. Science. 2010;328:593–599. PubMed PMC

Hendrickson TW, Yao J, Bhadury S, Corbett AH, Joshi HC. Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis. Mol Biol Cell. 2001;12:2469–2481. PubMed PMC

Lynch M, Force A. The probability of duplicate gene preservation by subfunctionalization. Genetics. 2000;154:459–473. PubMed PMC

Lam EW, Glassford J, Banerji L, Thomas NS, Sicinski P, et al. Cyclin D3 compensates for loss of cyclin D2 in mouse B-lymphocytes activated via the antigen receptor and CD40. J Biol Chem. 2000;275:3479–3484. PubMed

Kafri R, Levy M, Pilpel Y. The regulatory utilization of genetic redundancy through responsive backup circuits. Proc Natl Acad Sci U S A. 2006;103:11653–11658. PubMed PMC

DeLuna A, Springer M, KIrschner MW, Kishony R. Need-based up-regulation of protein levels in response to deletion of their duplicate genes. PLoS Biol. 2010;8:e1000347. PubMed PMC

Katsetos CD, Reddy G, Dráberová E, Šmejkalová B, Del Valle L, et al. Altered cellular distribution and subcellular sorting of γ-tubulin in diffuse astrocytic gliomas and human glioblastoma cell lines. J Neuropathol Exp Neurol. 2006;65:465–477. PubMed

Loh JK, Lieu AS, Chou CH, Lin FY, Wu CH, et al. Differential expression of centrosomal proteins at different stages of human glioma. Bmc Cancer. 2010;10:268. PubMed PMC

Hájková Z, Bugajev V, Dráberová E, Vinopal S, Dráberová L, et al. STIM1-directed reorganization of microtubules in activated cells. J Immunol. 2011;186:913–923. PubMed

Mimori-Kiyosue Y, Shiina N, Tsukita S. The dynamic behavior of the APC-binding protein EB1 on the distal ends of microtubules. Curr Biol. 2004;10:865–868. PubMed

Nováková M, Dráberová E, Schürmann W, Czihak G, Viklický V, et al. γ-Tubulin redistribution in taxol-treated mitotic cells probed by monoclonal antibodies. Cell Motil Cytoskel. 1996;33:38–51. PubMed

Dráberová E, Sulimenko V, Kukharskyy V, Dráber P. Monoclonal antibody NF-09 specific for neurofilament protein NF-M. Folia Biol (Prague) 1999;45:163–165. PubMed

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–685. PubMed

Sulimenko V, Sulimenko T, Poznanovic S, Nechiporuk-Zloy V, Böhm JK, et al. Association of brain γ-tubulins with αβ-tubulin dimers. Biochem J. 2002;365:889–895. PubMed PMC

Dráber P. Quantitation of proteins in sample buffer for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using colloidal silver. Electrophoresis. 1991;12:453–456. PubMed

Kukharskyy V, Sulimenko V, Macůrek L, Sulimenko T, Dráberová E, et al. Complexes of γ-tubulin with non-receptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells. Exp Cell Res. 2004;298:218–228. PubMed

Dráberová E, Dráber P. A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules. J Cell Sci. 1993;106:1263–1273. PubMed

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