Journal
JOURNAL OF CELLULAR PHYSIOLOGY
Volume 227, Issue 1, Pages 367-382Publisher
WILEY
DOI: 10.1002/jcp.22772
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Funding
- Grant Agency of the Czech Republic [204/09/1777, 204/09/H084, P302/10/1701]
- Ministry of Education, Youth and Sports of the Czech Republic [LC545, 1M6837805001]
- Grant Agency of the Czech Academy of Sciences [KAN200520701]
- Institutional Research Support [AVOZ 50520514]
- Grant Agency of Charles University [11109]
- St Christopher's Foundation for Children (Philadelphia) [203]
- Department of Cell Biology, Faculty of Science, Charles University, Prague, Czech Republic
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gamma-Tubulin is assumed to be a typical cytosolic protein necessary for nucleation of microtubules from microtubule organizing centers. Using immunolocalization and cell fractionation techniques in combination with siRNAi and expression of FLAG-tagged constructs, we have obtained evidence that gamma-tubulin is also present in nucleoli of mammalian interphase cells of diverse cellular origins. Immunoelectron microscopy has revealed gamma-tubulin localization outside fibrillar centers where transcription of ribosomal DNA takes place. gamma-Tubulin was associated with nucleolar remnants after nuclear envelope breakdown and could be translocated to nucleoli during mitosis. Pretreatment of cells with leptomycin B did not affect the distribution of nuclear gamma-tubulin, making it unlikely that rapid active transport via nuclear pores participates in the transport of gamma-tubulin into the nucleus. This finding was confirmed by heterokaryon assay and time-lapse imaging of photoconvertible protein Dendra2 tagged to g-tubulin. Immunoprecipitation from nuclear extracts combined with mass spectrometry revealed an association of gamma-tubulin with tumor suppressor protein C53 located at multiple subcellular compartments including nucleoli. The notion of an interaction between gamma-tubulin and C53 was corroborated by pull-down and co-immunoprecipitation experiments. Overexpression of gamma-tubulin antagonized the inhibitory effect of C53 on DNA damage G(2)/M checkpoint activation. The combined results indicate that aside from its known role in microtubule nucleation, gamma-tubulin may also have nuclear-specific function(s). J. Cell. Physiol. 227: 367-382, 2012. (C) 2011 Wiley Periodicals, Inc.
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