gamma-Tubulin in Leishmania: cell cycle-dependent changes in subcellular localization and heterogeneity of its isoforms

A panel of six anti-peptide antibodies recognizing epitopes in different regions of the gamma-tubulin molecule was used for the characterization and localization of gamma-tubulin during cell cycle in Leishmania promastigotes. Immunofluorescence microscopy revealed the presence of gamma-tubulin in the basal bodies, posterior pole of the cell, and in the flagellum. Furthermore, the antibodies showed punctuate staining in the subpellicular microtubule. This complex localization pattern was observed in both interphase and dividing cells, where staining of posterior poles and the subpellicular corset was more prominent. In posterior poles, gamma-tubulin co-distributed with the 210-kDa microtubule-interacting protein and the 57-kDa protein immunodetected with anti-vimentin antibody. Immunogold electron microscopy on thin sections of isolated flagella showed that gamma-tubulin was associated with the paraflagellar rod (PFR) that runs adjacent to the axonemal microtubules. Under different extraction conditions, gamma-tubulin in Leishmania was found only in insoluble cytoskeletal fractions, in contrast to tubulin dimers that were both in soluble and cytoskeletal pool. Two-dimensional electrophoresis revealed multiple charge variants of gamma-tubulin. Posttranslational modifications of Leishmania gamma-tubulin might therefore have an important role in the regulation of microtubule nucleation and interaction with other proteins. The complex pattern of gamma-tubulin localization and its properties indicate that gamma-tubulin in Leishmania might have other function(s) besides microtubule nucleation.     

The tubulin-depolymerising agent combretastatin-4 induces ectopic aster assembly and mitotic catastrophe in lung cancer cells H460

The relationship between microtubular dynamics, dismantling of pericentriolar components and induction of apoptosis was analysed after exposure of H460 non-small lung cancer cells to anti-mitotic drugs. The microtubule destabilising agent, combretastatin-A4 (CA-4) led to microtubular array disorganization, arrest in mitosis and abnormal metaphases, accompanied by the presence of numerous centrosome-independent “star-like” structures containing tubulin and aggregates of pericentrosomal matrix components like γ-tubulin, pericentrin and ninein, whereas the structural integrity of centrioles was not affected by treatment. On the contrary, in condition of prolonged exposure or high concentrations of CA-4 such aggregates never formed. Treatment with 7.5 nM CA-4, which produced a high frequency “star-like” aggregates, was accompanied by mitotic catastrophe commitment characterized by translocation of the proapoptotic Bim protein to mitochondria activation of caspases-3/9 and DNA fragmentation as a result of either prolonged metaphase arrest or attempt of cells to divide. Drug concentrations which fail to block cells at mitosis were also unable to activate apotosis. A detailed time-course analysis of cell cycle arrest and apoptosis indicated that after CA-4 washout the number of metaphases with “star-like” structures decreased as a function of time and arrested cells proceeded in anaphase. After 4 h, the multiple α- and γ-tubulin aggregates coalesced into two well-defined spindles in a bipolar mitotic spindle organization. Overall, our findings suggest that the maintenance of microtubular integrity plays a relevant role in stabilising the pericentriolar matrix, whose dismantling can be associated with apoptosis after exposure to microtubule depolymerising agents.     

Complexes of gamma-tubulin with nonreceptor protein tyrosine kinases Src and Fyn in differentiating P19 embryonal carcinoma cells

Nonreceptor protein tyrosine kinases of the Src family have been shown to play an important role in signal transduction as well as in regulation of microtubule protein interactions. Here we show that gamma-tubulin (gamma-Tb) in P19 embryonal carcinoma cells undergoing neuronal differentiation is phosphorylated and forms complexes with protein tyrosine kinases of the Src family, Src and Fyn. Elevated expression of both kinases during differentiation corresponded with increased level of proteins phosphorylated on tyrosine. Immunoprecipitation experiments with antibodies against Src, Fyn, gamma-tubulin, and with anti-phosphotyrosine antibody revealed that gamma-tubulin appeared in complexes with these kinases. In vitro kinase assays showed tyrosine phosphorylation of proteins in gamma-tubulin complexes isolated from differentiated cells. Pretreatment of cells with Src family selective tyrosine kinase inhibitor PP2 reduced the amount of phosphorylated gamma-tubulin in the complexes. Binding experiments with recombinant SH2 and SH3 domains of Src and Fyn kinases revealed that protein complexes containing gamma-tubulin bound to SH2 domains and that these interactions were of SH2-phosphotyrosine type. The combined data suggest that Src family kinases might have an important role in the regulation of gamma-tubulin interaction with tubulin dimers or other proteins during neurogenesis.     

Expression of gamma-tubulin during the development of nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Gamma-tubulin is a centrosomal protein found in microtubule organizing centres (MTOCs) in cells from many different organisms, and has several properties, which makes it a candidate for both the initiation of microtubule assembly and anchorage. Gamma-tubulin is encoded by a single gene tbg-1 in Caenorhabditis elegans. In this paper tbg-1 was studied to understand the essential role of gamma-tubulin in C. elegans. Essential role of tbg-1 expression was confirmed by the disruption of the gene expression by gamma-tubulin anti-sense RNA production in vivo under the heat shock promoter that caused lethality in the nematodes. Expression of tbg-1 deduced from Northern blot analysis during the development revealed differential expression in different developmental stages. Using tbg-1::lacZ fusion gene expression studies in the germ line transformed worms, it was further revealed that gamma-tubulin expression was observed through out the development from embryonic and post-embryonic stages.     

Polar cytoplasmic evaginations in dividing spermatocytes of the firebug,Pyrrhocoris apterus (Pyrrhocoridae,Hemiptera)

Summary The present fine structure and anti-tubulin immunofluorescence study deals with evaginations from the cell surface in metaphase I spermatocytes of the firebugPyrrhocoris apterus (Pyrrhocoridae, Hemiptera). The surface of spermatogonia and prophase spermatocytes was smooth throughout. Only in metaphase I and anaphase I, cytoplasmic threads projected from polar portions of the spermatocytes. In contrast, equatorial portions of these cells possessed a smooth surface. By mid to late telophase, the evaginations were no longer detectable in spermatocytes. Three ideas are at hand to explain the development of polar cytoplasmic evaginations. First, they could represent a membrane reserve used up during spindle elongation in telophase of meiosis. In order to test this idea, spindle structure was analyzed in meiosis I using simultaneously antibodies to -tubulin and -tubulin. -Tubulin represents a tubulin isoform prevalent in centrosomes. The observations showed that spindle elongation was not very prominent in meiosis of the bug. Although it cannot be ruled out, the formation of a polar membrane reserve prior to spindle elongation is not a likely explanation for the evaginations from the cell surface. Second, the development of surface extensions could bring about increased exchange of metabolites during a particularly active stage of meiosis. Third, the polar evaginations could be an inadvertent product of the aster microtubules protruding towards the plasma membrane.Abbreviations BSA bovine serum albumin - DAPI 4,6-diamidino-2-phenylindole.2 HCl - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N-tetraacetic acid - FITC fluorescein-isothiocyanate - PBS phosphate-buffered saline - PIPES piperazine-N,N bis (2-ethane sulfonic acid) - MT microtubule - MTOC microtubule-organizing centre