Localization of gamma-tubulin in interphase and mitotic cells of unicellular eukaryote, Giardia intestinalis

Giardia intestinalis, a bi-nucleated amitochondrial flagellate, possesses a complex cytoskeleton based on several microtubular systems (flagella, adhe sive disk, median body, funis, mitotic spindles). MTOCs of the individual s ystems have not been fully defined, By using monoclonal antibodies against a conserved synthetic peptide from the C-terminus of human gamma-tubulin we investigated occurrence and distribution of gamma-tubulin in interphase an d mitotic Giardia cells. On the immunoblots of Giardia cytoskeletal extract s the antibodies bound to a single polypeptide of approximately 50 kDa, Imm unostaining of the interphase cell demonstrated gamma-tubulin as four brigh t spots at the basis of four out of eight flagella, gamma-Tubulin label was associated with perikinetosomal areas of the ventral and posterolateral pa irs of flagella which are formed de novo during cell division. Basal body r egions of the anterolateral and caudal pairs of flagella which persist duri ng the division and are integrated into the flagellar systems of the daught er cells did not show gamma-tubulin staining, At early mitosis, gamma-tubul in spots disappeared reappearing again at late mitosis in accord with reori entation of parent flagella and reorganization of flagellar apparatus durin g cell division. The antibody-detectable gamma-tubulin epitope was absent a t the poles of both mitotic spindles. Albendazole-treated Giardia, in which spindle assembly was completely inhibited, showed the same gamma-tubulin s taining pattern thus confirming that the fluorescent label is exclusively l ocated in the basal body regions. Our results point to a role of gamma-tubu lin in nucleation of microtubules of newly formed flagella and indicate unu sual mitotic spindle assembly. Moreover, the demonstration of gamma-tubulin in Giardia shows ubiquity of this protein through the evolutionary history of eukaryotes.