Different roles of two [gamma]-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii Remodelling of interaction surfaces may enhance microtubule nucleation at low temperature

• Published in: The FEBS journal Vol. 275; no. 21; p. 5367
• Main Authors: Marziale, Francesca, Pucciarelli, Sandra, Ballarini, Patrizia, Melki, Ronald, Uzun, Alper, Ilyin, Valentin A, Detrich III, H W, Miceli, Cristina
• Format: Journal Article
• Language: English
• Published: Oxford Blackwell Publishing Ltd 01.11.2008
• Subjects: Amino acids; Biochemistry; Cellular biology; Comparative studies; Genetics
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2008.06666.x

[gamma]-Tubulin belongs to the tubulin superfamily and plays an essential role in the nucleation of cellular microtubules. In the present study, we report the characterization of [gamma]-tubulin from the psychrophilic Antarctic ciliate Euplotes focardii. In this organism, [gamma]-tubulin is encoded by two genes, [gamma]-T1 and [gamma]-T2, that produce distinct isotypes. Comparison of the [gamma]-T1 and [gamma]-T2 primary sequences to a Euplotes [gamma]-tubulin consensus, derived from mesophilic (i.e. temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in [gamma]-T2. Structural models of [gamma]-T1 and [gamma]-T2, obtained using the 3D structure of human [gamma]-tubulin as a template, suggest that these substitutions are responsible for conformational and/or polarity differences located: (a) in the regions involved in longitudinal 'plus end' contacts; (b) in the T3 loop that participates in binding GTP; and (c) in the M loop that forms lateral interactions. Relative to [gamma]-T1, the [gamma]-T2 gene is amplified by approximately 18-fold in the macronuclear genome and is very strongly transcribed. Using confocal immunofluorescence microscopy, we found that the [gamma]-tubulins of E. focardii associate throughout the cell cycle with basal bodies of the non-motile dorsal cilia and of all of the cirri of the ventral surface (i.e. adoral membranelles, paraoral membrane, and frontoventral transverse, caudal and marginal cirri). By contrast, only [gamma]-T2 interacts with the centrosomes of the spindle during micronuclear mitosis. We also established that the [gamma]-T1 isotype associates only with basal bodies. Our results suggest that [gamma]-T1 and [gamma]-T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that [gamma]-T1 and [gamma]-T2 have evolved sequence-based structural alterations that facilitate template nucleation of microtubules by the [gamma]-tubulin ring complex at cold temperatures. [PUBLICATION ABSTRACT]