Different roles of two γ‐tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii

• Published in: FEBS Journal
• Main Authors: Marziale, Francesca, Pucciarelli, Sandra, Ballarini, Patrizia, Melki, Ronald, Uzun, Alper, Ilyin, Valentin A., Detrich III, H. W., Miceli, Cristina
• Format: Article in Journal/Newspaper
• Language: English
• Published: Wiley 2008
• Subjects: Antarc*; Antarctic
• Online Access: http://dx.doi.org/10.1111/j.1742-4658.2008.06666.x; https://api.wiley.com/onlinelibrary/tdm/v1/articles/10.1111%2Fj.1742-4658.2008.06666.x; https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2008.06666.x

γ‐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 γ‐tubulin from the psychrophilic Antarctic ciliate Euplotes focardii. In this organism, γ‐tubulin is encoded by two genes, γ‐T1 and γ‐T2, that produce distinct isotypes. Comparison of the γ‐T1 and γ‐T2 primary sequences to a Euplotes γ‐tubulin consensus, derived from mesophilic (i.e. temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in γ‐T2. Structural models of γ‐T1 and γ‐T2, obtained using the 3D structure of human γ‐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 γ‐T1, the γ‐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 γ‐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 γ‐T2 interacts with the centrosomes of the spindle during micronuclear mitosis. We also established that the γ‐T1 isotype associates only with basal bodies. Our results suggest that γ‐T1 and γ‐T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that γ‐T1 and γ‐T2 have evolved sequence‐based structural alterations that facilitate template nucleation of microtubules by the γ‐tubulin ring complex at cold temperatures.