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: FEBS Journal
• Main Authors: Marziale, Francesca, Pucciarelli, Sandra, Ballarini, Patrizia, Melki, Ronald, Uzun, Alper, Ilyin, Valentin A, Detrich, H W, Miceli, Cristina
• Other Authors: School of biosciences and biotechnology, University of Camerino, Università degli Studi di Camerino = University of Camerino (UNICAM), Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Department of Biology, Northeastern University, Northeastern University Boston
• Format: Article in Journal/Newspaper
• Language: English
• Published: HAL CCSD 2008
• Subjects: MESH: Amino Acid Sequence; MESH: Animals; MESH: Ciliophora; MESH: Cold Temperature; MESH: Cytoskeleton; MESH: Euplotes; MESH: Microtubules; MESH: Protein Conformation; MESH: Tubulin; [SDV]Life Sciences [q-bio]; Antarc*; Antarctic
• Online Access: https://hal.science/hal-01183800; https://doi.org/10.1111/j.1742-4658.2008.06666.x

International audience 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 Euplotesgamma-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 ...