title: 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.
abstract: 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 temperatures.
authors.affiliation: Dipartimento di Biologia Molecolare, Cellulare e Animale, University of Camerino, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Marziale
authors.forename: Sandra
authors.initials: S
authors.lastname: Pucciarelli
authors.forename: Patrizia
authors.initials: P
authors.lastname: Ballarini
authors.forename: Ronald
authors.initials: R
authors.lastname: Melki
authors.forename: Alper
authors.initials: A
authors.lastname: Uzun
authors.forename: Valentin A
authors.initials: VA
authors.lastname: Ilyin
authors.forename: H W
authors.initials: HW
authors.lastname: Detrich
authors.forename: Cristina
authors.initials: C
authors.lastname: Miceli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016798:Ciliophora; D003080:Cold Temperature; D003599:Cytoskeleton / Q000737:chemistry*; D016812:Euplotes / Q000737:chemistry* / Q000648:ultrastructure; D008870:Microtubules / Q000378:metabolism; D011487:Protein Conformation; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
chemical_list: D014404:Tubulin