Different roles of two c-tubulin isotypes in the cytoskeleton of the Antarctic ciliate Euplotes focardii Remodelling of interaction surfaces may enhance microtubule nucleation at low temperature Francesca Marziale1, Sandra Pucciarelli1, Patrizia Ballarini1, Ronald Melki2, Alper Uzun3, Valentin A Ilyin3, H W Detrich III3 and Cristina Miceli1 Dipartimento di Biologia Molecolare, Cellulare e Animale, University of Camerino, Italy Laboratoire d’Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France Department of Biology, Northeastern University, Boston, MA, USA Keywords microtubule nucleation; molecular coldadaptation; psychrophilic microorganism; quantitative PCR; tubulin genes Correspondence C Miceli, Dipartimento di Biologia Molecolare, Cellulare e Animale, University of Camerino, Via Gentile III da Varano, 62032 Camerino (MC), Italy Fax: +39 0737 40 32 90 Tel: +39 0737 40 32 55 E-mail: cristina.miceli@unicam.it (Received 22 July 2008, revised 27 August 2008, accepted September 2008) doi:10.1111/j.1742-4658.2008.06666.x c-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 c-tubulin from the psychrophilic Antarctic ciliate Euplotes focardii In this organism, c-tubulin is encoded by two genes, c-T1 and c-T2, that produce distinct isotypes Comparison of the c-T1 and c-T2 primary sequences to a Euplotes c-tubulin consensus, derived from mesophilic (i.e temperate) congeneric species, revealed the presence of numerous unique amino acid substitutions, particularly in c-T2 Structural models of c-T1 and c-T2, obtained using the 3D structure of human c-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 c-T1, the c-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 c-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 c-T2 interacts with the centrosomes of the spindle during micronuclear mitosis We also established that the c-T1 isotype associates only with basal bodies Our results suggest that c-T1 and c-T2 perform different functions in the organization of the microtubule cytoskeleton of this protist and are consistent with the hypothesis that c-T1 and c-T2 have evolved sequencebased structural alterations that facilitate template nucleation of microtubules by the c-tubulin ring complex at cold temperatures Microtubule assembly in metazoan cells is nucleated by organizing centers, which include centrioles, basal bodies, and other structures Mitotic centrosomes contain a pair of centrioles and associated pericentriolar material, whereas basal bodies recruit other accessory structures [1,2] Both centrioles and basal bodies require c-tubulin, the ubiquitous third member of the ‘tubulin superfamily’ [3–5], for their assembly and maintenance [6–8], and for their capacity to nucleate microtubules [9] This tubulin variant associates with Abbreviations qPCR, quantitative PCR; RATE, rapid amplification of telomeric ends; TuRC, tubulin ring complex FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5367 c-Tubulin isotypes in E focardii F Marziale et al other proteins to form two macromolecular structures, the c-tubulin small complex, which possesses a weak microtubule nucleating activity [10,11], and the c-tubulin ring complex (TuRC) [12], which nucleates strongly c-TuRC resembles a lock washer and is considered to be the fundamental unit required for microtubule nucleation Two models have been proposed to explain microtubule nucleation by c-TuRC: (a) the ‘protofilament’ model, in which the c-tubulin subunits of c-TuRC associate longitudinally with ab-tubulin dimers [13], and (b) the ‘template’ model, in which the c-TuRC ring mimics the end of a microtubule, and c-tubulin interacts both longitudinally and laterally with a-tubulin but only laterally with b-tubulin [14,15] Microtubule assembly is entropically driven, predominantly via hydrophobic interactions, and therefore is sensitive to environmental temperature both in vitro and in vivo [16,17] The ab-tubulin dimers of mammals, for example, form microtubules in vitro at temperatures near 37 °C, and these polymers dissociate at low temperature (4 °C) to yield tubulin dimers and ring-shaped oligomers [18–20] Ectothermic (coldblooded) Antarctic fishes, by contrast, possess tubulins that polymerize at temperatures as low as )1.8 °C, which is the freezing point of their chronically cold marine habitat [16,17] Detrich and colleagues have shown that thermal compensation of microtubule assembly and dynamics in these fishes results from the evolution of structural changes intrinsic to the a- and b-tubulins [21–24] The nucleation of cytoplasmic microtubules by centrosomes requires productive binding reactions between c-tubulin and the ab-tubulin dimer, but the molecular alterations that conserve nucleation in coldliving organisms have not been studied Data indirectly relevant to temperature compensation of microtubule nucleation were obtained from alanine-scanning mutagenesis of the c-tubulins of Tetrahymena thermophila [25] and Aspergillus nidulans [26] Substitution of alanine at sites in the lateral surfaces (the H3 helix and the M loop) of these c-tubulins causes cold-sensitivity of cell growth and ⁄ or loss of basal bodies [25,26] In light of this evidence, we propose that the capacity of c-tubulin to perform efficient microtubule nucleation at cold temperatures reflects evolved molecular alterations to its interaction surfaces Psychrophilic ciliated protozoa are uniquely suited to an investigation of this issue As single cells, ciliates are directly exposed to environmental factors throughout their life cycle, and modifications of the primary sequences of many of their proteins are likely to reflect adaptive mutations that increase the fitness of the organism at cold temperatures In ciliates, microtubule 5368 nucleation is promoted mainly by basal bodies, which are positioned precisely in organized rows in the somatic cell cortex and in the oral apparatus [8] The assembly and maintenance of basal bodies were both shown to require c-tubulin [7,8] The ciliate Euplotes focardii, which is endemic to Antarctic coastal seawaters, shows strictly psychrophilic phenotypes, including optimal survival and multiplication rates at 4–5 °C [27], the lack of a transcriptional response of the Hsp70 genes to thermal shock [28], and modifications in the primary structures of the a- and b-tubulin [29–31] and of the proteins that form the ribosomal stalk [32] In the present study, we characterized the two c-tubulin isotypes, c-T1 and c-T2, of E focardii, model their 3D structures, and examined their differential expression and cellular localization We suggest that novel amino acid substitutions located at the plus ends, near the GTP-binding sites, and within the M loops of the E focardii c-tubulins, preserve their microtubulenucleating activities at cold temperatures and ⁄ or confer different functions on the two isotypes Results Sequence analysis of E focardii c-tubulin genes Two c-tubulin genes (nanochromosomes), designated c-T1 (1623 bp; GenBank accession number EF189704) and c-T2 (1619 bp; GenBank accession number EF189705), were obtained by our rapid amplification of telomeric ends (RATE)-PCR-based cloning strategy The existence of more than two c-tubulin genes in E focardii was excluded by restriction analysis of macronuclear DNA Figure 1A shows that undigested macronuclear DNA gave a single band of approximately 1.6 kb (lane 1) when hybridized at low stringency to a probe derived from the c-T2 gene Co-digestion of macronuclear DNA by EcoRI and HindIII (lane 2) gave strongly hybridizing fragments of approximately 640, 480, and 300 bp, and weakly hybridizing bands of approximately 750 and 200, consistent with the lengths and restriction maps of the two c-tubulin nanochromosomes (Fig 1B) The restriction maps and relative abundances of the DNA fragments suggest that the c-T2 nanochromosome is amplified to a greater extent than the c-T1 nanochromosome Both isotypes are expressed, as shown by the recovery of distinct c-T1 and c-T2 cDNAs of approximately 1.4 kb Furthermore, northern blot analysis of mRNA extracted from exponentially growing E focardii cells, when hybridized at low stringency to the c-T2 probe, indicated that the two mRNAs were comparable in size (1.4 kb; not shown) FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS F Marziale et al c-Tubulin isotypes in E focardii Comparative structural modelling of E focardii c-tubulins to human c-tubulin The 3D structures of the E focardii c-tubulins were modeled comparatively with respect to human c-tubulin [35] The predicted structures of c-T1 and c-T2 were remarkably similar to that of the human protein (Fig S1) Structural features of E focardii c-tubulin isotypes Plus ends Fig The macronucleus of Euplotes focardii contains two different c-tubulin nanochromosomes (A) Southern blot analysis of the two c-tubulin genes of E focardii using the c-T2 gene as probe Lane 1, undigested DNA; lane 2, EcoRI- and HindIII-digested DNA The sizes (bp) of DNA standards are indicated on the left The sizes of the two c-tubulin nanochromosomes (1600 bp) and their digestion products are indicated on the right (B) Structural features and EcoRI ⁄ HindIII restriction maps of the E focardii c-T1 and c-T2 nanochromosomes Coding, noncoding regions, introns, and telomeres (C4A4 ⁄ G4T4) are indicated in the key The coding sequences of the E focardii c-T1 and c-T2 nanochromosomes were interrupted by two introns located in identical positions (Fig 1B) The first intron included nucleotides 50–96 and the second intron included nucleotides 210–253 in each gene Excluding introns and stop codons, the c-T1 and c-T2 coding regions were each 1383 bp in length and predicted proteins of 461 amino acids The nucleotide sequence identity between c-T1 and c-T2 was 94.6% Two in-frame UGA codons, which are known to code for cysteine in other Euplotes species [33,34], were present at residue positions 109 and 185 in each of the genes The deduced amino acid sequences of the c-T1 and c-T2 isotypes were aligned with respect to a Euplotes c-tubulin consensus sequence and mapped onto the consensus secondary structure of the tubulin monomer [35,36] (Fig 2) c-T1 and c-T2 were 95.4% identical in amino acid sequence The main differences of the two isotypes compared to the Euplotes c-tubulin consensus were found in two regions, 390–403 and 70–95 (Fig 2), both of which are located at the plus end (Fig 3) In the former, c-T2 contained several polar-for-charged substitutions (K394S, R395N, D396N, and K403Q) with respect to the consensus (consensus residue ⁄ residue position ⁄ c-T2 residue) c-T1 displayed substitutions of bulky residues with respect to the consensus sequence (T391I, K394R; consensus ⁄ position ⁄ c-T1), reciprocal changes of polar and charged amino acids (D396N, N400D), and one polar-for-hydrophobic alteration (I401N) Notable amino acid substitutions in the second region (70–95) of c-T1 and c-T2 with respect to the Euplotes consensus included the V of c-T1 and K of c-T2 for G at position 76, A of c-T1 ⁄ c-T2 for the consensus P at position 81, G for S at position 84, F for Y at position 92, and S for A at position 94 Together, these results show that the plus-end surfaces of the two E focardii c-tubulins have diverged considerably from those of mesophilic Euplotes species, with an overall tendency toward greater hydrophobicity By contrast, very few changes were observed in sequences that contribute to the c-tubulin minus end (Figs and 3) Isotypic substitutions E focardii c-T1 and c-T2 differed considerably between themselves at their plus ends Major residue changes included R72G, V76K, R394S, R395N, Y398F, D400T, N401T, and K403Q (c-T1 ⁄ residue position ⁄ c-T2) This suite of residue substitutions may confer unique functions upon each isotype FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5369 c-Tubulin isotypes in E focardii F Marziale et al Fig Sequence comparisons of Euplotes focardii c-T1 and c-T2 with the Euplotes c-tubulin consensus The unique substitutions of E focardii c-T1 and c-T2 are shown as a single-letter code underneath the Euplotes c-tubulin consensus sequence; conserved residues are indicated by dots Predicted secondary structural elements, H for helices and S for strands [37], are represented by white cylinders and black arrows, respectively T1 to T7 indicate loops that are involved in contacts with the bound GTP [37] Residues involved in longitudinal contacts at the ‘plus’ and ‘minus’ ends are indicated by ‘+’ and ‘)’, respectively, whereas those involved in the lateral contacts of the H3 and M-loop are shown by ‘H’ and ‘M’ Regions thought to participate in binding to ab tubulin heterodimers [68] are underlined Nucleotide-binding sites Human c-tubulin binds GTP in a plus-end cleft enclosed by residues G11, Q12, C13, Q16, G101, 5370 N102, S140, A142, G143, G144, T145, V171, P173, N207, F225, I228, and N229 (where the residues shown underlined form main- and ⁄ or side-chain hydrogen bonds with atoms of the nucleotide) [35] FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS F Marziale et al c-Tubulin isotypes in E focardii Fig 3D mapping of the sequence substitutions of Euplotes focardii c-T1 and c-T2 with respect to the Euplotes c-tubulin consensus (A, B) c-T1 viewed from the side and from the plus end, respectively (C, D) c-T2 viewed from the side and from the plus end, respectively Ribbon diagrams of c-T1 and c-T2 were obtained by comparative modelling to human c-tubulin using MODELLER, version 9.1 (http://www salilab.org/modeller/) [64] Residues that distinguish the E focardii c-tubulins from the Euplotes consensus are shown in red and annotated as consensus residue ⁄ position ⁄ c-T1 or c-T2 residue The GTP molecule is shown in green The plus and minus ends, the H3 helix, and the M-loop are indicated These residues are all conserved in the E focardii c-tubulins Near the entrance to the nucleotide pocket within the H2 helix, c-T2 possessed a striking substitution at position 72: glycine in place of the c-T1 ⁄ consensus arginine (Figs 2–4) The presence of glycine at this position in c-T2 has only been observed in E focardii and in the psychrotolerant Euplotes crassus [34, present study] By contrast, substitution of alanine for arginine 72 in the c-tubulins of T thermophila and A nidulans produces a lethal phenotype [25,26], which suggests that this basic residue is important for c-tubulin function at moderate temperature The sequences of the nucleotide-binding T3 and T5 loops of c-T1 and c-T2 were highly conserved across all Euplotes species; the former perfectly, whereas the latter contained a single polarfor-hydrophobic change, I174N (consensus ⁄ position ⁄ c-T1 and c-T2) FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5371 c-Tubulin isotypes in E focardii F Marziale et al Fig Comparison of the tertiary structures of Euplotes focardii c-T1 and c-T2 Ribbon diagrams of the two proteins, obtained by comparative modelling to human c-tubulin using MODELLER, version 9.1 (http://www.salilab.org/modeller/) [64], are superimposed to highlight structural differences The c-T1 and c-T2 loops are shown in yellow and cyan, respectively Residue substitutions that differentiate the two E focardii c-tubulins are colored violet and designated as c-T1 ⁄ residue position ⁄ c-T2 Notable loop displacements are indicated by double arrows The GTP molecule is shown in green The Mg2+ ion (blue sphere) is shown bound to the b- and c-phosphates of GTP (dark green) (A) Side view (A¢, A¢¢) Show close-up side views (generated using Chimera; http://www.cgl.ucsf.edu/chimera/) [69] of the H9-H9¢ loop (dashed box in A), which contains a proline at position 303 in c-T1 (A¢) in contrast to the serine of c-T2 (A¢¢) The proline substitution of c-T1 eliminates the hydrogen bond between Ser303 and Asn205 in c-T2 (B) Plus-end view (B¢) An enlargement of the nucleotide-binding pocket, shown boxed in (B) Near the entrance to the pocket, c-T2 contains glycine (cyan) in place of the arginine (yellow) normally found at position 72 in helix H2 M loops The ‘extended’ M loop, which we define as encompassing the S7-H9 (M) loop, H9, and the H9-S8 loop, and the H3 surfaces of c-tubulin are involved in lateral contacts [35–37] Amino acid substitutions with respect to the Euplotes consensus were found in the extended M loop in E focardii c-T1 and c-T2 (Figs and 3) Two hydrophobic-for-hydrophobic changes occurred near position 280 (F279L, V282I, consensus ⁄ position ⁄ c-T1 and c-T2) and c-T1 possessed an alanine at 280 in place of consensus threonine The changes in the H9-H9¢ loop were more dramatic Both c-T2 and c-T1 contained proline-for-hydroxyl substitutions (T297P and T303P, respectively) 5372 Tertiary structural differences between E focardii c-T1 and c-T2 Figure shows the superimposition of the 3D structures of c-T1 and c-T2 from the side and the plus end, respectively The comparison demonstrates that the differences between c-T1 and c-T2 (c-T1 ⁄ residue position ⁄ c-T2) mapped largely to exposed areas (plus-end loops and helices, extended M loop) of the polypeptides The valine at S3 position 93 of c-T2 appears to confer a conformational change in the adjacent T3 loop (Fig 4B, double arrow), which is directly involved in the formation of the GTP-binding site The alanine at 280 in c-T1 apparently causes a conformational change in the M loop (S7-H9), which may influence lateral interactions (Fig 4A, double arrow) The substitutions FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS F Marziale et al c-Tubulin isotypes in E focardii of prolines for threonine at position 297 of c-T2 and for serine at 303 in c-T1 not alter significantly the conformation of the H9-S8 loop (Fig 4), although they are likely to restrict its mobility However, the Pro303 substitution of c-T1 eliminates the bent hydrogen bond that forms between Ser303 and Asn205 in c-T2 (compare Fig 4A¢,A¢¢) The cluster of substitutions in H11 and the H11-H12 loop of the two c-tubulins cause polarity changes at the plus end (Fig 4) that would differentiate the longitudinal interactions formed by c-T1 and c-T2 Finally, c-T2 possesses a glycine at position 72 in place of consensus ⁄ c-T1 arginine (Fig 4B¢) This substitution may ‘open’ the nucleotide-binding site to facilitate exchange We have not attempted to quantify the loop dis˚ placements because the T3 and M loops of the 3.0 A crystal structure of GTP-bound tubulin are disordered [35] Hence, we consider the modeled loop displacements of the E focardii c-tubulins to be provisional and to require future validation Transcription of the E focardii c-T1 and c-T2 nanochromosomes To gain insight into the roles of the E focardii c-tubulin isotypes, we measured the steady-state levels of macronuclear mRNAs transcribed from the c-T1 and c-T2 nanochromosomes of starvation-synchronized cultures by quantitative PCR (qPCR) During starvation, the transcript levels for both isotypes were low (Fig 5A) After feeding, the amounts of c-T1 and c-T2 mRNAs increased, with the latter being two- to three-fold higher than the former at 18 h At 36 h post-feeding, c-T2 mRNA increased 16-fold relative to its abundance at 18 h (53-fold increase with respect to t = h), whereas the level of the c-T1 transcript remained unchanged Ninety-eight percent of the cells were undergoing mitosis ⁄ cytokinesis at this time (as determined by counting of cells using a stereomicroscope) By 54 h, c-T2 mRNA returned to a value similar to that at 18 h, whereas the amount of the c-T1 transcript was comparable to that observed at 18 and 36 h Thus, the amount of the c-T2 transcript varies widely during the cell cycle, whereas the c-T1 mRNA is expressed at low, almost constant levels The disparity between c-T1 and c-T2 transcript levels could result from differential amplification of the corresponding macronuclear nanochromosomes, as has been reported for other Euplotes genes [38], from different rates of transcription initiation and elongation between the two genes, and ⁄ or from variation in the rates of degradation of the two messages To test the first hypothesis, the gene copy number of c-T1 and c-T2 was estimated by qPCR c-T1 and c-T2 nanochromosomes were present at approximately 175 and 3600 copies per cell, respectively (Fig 5B) Thus, the c-T2 template was approximately 21-fold more abundant than the c-T1 template To evaluate the second hypothesis, the 5¢ and 3¢ noncoding sequences of the two c-tubulin genes were compared Figure 5C shows that the 5¢-UTR of c-T1 contained the sequence TGATAC ()26 to )21; gray shading), which matches the consensus sequence for GATA-binding transcription factors (WGATAR), whereas the c-T2 5¢-UTR possessed two tandem repeats ()32 to )27, )24 to )19; gray shading) of the same motif in essentially the same A Fig Macronuclear amplification and transcription of Euplotes focardii c-T1 and c-T2 genes (A) Cell-cycle-dependence of steadystate c-T1 and c-T2 mRNA levels determined by qPCR Values are the mean ± SD (n = 4) (B) Determination of macronuclear gene copy-number of c-T1 and c-T2 by qPCR Values are the mean ± SD (n = 4) (C) Sequences of the 5¢- and 3¢ noncoding regions of c-T1 and c-T2 putative GATA transcription factor-binding motifs are shown in gray B C FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5373 c-Tubulin isotypes in E focardii F Marziale et al location GATA-binding transcription factors are known to regulate the transcription of some genes in protists [39] The 3¢-UTR of c-T2 was three nucleotides shorter than that of c-T1 but, otherwise, these two sequences were quite similar We have not yet investigated the role of message degradation with respect to the control of c-tubulin transcript abundance With the latter caveat, we propose that the quantities of c-T1 and c-T2 mRNAs are regulated, at least in part, by differential gene amplification and by the number of GATA-factor promoter motifs Distribution of c-tubulins in E focardii cells To examine the cellular distribution of c-tubulin, we used polyclonal anti-(human c-tubulin) serum [40] and a polyclonal antibody that we prepared against the most divergent peptide [(390)RIFRRRNAYIDNYK (403)] of E focardii c-T1 Figure presents confocal microscopic images of three E focardii cells after staining with antibodies directed against a- and c-tubulins (cell 1, Fig 6A–H; cell 2, Fig 6I–L; cell 3, Fig 6M–P) The anti-(human c-tubulin) serum stained all classes of basal bodies (Fig 6B,F, red), including those of: (a) the adoral membranelles that nucleate the microtubules of the cytostomal ciliature (labeled green by DM1A in Fig 6A,C); (b) the paraoral membrane that surrounds the cytostomal area; (c) the four groups of locomotory cirri [frontoventral (numbered 1–10), transverse, caudal, and marginal]; and (d) the nonmotile cilia of the dorsal surface [41], which are arranged in longitudinal rows (kineties) Interestingly, dorsal ciliary microtubules were absent in the equatorial area (Fig 6E,G), which suggests that this cell is entering mitosis and that duplication of basal bodies at the dorsal surface requires the disassembly of dorsal cilia In mitotic E focardii cells (Fig 6I–P), the anti(human c-tubulin) serum stained newly-formed basal bodies (Fig 6J,L, red, arrows) and the poles of the micronuclear mitotic spindle (Fig 6J, solid arrow- head), but macronuclear staining was never observed The basal bodies indicated by the upper arrow in Fig 6J will form the transverse, caudal and marginal cirri of the anterior daughter cell, which also inherits the frontoventral cirri of the parental cell Conversely, the basal bodies marked by the lower arrow will produce the frontoventral cirri of the posterior daughter cell [42,43] and its transverse, caudal, and marginal cirri derive from the parent As division proceeds, the duplicated basal bodies nucleate new ciliary microtubules of the nascent cirri, as shown by the DMIA staining in Fig 6I To determine the subcellular localization of c-T1 and c-T2, we attempted to prepare rabbit polyclonal antibodies specific for the two peptides that clearly distinguish c-T1 [(390)RIFRRRNAYIDNYK(403)] and c-T2 [(390)KKLRSNNAFITTYQ(403)] We obtained an antibody specific for c-T1 The c-T2 peptide was, however, not immunogenic Fig 6M–O shows that the anti-c-T1 serum gave staining identical to that observed with anti-(human c-tubulin), with the exception that the micronuclear spindle poles were not recognized Therefore, we conclude that c-T2, but not cT1, participates in the assembly of the mitotic spindle of E focardii and that both isotypes are involved in the nucleation of other microtubule structures Finally, we examined the distribution of E focardii c-tubulins in total cell extracts and in subfractions enriched in basal bodies or in micronuclei using anti(human c-tubulin) and anti-c-T1 sera Figure shows that the human antibody recognized c-tubulins in all three samples, whereas the c-T1 antibody gave positive signals only for the total cell extracts and basal bodies These results confirm that c-T2 alone nucleates microtubules in the micronucleus Discussion In the present study, we have shown that the psychrophilic ciliate E focardii possesses two c-tubulin genes Fig Spatial distribution of c-tubulins in Euplotes focardii cells Confocal immunofluorescence microscopic images of three E focardii cells were recorded after staining with antibodies directed against a- and c-tubulins Six optical sections, separated by intervals of lm, were collected and merged for each cell ⁄ antigen combination (A–D) Ventral view of cell in late vegetative stage; (E–H) dorsal view of cell 1; and (I–L) ventral view of cell in mitosis The arrowhead in (J) indicates the micronuclear mitotic spindle, and the arrows show the newly-formed basal bodies (M–P) Ventral view of cell in mitosis The arrows in (M) and (O) indicate the micronuclear mitotic spindle (A–C, E–G, I–K) Cells were co-stained with mouse monoclonal anti-a-tubulin serum DM1A (Amersham) and rabbit polyclonal anti-(human c-tubulin) serum The primary antibodies were detected using Alexa Flour 488 goat anti-(mouse IgG) (green signal indicates microtubules) and Alexa Fluor 594 goat anti-(rabbit IgG) (red signal indicates c-tubulin in basal bodies) (M–O) Cell was stained for microtubules with the primary antibody DM1A and for c-tubulins with rabbit polyclonal anti-(E focardii c-T1); secondary antibodies were as before Co-localization of a- and c-tubulins is shown by the yellow signals in merged images (C, G, K, O) (D, H, L, P) Black-and-white versions of the merged images are labeled to identify cytoskeletal structures am, adoral membranelles; pm, paraoral membrane; 1–10, frontoventral cirri involved in locomotion; tc, transverse cirri; cc, caudal cirri; mc, marginal cirri; mb, microtubule bundles that elongate from the basal bodies of each transverse cirrus into the cytoplasm Scale bar = 10 lm 5374 FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS F Marziale et al c-Tubulin isotypes in E focardii B C D E F G H Eq ua t are orial a A Dorsal cirri Kinety I J K L Mitotic spindle Newly formed basal bodies M N O P Mitotic spindle that encode distinct isotypes, c-T1 and c-T2 The amino acid sequences of the two isotypes have diverged from those of mesophilic Euplotes species primarily in two regions that are involved in protein– protein quaternary interactions: (a) the plus end, which forms longitudinal contacts, and (b) the extended FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5375 c-Tubulin isotypes in E focardii A F Marziale et al Recently, we have shown that activation of the heatshock response in the ciliate T thermophila requires GATA motifs, heat-shock transcription elements, and their cognate transcription factors [39] Taken together, our results with protistan genera support the hypothesis that the GATA gene-regulatory system arose early in metazoan evolution B Fig Distribution of c-tubulins in Euplotes focardii nuclei, basal bodies, and total cell extracts (A) Total cell extracts (TCE) and subfractions enriched in basal bodies (BB) and in micronuclei (N) were prepared as described in the Experimental procedures Western blots of the extracts and fractions were incubated with (A) polyclonal antibodies against human c-tubulin and (B) polyclonal antibodies specific for E focardii c-T1 M loop, which participates in lateral bonding The extensive alterations of sequence elements that form these surfaces are likely to be adaptations that preserve c-tubulin function at cold temperatures Moreover, c-T1 and c-T2 differed substantially in their sequences at these locations, consistent with the possibility that the functions of the single c-tubulin found in most organisms may be partitioned between the two protistan isotypes Together, our results suggest strongly that E focardii has evolved c-tubulins that are able to nucleate microtubule structures at low temperature while individually performing specialized subfunctions The E focardii c-tubulin gene family – regulation of expression The two c-tubulin genes of E focardii appear to be a feature characteristic of this protistan genus Two c-tubulin genes have also been reported for Euplotes octocarinatus [44] and for E crassus [34] In the former case, the c-tubulin genes produce identical proteins, whereas, in the latter, they encode two different isotypes whose functional differences, if any, are unknown [34] Transcription of the E focardii c-T2 gene was robust and cell-cycle dependent, whereas synthesis of the c-T1 mRNA occurred at low, almost constant levels The differential transcription of the two c-tubulin genes appears to be due to the greater copy number of the c-T2 nanochromosome in the macronucleus (i.e 20-fold larger than that of c-T1) and to the duplication of a GATA-transcription factor binding site in the c-T2 promoter, although other processes might also be involved In multicellular organisms, GATA-binding factors play critical roles in development, including cell-fate specification, regulation of differentiation, and control of cell proliferation and movement [45] 5376 Sequence changes in relation to the tertiary and quaternary structures of the E focardii c-tubulins – implications for cold adaptation of tubulins Although the c-tubulins of E focardii are strikingly similar in overall 3D organization to human c-tubulin, the former contain divergent sequence elements that are likely to affect the mobility of their domains and their interactions with partner proteins That the plus-end surfaces of c-T1 and c-T2 differ physicochemically from each other and from that of the mesophilic Euplotes consensus is clear The extended M loops of c-T1 and c-T2 are more hydrophobic than the Euplotes consensus and contain proline substitutions whose role may be to constrain the lateral contact residues to a conformation that is favorable for formation of the c-TuRC nucleation complex from multiple c-tubulin small complexes [12] Similarly, the lateral surfaces of a-tubulins from E focardii and from two psychrophilic algae of the genus Chloromonas contain hydrophobic substitutions with respect to the corresponding a-isotypes of temperate congeners [31,46] Detrich et al [27] have shown that a small number of hydrophobic substitutions in Antarctic fish tubulins appear to be important for compensatory adaptation of microtubule assembly at cold body temperatures; one such change, F200Y (Antarctic fish residue ⁄ position ⁄ mesophilic residue), which is located at the interface between the nucleotide-binding and intermediate domains of b-tubulin, clearly affects microtubule dynamics when mutated in Schizosaccharomyces pombe [47] Thus, increased hydrophobicity of tubulins, both at surface interaction sites and at internal domain interfaces, emerges as a common theme for psychrophilic organisms Sequence alterations near the nucleotide-binding pocket of E focardii c-tubulins are also candidates for adaptive compensation Amino acid changes adjacent to the T3 loop and within the T5 loop may influence the binding affinity and hydrolysis of GTP and ⁄ or the egress of GDP and Pi, which may in turn control the assembly and stability of new basal bodies [25] The most striking of these is the G72R substitution of c-T2 near the entrance to the FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS F Marziale et al nucleotide pocket This replacement has only been observed in E focardii and in the psychrotolerant E crassus [34, present study] By contrast, the similar substitution of alanine for arginine 72 in the c-tubulins of T thermophila and A nidulans yields a lethal phenotype [25,26] Together, these results implicate position 72 as being important for thermal adaptation of c-tubulins and motivate a detailed examination of the tertiary interactions that surround the nucleotide-binding site Finally, GTP hydrolysis may be also affected by the unique substitution M250L, which is located adjacent to the motif GxxNxD that has been proposed to act as a ‘synergistic’ loop in nucleotide cleavage [37,48,49] Functions of the E focardii c-tubulin isotypes The results obtained in the present study demonstrate that one or both of the c-tubulins of E focardii associate permanently with basal bodies, consistent with prior observations that c-tubulin is present in these microtubule organizing centers in the ciliates E octocarinatus [50], T thermophila [8], Tetrahymena pyriformis [51], and Paramecium tetraurelia [52] Furthermore, we show that duplication of the basal bodies of the dorsal surface during cell division in E focardii is associated, perhaps causally, with disassembly of the equatorial cilia and their microtubules It is tempting to speculate that the ciliary tubulins are recycled to form the cytoplasmic microtubule bundles [42,43] that guide the positioning of basal bodies in the nascent daughter cells During cell division, the poles of the micronuclear mitotic spindle of E focardii stained with an antibody prepared against human c-tubulin but not with an antibody specific for the c-T1 isotype, which recognized only basal bodies Thus, we conjecture that E focardii c-T2, whose mRNA levels peak in mitosis, is the only isotype required for centrosome function in the closed orthomitosis of the micronucleus We did not detect c-tubulin or microtubules in the macronucleus of E focardii, in contrast to reports that c-tubulin and microtubules are present in the amitotically dividing macronucleus of T thermophila [8,53] This result indicates either that c-tubulin and microtubules are not required for macronuclear division during vegetative growth in E focardii or that the anti-a-tubulin serum and the anti-c-tubulin sera used in the present study are unable to recognize macronuclear a- and c-tubulin isotypes in the E focardii macronucleus, perhaps due to post-translational modifications that block the corresponding tubulin epitopes We regard the second explanation as being unlikely c-Tubulin isotypes in E focardii Cold adaptation of E focardii c-tubulins – implications for the mechanism of microtubule nucleation The suite of amino acid substitutions observed in c-T1 and c-T2 with respect to the Euplotes c-tubulin (generally small polar residues replacing bulky and charged residues) is similar to those that transform mesophilic subtilisin-like proteases into cold-active variants [54] Similar trends in sequence substitutions [55] have also been observed in the cold-adapted variants of lactate dehydrogenase [56], seralysin [57], the pheromone En-1 from Euplotes nobilii [58], the ribosomal proteins P0 and P2 [32], two subunits of the chaperonin containing TCP-1 [59], and a theromlysinlike enzyme [60] The overall effect of this substitution pattern is to introduce greater flexibility in the cold-functioning proteins, particularly in surface loops whose conformational adjustments are required to facilitate attaining the active-site transition state in cold thermal regimes How the unique features of the E focardii c-tubulins enhance microtubule nucleation by the c-TuRC complex at low temperature, and what these changes imply regarding the mechanism of nucleation? Based on the evidence presented in the present study, we propose that the conformational differences and increased hydrophobicity of the M loops of c-T1 and c-T2 promote the lateral interactions necessary to form the c-TuRC ring template at cold temperatures [12,35] and that the residue substitutions and conformational changes at the plus-end facilitate longitudinal interaction with the a-tubulin subunit located at the minus-end of the tubulin heterodimer [12] or lateral interactions with the b-tubulin subunit [61] These hypotheses are readily amenable to testing via site-directed mutagenesis and functional analysis of c-tubulins in several model systems, including the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe Thus, our comparative analysis of the c-tubulins of psychrophilic and mesophilic Euplotes species should contribute to resolving the validity of the template versus protofilament models of c-TuRC-mediated nucleation of microtubules Experimental procedures Cell culture and cell cycle synchronization Cultures of E focardii strains TN1 and TN15 were used [27]; they represent type-species material chosen from a number of wild-type strains isolated from sediment and seawater samples collected in Antarctica (Terra Nova Bay FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5377 c-Tubulin isotypes in E focardii F Marziale et al coastal waters) They were cultivated in a cold room at °C using the green alga Dunaliella tertiolecta as food Cells from logarithmic phase cultures were synchronized by three consecutive treatments of starvation and re-feeding Isolation of E focardii c-tubulin nanochromosomes from macronuclear DNA via PCR and RATE E focardii macronuclear DNA was purified as described previously [31] To obtain partial c-tubulin gene sequences, we based our PCR strategy on degenerate oligonucleotide primers designed from a Euplotes c-tubulin consensus sequence (obtained by the alignment of genes from three mesophilic Euplotes; GenBank accession numbers: Euplotes aediculatus, X85233.1; Euplotes otocarinatus, Y09553.1; E crassus, X85234.1 and X85235.1) The forward primer, 5¢-ATGCCA AGAGAAATYATYACTTG-3¢, covered codons 1–7 plus two nucleotides from 8, and the reverse primer, 5¢-TGAA CTTGAGTTGGRTCAACRTC-3¢, corresponded to two nucleotides of codon 328 plus triplets 327–321 Amplification was performed using standard conditions: 30 cycles at 94 °C for 50 s, 45 °C for min, and 72 °C for A final incubation at 72 °C for was added to the last cycle The amplification products were expected to contain 328 codons E focardii macronuclear DNA is composed of small nanochromosomes, each usually containing a single gene, which are always terminated by telomeres consisting of four repetitions of the motif C4A4 [62] This stereotypic organization facilitated obtaining the sequences of the C-terminal coding region and the 5¢- and 3¢-UTRs using the RATEPCR technique as described previously [31,32] We used the forward and reverse primers (see above) individually in combination with the telomeric oligonucleotide 5¢-(C4A4)43¢ Amplified products were cloned into the pCR2.1-TOPO vector of the TOPO TA CloningÒ kit (Invitrogen, San Diego, CA, USA) following the manufacturer’s recommendations Colony blotting and double-strand DNA labeling by the random priming method were performed as described previously [63] Clones containing c-tubulinrecombinant plasmids were sequenced in both strands (ABI Prism sequence analyzer Model 373A and Big Dye Terminator Methodology; PE Applied Biosystems, Foster City, CA, USA) DNA sequence analysis of c-tubulin nanochromosomes and prediction of the encoded amino acid sequences DNA sequence analysis, amino acid sequence prediction, and sequence alignments were performed with lasergeneÒ, version 7.2 (DNASTAR Inc., Madison, WI, USA) 5378 Comparative protein structure modelling of E focardii c-tubulins Comparative homology models of the two E focardii c-tubulins were obtained by use of modeller (version 9.1) [64] ˚ and the friend interface [65] The 3.0 A structure of human c-tubulin containing bound GTP (Protein Databank 1z5w) [66] was used as a template for comparative modelling Structural alignments between the template and modeled sequences were performed with topofit [66] and models were analyzed under friend The percentage similarities between modeled and template sequences were 68.36% for c-T1 and 69.28% for c-T2, and the length of alignment was 433 residues for both models Based on these values, we estimate that the accuracies of the modeled ˚ structures of c-T1 and c-T2 approach A [62] Poly[A] + RNA purification, cDNA synthesis and cloning, and Southern and northern blotting Poly[A] + RNA was purified from E focardii using the Quick-PrepÒ mRNA purification kit (GE Healthcare Life Sciences, Milan, Italy) For cDNA synthesis, the poly[A] + RNA (4 lg) was treated with 10 U of RNase-free DNaseI (Bethesda Research Laboratories, Bethesda, MD, USA), in the presence of 40 U of RiboLockÒ (Fermentas, Milan, Italy) and mm MgCl2 for h at 37 °C DNase-treated RNA was incubated with Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories) as recommended by the manufacturer The resulting cDNA was then precipitated in ethanol, collected by centrifugation, resuspended in distilled water, and used as template for PCR; the program was 30 cycles at 94 °C for 50 s, 48 °C for min, and 72 °C for A final incubation at 72 °C for was added to the last cycle cDNA was cloned into pCR2.1-TOPO as described above Southern and northern blotting were performed according to standard procedures on Hybond-N filters from Amersham (Milan, Italy) Filters were prehybridized, hybridized to DNA probes, and washed to remove nonspecifically bound probe according to the manufacturer’s recommendations Filters were stripped for reuse by boiling in distilled water for 15 s Estimation of macronuclear gene copy number and gene transcription To estimate gene copy number, qPCR was performed on total macronuclear DNA using the SYBR green DNAbinding method (TaKaRa Biotech, Dalian, China) The two E focardii c-tubulin genes were distinguished by use of the following primer pairs: c-T1, EfgT1_FW (5¢-ATGCGT CGTTTATTGCAGACT-3¢) as forward primer and EfgT1_REV (5Â-TGTTTTAGATAGAGCAACTTGGATT-3Â) FEBS Journal 275 (2008) 53675382 ê 2008 The Authors Journal compilation ª 2008 FEBS F Marziale et al as reverse primer; and c-T2, EfgT2_FW (5¢-ATGCGTCGT TTATTGCAACCC-3¢) as forward primer and Efg-T2_ REV (5¢-TGTTTCCGCTATCGATGTATGGTGA-3¢) as reverse primer 12.5 lL of SYBR Premix Ex Taq (2·) buffer, pg of each primer, and water were added to 100 ng of E focardii macronuclear DNA to reach a final volume of 25 lL The PCR parameters were initial denaturation at 95 °C for to activate the polymerase followed by 45 cycles of denaturation at 95 °C for 30 s and annealing and extension at 60 °C for 15 s each Following amplification, melting curve analysis of the DNA was performed at temperatures in the range 50–95 °C, with the temperature increasing at a rate of 0.5 °C every 10 s All PCR reactions were performed in a Multicolor qPCR Detection System iCycleriQ (Bio-Rad, Milan, Italy) During the primer annealing ⁄ extension step, the increase in the fluorescence from the amplified DNA was recorded by using the SYBR Green optical channel set at a wavelength of 495 nm The initial threshold value was set at 30 fluorescent units To analyse transcription of the two c-tubulin mRNAs, the same qPCR protocol was performed using 100 ng of E focardii cDNA prepared from total RNA Preparation of antibodies to E focardii c-tubulins Peptides that distinguish c-T1 [(390)RIFRRRNAYIDNYK(403)] and c-T2 [(390)KKLRSNNAFITTYQ(403)] were synthesized by Sigma Genosys (Milan, Italy) and used as antigens to obtain rabbit polyclonal anti-peptide sera The c-T1 peptide was immunogenic, whereas the c-T2 peptide was not c-Tubulin isotypes in E focardii (Diaphot-TMD equipped with an Apoplan ·60 objective; Nikon, Tokyo, Japan) Preparation of whole cell extract, basal bodies, and nuclei Whole cell extract preparations were obtained as described previously [29,31] Basal bodies were extracted by the following procedure: (a) E focardii cells were washed with MT buffer [30 mm Tris–acetate (pH 7.3), mm MgSO4, mm EGTA, 25 mm KCl, mm dithiothreitol], collected by low-speed centrifugation, and resuspended by extensive stirring in two volumes of MT buffer containing 2% NP-40 and protease inhibitors (0.01% aprotinin, 0.005% phenylmethanesulfonyl fluoride) An equal volume of 50% (w ⁄ v) Percoll was then added, and the mixture was centrifuged for 30 at 14 500 g in a fixed-angle rotor The fraction containing the basal bodies was recovered from the interface between the Percoll and aqueous phases, diluted in MT buffer, and centrifuged for 15 at 14 500 g The resulting pellet was washed twice in MT buffer Nuclei were prepared by resuspension of E focardii cell pellets in two volumes of lysis buffer [10 mm Tris–HCl (pH 6.8), 0.25 m sucrose, 10 mm MgCl2, mm phenylmethanesulfonyl fluoride, 0.5% NP-40] with gentle stirring on ice for min; two volumes of washing buffer (0.25 m sucrose, 10 mm MgCl2) were added to stop lysis The nuclear suspension was centrifuged at 1000 g for (4 °C), the supernatant was transferred to glass tubes, and nuclei were collected by centrifugation (9000 g, min, °C) SDS ⁄ PAGE and immunoblotting Immunofluorescence microscopy E focardii cells in logarithmic phase were washed, placed on a polylysine-coated (0.5 mgỈmL)1) coverslip, and permeabilized with 0.2% Triton X-100 in PHEM buffer (60 mm Pipes, 25 mm Hepes, 10 mm EGTA, mm MgCl2, final pH adjusted to 6.9 with NaOH) Cells were fixed with 2% paraformaldehyde in PHEM for approximately 30– 60 min, washed once with NaCl ⁄ Pi (130 mm NaCl, mm KCl, mm Na2HPO4, mm KH2PO4, pH 7.2), and then twice with NaCl ⁄ Pi plus 0.1% BSA; washes were 10 each Cells were incubated with rabbit polyclonal anti(human c-tubulin) serum (1 : 100 dilution in NaCl ⁄ Pi) and mouse monoclonal anti-a-tubulin serum DM1A (1 : 50; Sigma Genosys) overnight at °C, washed, and then incubated with fluorescent secondary antibodies [Alexa Fluor 594 goat anti-(rabbit IgG) and Alexa Flour 488 goat anti(mouse IgG)] at : 200 dilutions for h at 37 °C; in some experiments, rabbit polyclonal anti-(E focardii c-T1) was substituted for the anti-(human c-tubulin) serum Finally, cells were washed and suspended in 0.5% propyl gallate in glycerol Images were collected using an MRC600 Bio-Rad confocal system connected to a Nikon inverted microscope Denaturing SDS ⁄ PAGE was performed according to the method of Laemmli [67] After electrophoresis, gels were subjected to immunoblotting as described previously [29] Blots were incubated either with a rabbit polyclonal anti(human c-tubulin) primary serum [40] at a : 1000 dilution or with a rabbit polyclonal antibody directed against the c-T1 peptide (see above) at the same dilution Blots were washed extensively in NaCl ⁄ Tris ⁄ Tween buffer [5 mm Tris– HCl (pH 7.5), 0.138 m NaCl, 0.1% Tween-20] and then incubated with peroxidase-conjugated secondary anti-rabbit serum (1 : 1000 dilution) After washing with NaCl ⁄ Tris ⁄ Tween, bound primary antibody was detected by enhanced chemiluminescence (ECL Western Blotting Analysis System; GE Healthcare) Chemicals, materials, and reagents DNA modifying and restriction enzymes, RNAse A, 32 P-dATP, and Hybond-N filters were purchased from GE Healthcare Taq polymerase was from PE Applied Biosystems Oligonucleotides were synthesized by Labtek FEBS Journal 275 (2008) 5367–5382 ª 2008 The Authors Journal compilation ª 2008 FEBS 5379 c-Tubulin isotypes in E focardii F Marziale et al Eurobio (Milan, Italy) All routine chemicals were of analytical grade and supplied by Sigma Aldrich (Milan, Italy) Acknowledgements We are grateful to Professor Piero Luporini for providing conceptual and 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How the unique features of the E focardii c-tubulins enhance microtubule nucleation by the c-TuRC complex at low temperature, and what these changes imply regarding the mechanism of nucleation? ... that c-T2, but not cT1, participates in the assembly of the mitotic spindle of E focardii and that both isotypes are involved in the nucleation of other microtubule structures Finally, we examined... located at the plus ends, near the GTP-binding sites, and within the M loops of the E focardii c-tubulins, preserve their microtubulenucleating activities at cold temperatures and ⁄ or confer different