Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies

BMC Cell Biology (2001) 2:4 http://www.biomedcentral.com/1471-2121/2/4 BMC Cell Biology Research article (2001) 2:4 Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies Nicole Garreau de Loubresse, Françoise Ruiz, Janine Beisson and Catherine Klotz* Address: Centre de Génétique Moléculaire du C.N.R.S., Allée de la Terrasse, 91190 Gif- sur-Yvette, France E-mail: Nicole Garreau de Loubresse - ; Françoise Ruiz - ; Janine Beisson - ; Catherine Klotz* - *Corresponding author Published: 7 March 2001 BMC Cell Biology 2001, 2:4 Received: 29 January 2001 Accepted: 7 March 2001 This article is available from: http://www.biomedcentral.com/1471-2121/2/4 (c) 2001 Garreau de Loubresse et al, licensee BioMed Central Ltd. Abstract Background: A breakthrough in the understanding of centriole assembly was provided by the characterization of the UNI3 gene in Chlamydomonas. Deletion of this gene, found to encode a novel member of the tubulin superfamily, delta-tubulin, results in the loss of the C-tubule, in the nine microtubule triplets which are the hallmark of centrioles and basal bodies. Delta-tubulin homologs have been identified in the genomes of mammals and protozoa, but their phylogenetic relationships are unclear and their function is not yet known. Results: Using the method of gene-specific silencing, we have inactivated the Paramecium deltatubulin gene, which was recently identified. This inactivation leads to loss of the C-tubule in all basal bodies, without any effect on ciliogenesis. This deficiency does not directly affect basal body duplication, but perturbs the cortical cytoskeleton, progressively leading to mislocalization and loss of basal bodies and to altered cell size and shape. Furthermore, additional loss of B- and even Atubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry. Conclusions: The fact that in two distinct phyla, delta-tubulin plays a similar role provides a new basis for interpreting phylogenetic relationships among delta-tubulins. The role of delta-tubulin in C-tubule assembly reveals that tubulins contribute subtle specificities at microtubule nucleation sites. Our observations also demonstrate the existence of a prepattern for the ninefold symmetry of the organelle which is maintained even if less than 9 triplets develop. Background In addition to the alpha-, beta- and gamma-tubulins, essential for microtubule assembly in all eukaryotes, several new tubulin subfamilies have been recently identified in a cascade of discoveries [1]. Complementation cloning of the UNI3 mutation in Chlamydomonas led to the characterization of delta-tubulin, an unexpected fourth member of the tubulin subfamily, involved in assembly of basal bodies [2]. Genome search for delta-tubulin led to identify not only deltas in mammals and protozoa, but also to disclose further new divergent tubulins, epsilon and zeta [3,4,5]. A sixth subfamily, eta, was characterized in Paramecium [6] by complementation cloning of the sm19 mutation affecting basal body duplication [7]. In contrast to alpha-, beta- and gamma-tubulins, the new tubulins, of which a few sequences only are availa- BMC Cell Biology (2001) 2:4 ble, do not seem to be present in all eukaryotes and their function might concern such elaborate microtubule arrays as centrioles and basal bodies. For each of the new subfamilies, sequence conservation is weak and, as only a few sequences are presently available, their phylogenetic relationships are unclear [1,6]. It is therefore important to ascertain if the members of a given presumed subfamily have the same function. For the delta-tubulin subfamily, the function is known only for Chlamydomonas: deletion of this gene results in the loss of the C-tubule, in each of the nine microtubule triplets which are the hallmark of centrioles and basal bodies. In the absence of a functional assay, a similar role of the delta-tubulin homologs identified in mammals [3,4] and protozoa [5,6] had not yet been demonstrated. We report here a functionnal analysis of delta-tubulin in Paramecium, a favourable organism because of the high number of its basal bodies and of their differentiated duplication pattern. Results In the course of random sequencing (see http://caroll.vjf.inserm.fr/pt/) of clones from the recently described indexed genomic library of Paramecium [8], a likely δ-tubulin homolog was characterized [6]. Southern blots (not shown) demonstrate that this sequence corresponds to a unique gene, designated as δPT1. The deduced polypeptide, 397 amino acids long, is shorter than tubulins; alignment with the known δ-tubulin sequences shows (Figure 1) that 43% of its residues are identical to the corresponding residue in at least one of the other sequences. After those of Chlamydomonas [2], man [4], Basal body ultrastructure was examined on cells just beginning to display cortical disorders and shape changes, around the 5th division. At this early stage of expression of the transformant phenotype, a striking defect, lack of the C-tubule, was found in practically all basal bodies (156 out of 171 unambiguous basal body cross-sections), corresponding to at least 20 different cells from several transformed clones (Figure 3). It must be pointed out that this effect is highly specific: loss of the C-tubule was never observed in other experimental or genetical conditions interfering with basal body properties or duplication, such as γ-tubulin or centrin inactivation [10, 11; Garreau de Loubresse, unpublished] or in basal body defective mutants such as sm19 cells, mutated in -tubulin [6], or sm2 cells [7]. In some basal bodies, both triplets and doublets coexisted (Figure 3c', e'), suggesting, as demonstrated in the case of other genes [9], that inactivation of the δPT1 gene was not total. In several instances, a mother "all doublets"basal body had produced a daughter basal body with more pronounced tubule deficiencies (Figure 3c'; http://www.biomedcentral.com/1471-2121/2/4 mouse [3] and Trypanosoma [5], δPT1 is the fifth δ or putative δ-tubulin identified to date. To ascertain whether δPT1 had a similar function, we took advantage of the phenomenon of homology-dependent gene silencing operating in Paramecium, previously discribed [9] and used to demonstrate the role of δtubulin in basal body duplication [10]. Microinjection of the coding sequence of a gene, at high copy number, into the macronucleus results in the inactivation of the corresponding endogenous gene. The effect of δPT1 inactivation was studied on the individual progenies of 132 microinjected cells from two independent series of 33 and 99 cells respectively. In both sets of clones, growth rate, cell morphology and cytoskeletal organization remained normal over the four first divisions after injection. Then, over the next 2-3 divisions, the same syndrome progressively develop (...truncated)