Role of delta-tubulin and the C-tubule in assembly of Paramecium basal bodies
© Garreau de Loubresse et al; licensee BioMed Central Ltd. 2001
Received: 29 January 2001
Accepted: 7 March 2001
Published: 7 March 2001
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.
Using the method of gene-specific silencing, we have inactivated the Paramecium delta-tubulin 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 A-tubules at one or more triplet sites are observed: around these incomplete cylinders, the remaining doublets are nevertheless positioned according to the native ninefold symmetry.
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.
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 . 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 . 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  by complementation cloning of the sm19 mutation affecting basal body duplication . In contrast to alpha-, beta- and gamma-tubulins, the new tubulins, of which a few sequences only are available, 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.
To ascertain whether δPT1 had a similar function, we took advantage of the phenomenon of homology-dependent gene silencing operating in Paramecium, previously discribed  and used to demonstrate the role of δ-tubulin in basal body duplication . Microinjection of the coding sequence of a gene, at high copy number, into the macronucleus results in the inactivation of the corresponding endogenous gene.
Cytoskeletal abnormalities induced by binactivation of the δ PT1 gene
Total number of
Number of cells
Inactivation of the δPT1 gene results in loss of the C-tubule, specific of the centriolar structure, as previously observed in the UNI3 mutant of Chlamydomonas, carrying a total deletion of the δ-tubulin gene . In Paramecium, further loss, namely of B- and even A-tubules, is also occasionally observed. The latter observation may imply either that the function of δ-tubulin is not restricted to C-tubule assembly, or that absence of the C-tubule destabilizes B- and, in turn, A-tubules. While δ-tubulin function remains to be characterized, the available observations in the two organisms at least delineate likely functions of the C-tubule. First, in both organisms, the C-tubule plays a role in anchoring, development and positioning of basal body appendages which form the cortical cytoskeleton; in Paramecium, these defects also correlate with mislocalization and internalization of basal bodies, yielding cortical disorders and reduction of basal body number and cell size. Second, the C-tubule does not play a direct role in ciliogenesis: axoneme structure and cilary activity seem normal in Paramecium ; in Chlamydomonas, doublet basal bodies can form flagella, although after some maturation delay . Third, the C-tubule is not directly required for basal body duplication: doublet basal bodies duplicate normally in the algae and in the ciliate; however, in Paramecium, further tubule loss may appear (Figure 3c', Figure 4). This difference may be due to the fact that in Chlamydomonas, basal bodies duplicate once per cell cycle, while in Paramecium, more than half of the ca 3500 cortical basal bodies, and all those destined to the neo-formed oral apparatus, undergo two or three fast cycles of duplication at each division [12,13]: the C-tubule might then contribute to stability of the microtubule scaffold (see Figure 4) and in turn to the efficiency of the "duplication complex" assumed to relay the structural information from mother to daughter basal body [14,15]. Conversely, the striking fact that absence of one or more triplets does not alter the underlying ninefold symmetry of the organelle, as also observed in Chlamydomonas mutants , provides an experimental argument in favour of the existence of a complex providing pattern information.
Finally, considering that addition of the C-tubule (a hemi-tubule, like the B-tubule) is the last step in basal body/centriole assembly [17,18], its absence upon depletion of δ-tubulin reveals not only the existence of subtle specificities at microtubule nucleation sites, but also a role of tubulin isotype diversity in generating microtubule arrays, well documented in Drosophila [19,20].
Material and Methods
Strain and Culture conditions
The wild type strain used was stock d4-2 of Paramecium tetraurelia. Cells were grown at 27°C as previously discribed .
As previously demonstrated, a specific gene can be silenced by microinjecting large quantities of its coding sequence in the Paramecium macronucleus . This results in a significant decrease in the amount of the corresponding mRNA or of the corresponding polypeptide measured by immunological reactivity on Western blots or in situ [9,10]. Furthermore, this inactivation is highly sequence-specific, and within multigenic families, effective only within a sub-family sharing at least 85% identity, as in the case of the two γ-tubulin genes . In order to inactivate δ-tubulin gene expression, PCR amplification of macronuclear genomic DNA, as previously discribed was carried out using the primers δ-ATG (5'-ATGTCTTTAGGTTTTATTTAATTAGGATAATGTGG-3') and δ-TGA (5'-TCATTAAATTAATTATTCATAATC-3'), resulting in amplification of the gene precisely limited by its initiation and stop codons. After primer removal, the DNA was concentrated and microinjected in the macronucleus. Previous experiments allowed us to verify that the efficiency of gene silencing obtained after microinjection of PCR amplified genes was equivalent to that obtained with plasmid cloned genes and that silencing efficiency was correlated with the amount of microinjected DNA, which unavoidably varies from cell to cell.
Immunolabeling of whole cells was performed as previously described [6,10] using an anti-tubulin antibody ID5  which allows precise observation of the pattern of basal bodies both on the cell cortex and in the oral apparatus. This antibody also decorates the post-oral fibers, a massive microtubule bundle nucleated on the right side of the oral apparatus.
For electron microscopy, pooled cells from transformed clones were fixed in 2% glutaraldehyde in 0.05 M cacodylate buffer, pH 7.2, for 90 min at 4°C. After washing in the same buffer, the samples were postfixed in 1% osmium tetroxide in 0.05 M cacodylate buffer, for 60 min at 4°C. After dehydratation, thin sections were contrasted with ethanolic uranyl acetate and lead citrate, and examined with a Philips 410 electron microscope.
We thank André Adoutte, Jean Cohen, France Koll, Linda Sperling and Michel Wright for discussions and critical reading of the manuscript and Annie Le Berre for excellent technical assistance. This work was supported by the Centre National de la Recherche Scientifique and the Association pour la Recherche sur le Cancer (Contrat #5425).
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