Conserved molecular interactions in centriole-to-centrosome conversion.

Centrioles are required to assemble centrosomes for cell division and cilia for motility and signalling. New centrioles assemble perpendicularly to pre-existing ones in G1-S and elongate throughout S and G2. Fully elongated daughter centrioles are converted into centrosomes during mitosis to be able to duplicate and organize pericentriolar material in the next cell cycle. Here we show that centriole-to-centrosome conversion requires sequential loading of Cep135, Ana1 (Cep295) and Asterless (Cep152) onto daughter centrioles during mitotic progression in both Drosophila melanogaster and human. This generates a molecular network spanning from the inner- to outermost parts of the centriole. Ana1 forms a molecular strut within the network, and its essential role can be substituted by an engineered fragment providing an alternative linkage between Asterless and Cep135. This conserved architectural framework is essential for loading Asterless or Cep152, the partner of the master regulator of centriole duplication, Plk4. Our study thus uncovers the molecular basis for centriole-to-centrosome conversion that renders daughter centrioles competent for motherhood.J.F., Z.L., S.S. and N.S.D. are supported from Programme Grant to D.M.G. from Cancer Research UK. H.R. is supported from MRC Programme Grant to D.M.G. J.F. thank the British Academy and the Royal Society for Newton International Fellowship and Z.L. thanks the Federation of European Biochemical Societies for the Long-Term postdoctoral Fellowship. The authors thank Nicola Lawrence and Alex Sossick for assistance with 3D-SIM.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb327

The Effects of Lithium Chloride on Pattern Formation in Tetrahymena thermophila

AbstractLithium ions have long been known to exert dramatic effects on the specification of cell fates in multicellular systems. We have analyzed the effects of Li+ on intracellular patterning in a complex unicellular organism, the ciliate Tetrahymena thermophila. LiCl does not affect the locations of major structural landmarks in the cortical region of wild-type cells and does not modify the phenotype of pattern-mutant cells. However, in all strains studied LiCl differentially affects early stages of oral development. It initially triggers a slow regression of oral primordia, which is followed by an excessive proliferation of basal bodies that leads to a hypertrophy of the ciliature of the cell's feeding organelle. This hypertrophy mimics the effects of the membranellar-pattern-D mutation, the phenotype of which is enhanced in the presence of LiCl. These effects were partially reversed by myo-inositol; however, neomycin failed to mimic the effects of LiCl. Thus, although lithium ions have major cellular effects on Tetrahymena , they do not influence the specification of the body plan in a manner analogous to that observed in multicellular organisms and may work in part through mechanisms other than the now-classical inositol-phosphate cycle

Approches génétique et moléculaire de la duplication des corps basaux chez paramecium tetraurelia

La paramécie est un organisme recouvert de cils, chacun ancré sur un corps basal, structure polarisée se dupliquant à chaque cycle cellulaire et analogue structural des centrioles du centrosome. Chez le protozoaire Paramécie, plusieurs mutations affectant la duplication ont été décrites. Le mutant kin241-1, présente une hyperduplication des corps basaux, des anomalies de leur polarité et des zones morphogénétiques et de la différenciation nucléaire durant les processus sexuels. Le gène KIN241 a été isolé et cloné par complémentation fonctionnelle. Il code une protéine contenant 4 domaines: une isomérase-cyclophiline, un domaine d'interaction avec l'ARN, des répétition d'un dipeptide E-K et un C-terminal riche en sérines. Cette protéine, nommée CRIP (Cyclophiline RNA Interacting Protein), fusionnée avec la GFP, est localisée dans le noyau conformément aux signaux NLS (Nuclear Localisation Signal) de la partie Ct. L'introduction d'une délétion provoquant la disparition de la moitié des signaux NLS, diminue l'efficacité de l'adressage: la localisation est nucléaire et cytoplasmique. La mutation originale, kin241-1, (insertion de 2 nucléotides) introduit prématurément un signal de fin de traduction et donne une isoforme dépourvue de sa partie Ct. Cette isoforme n'est retrouvée ni dans le noyau ni dans le cytoplasme. Le phénotype de la mutation kin241-1 est donc dû à l'absence de la protéine dans la cellule. Ceci a été confirmé par des expériences d'extinction génique consistant à inactiver l'ARNm endogène et donc à prévenir la synthèse de la protéine CRIP. Nous concluons que CRIP est un facteur de processing d'une classe d'ARNm spécifiques des processus morphogénétiques. CRIP existe chez de nombreux organismes: levure, plantes, insectes et mammifères. Aucune de ces protéines n'a encore été étudiées. Cette thèse est un premier pas dans l'étude d'une nouvelle famille de protéines pouvant jouer un rôle dans le processing d'ARNm impliqués dans la morphogénèse.Paramecium is a unicellular organism covered by numerous cilia. Each of them is anchored on a basal body, a polarized structure, which duplicates during the cell cycle and is analogous to centrioles of the centrosome. In Paramecium, several mutants defective in basal body duplication have already been described. The kin241-1 mutation displays a highly pleiotropic effect on cortical organization, inducing hyperduplication of basal bodies and affecting nuclear reorganization during sexual processes. I have cloned the KIN241 gene by functional complementation. It encodes a new protein with 4 domains: a cyclophilin type isomerase, an RNA recognition motif, a domain rich in the dipeptide E-K and a C-terminal string of serines. This protein, named CRIP (Cyclophilin-RNA Interacting Protein) is localized in the nucleus via nuclear localization signals (NLS). A deletion which eliminates half of the NLS, decreases nuclear transport, so that the protein is localized in both the nucleus and the cytoplasm. The original mutation kin241-1 (insertion of 2 nucleotides) introduces a premature signal to end translation and leads to a protein truncated of its C-terminal domain. This isoform is unstable in vivo. This allowed me to conclude that the phenotype of the kin241-1 mutation is caused by the jack of CRIP protein. This hypothesis was confirmed by gene silencing experiments. In these experiments, the endogenous mRNA is degraded precluding the wild type protein synthesis. We have suggested that CRIP could be a factor involved in processing of a subclass of mRNA specific for morphological processes. The protein CRIP is present in different organisms including: yeast, plants, insects and mammals. These proteins have not been yet studied. This thesis thus raports the identification and first steps in investigation of the role this new family of proteins.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Cell Context-specific Effects of the β-Tubulin Glycylation Domain on Assembly and Size of Microtubular Organelles

Tubulin glycylation is a posttranslational modification found in cells with cilia or flagella. The ciliate Tetrahymena has glycylation on ciliary and cortical microtubules. We showed previously that mutating three glycylation sites on β-tubulin produces immotile 9 + 0 axonemes and inhibits cytokinesis. Here, we use an inducible glycylation domain mutation and epitope tagging to evaluate the potential of glycylation-deficient tubulin for assembly and maintenance of microtubular systems. In axonemes, the major defects, including lack of the central pair, occurred during assembly, and newly made cilia were abnormally short. The glycylation domain also was required for maintenance of the length of already assembled cilia. In contrast to the aberrant assembly of cilia, several types of cortical organelles showed an abnormally high number of microtubules in the same mutant cells. Thus, the consequences of deficiency in tubulin glycylation are organelle type specific and lead to either insufficient assembly (cilia) or excessive assembly (basal bodies and cortical microtubules). We suggest that the diverse functions of the β-tubulin glycylation domain are executed by spatially restricted microtubule-associated proteins

A functional study of genes essential for autogamy and nuclear reorganization in Paramecium

Like all ciliates, Paramecium tetraurelia is a unicellular eukaryote that harbors two kinds of nuclei within its cytoplasm. At each sexual cycle, a new somatic macronucleus (MAC) develops from the germline micronucleus (MIC) through a sequence of complex events, which includes meiosis, karyogamy and assembly of the MAC genome from MIC sequences. The latter process involves developmentally programmed genome rearrangements controlled by non-coding RNAs and a specialized RNA interference machinery. We describe our first attempts to identify genes and biological processes that contribute to the progression of the sexual cycle. Given the high percentage of unknown genes annotated in the P. tetraurelia genome, we applied a global strategy to monitor gene expression profiles during autogamy, a self-fertilization process. We focused this pilot study on the genes carried by the largest somatic chromosome and designed dedicated DNA arrays covering 484 genes from this chromosome (1.2% of all genes annotated in the genome). Transcriptome analysis revealed four major patterns of gene expression, including two successive waves of gene induction. Functional analysis of 15 up-regulated genes revealed four that are essential for vegetative growth, one of which is involved in the maintenance of MAC integrity and another in cell division or membrane trafficking. Two additional genes, encoding a MIC-specific protein and a putative RNA helicase localizing to the old, then to the new MAC, are specifically required during sexual processes. Our work provides a proof of principle that genes essential for meiosis and nuclear reorganization can be uncovered following genome-wide transcriptome analysis

Glutamylation on α-Tubulin Is Not Essential but Affects the Assembly and Functions of a Subset of Microtubules in Tetrahymena thermophila▿ †

Tubulin undergoes glutamylation, a conserved posttranslational modification of poorly understood function. We show here that in the ciliate Tetrahymena, most of the microtubule arrays contain glutamylated tubulin. However, the length of the polyglutamyl side chain is spatially regulated, with the longest side chains present on ciliary and basal body microtubules. We focused our efforts on the function of glutamylation on the α-tubulin subunit. By site-directed mutagenesis, we show that all six glutamates of the C-terminal tail domain of α-tubulin that provide potential sites for glutamylation are not essential but are needed for normal rates of cell multiplication and cilium-based functions (phagocytosis and cell motility). By comparative phylogeny and biochemical assays, we identify two conserved tubulin tyrosine ligase (TTL) domain proteins, Ttll1p and Ttll9p, as α-tubulin-preferring glutamyl ligase enzymes. In an in vitro microtubule glutamylation assay, Ttll1p showed a chain-initiating activity while Ttll9p had primarily a chain-elongating activity. GFP-Ttll1p localized mainly to basal bodies, while GFP-Ttll9p localized to cilia. Disruption of the TTLL1 and TTLL9 genes decreased the rates of cell multiplication and phagocytosis. Cells lacking both genes had fewer cortical microtubules and showed defects in the maturation of basal bodies. We conclude that glutamylation on α-tubulin is not essential but is required for efficiency of assembly and function of a subset of microtubule-based organelles. Furthermore, the spatial restriction of modifying enzymes appears to be a major mechanism that drives differential glutamylation at the subcellular level