Etude par mRNA differential display de l'enkystement et de la vie kystique du cilié Sterkiella histriomuscorum (caractérisation d'une Ca++-ATPase et implication de l'ion calcium dans la signalisation du dékystement)

Le cycle d'enkystement-dékystement du cilié Sterkiella histriomuscorum, induit par l'absence de nourriture, constitue un excellent modèle unicellulaire eacaryote de différenciation cellulaire. En effet, l'enkystement correspond à une phase de profonde dédifférenciation cellulaire. La recherche des gènes exprimés de façon différentielle au cours de l'enkystement et de la vie kystique a été effectuée par mRNA differential display. Ce travail a montré que l'enkystement et la maturation du kyste sont associés à l'accumulation de transcrits, suggérant leur implication intérieure lors du dékystement. Parmi quatre gènes caractérisés, l'un code une Ca++-ATPase de la famille IIB. Ces protéines, impliquées dans le contrôle de l'homéostasie calcique, peuvent être localisées dans la membrane plasmique ou dans celle de compartiments intracellulaires stockant les ions calcium. Un anticorps spécifique de cette protéine est en cours de réalisation, ce qui permettra de déterminer sa localisation cellulaire. La fonction de cette protéine a été abordée, via la complémentation fonctionnelle de différents mutants de levure. Nous avons montré qu'une augmentation artificielle de la concentration calcique intracellulaire dans des kystes induit le processus du dékystement en absence de tout stimulus naturel, ce qui suggère fortement une implication de l'ion calcium comme second messager lors du dékystement. Les essais préliminaires permettent la visualisation d'éventuels flux calciques lors du dékystement au moyen d'une sonde fluorescente sont décrits. Par analogie avec la fécondation chez les pluricellulaires, un modèle des mécanismes mis en oeuvre lors de la sortie de dormance chez les unicellulaires est proposé. Par ailleurs, une compilation des gènes de S. histriomuscorum (dont les quatre caractérisés au cours de cette étude) nous permet d'apporter des données nouvelles sur la structure des gènes macronucléaires et l'organisation génomique en mini-chromosomes de cet organisme.The encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, triggered by food depletion, provides a good model to study cell differentiation within a single cell. Indeed, during encystment, extensive morphological changes occur leading to a highly dedifferentiated, encysted cell. The search for differentially expressed genes during the encystment and the cystic life has been undertaken by mRNM differential display. This study showed that encystement and cyst maturation are associated with the accumulation of a pool of transcripts, probably translated latter during the excystment. Among those genes, one encodes a type IIB Ca++-ATPase. These proteins, implied in the calcium homeostasis control, can be located in the plasma membrane or in intracellular organelles that sequester calcium. Specific antibody against this protein and complementation of differents yeast mutants, currently investigated, will help us to determine the precise cellular location and function of this Ca++-ATPase. We have also demonstrated that an artificial increase in the intracellular calcium concentration within cysts induces the excystment process, even in the absence of any natural stimuli, strongly suggesting an implication of calcium ions as second messenger during excystment. Preliminary experiments allowing the visualization of a transitory increase in the cytosolic calcium concentration using a fluorescent dye are described. We finally propose a model concerning the mecanisms triggering dormancy exit in unicellular organisms, based on what happens during pluricellular organisms fertilization. Besides, a compilation of many S. histriomuscorum genes, in addition to the 4 recently caracterized, increases our knowledge on macronuclear genes structure and chromosomal DNA genomic organization in this organism.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

End-joining inhibition at telomeres requires the translocase and polySUMO-dependent ubiquitin ligase Uls1

International audienceIn eukaryotes, permanent inhibition of the nonhomologous end joining (NHEJ) repair pathway at telomeres ensures that chromosome ends do not fuse. In budding yeast, binding of Rap1 to telomere repeats establishes NHEJ inhibition. Here, we show that the Uls1 protein is required for the maintenance of NHEJ inhibition at telomeres. Uls1 protein is a non-essential Swi2/Snf2- related translocase and a Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) with unknown targets. Loss of Uls1 results in telomere-telomere fusions. Uls1 requirement is alleviated by the absence of poly-SUMO chains and by rap1 alleles lacking SUMOylation sites. Furthermore, Uls1 limits the accumulation of Rap1 poly-SUMO conjugates.We propose that one of Uls1 functions is to clear non-functional poly- SUMOylated Rap1 molecules from telomeres to ensure the continuous efficiency of NHEJ inhibition. Since Uls1 is the only known STUbL with a translocase activity, it can be the general molecular sweeper for the clearance of poly- SUMOylated proteins on DNA in eukaryotes

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell Cycle Checkpoint

The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3′ strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang

Proposed Function of the Accumulation of Plasma Membrane-Type Ca(2+)-ATPase mRNA in Resting Cysts of the Ciliate Sterkiella histriomuscorum

From an mRNA differential-display analysis of the encystment-excystment cycle of the ciliate Sterkiella histriomuscorum, we have isolated an expressed sequence tag encoding a plasma membrane-type Ca(2+)-ATPase (PMCA). PMCAs are located either in the plasma membranes or in the membranes of intracellular organelles, and their function is to pump calcium either out of the cell or into the intracellular calcium stores, respectively. The S. histriomuscorum macronuclear PMCA gene (ShPMCA) and its corresponding cDNA were cloned; it is the first member of the Ca(2+)-ATPase family identified in Sterkiella. The predicted protein of 1,065 amino acids exhibits 37% identity with PMCAs of diverse organisms. A phylogenetic analysis showed its relatedness to homologs of two alveolates: the ciliate Paramecium tetraurelia and the apicomplexan Toxoplasma gondii. Overexpression of the protein ShPMCA failed to rescue the wild-type phenotype of three Ca(2+)-ATPase-defective mutant strains of Saccharomyces cerevisiae; this failure contrasts with the reported ability of the PMCAs of parasites to complement defects in yeast. ShPMCA mRNA is markedly accumulated during encystment and in resting cysts, suggesting a function during excystment. To address the possibility of a signaling role for calcium at excystment, the capacity of calcium to induce excystment was examined

The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA

International audienceRap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere

The orientation of the C-terminal domain of the Saccharomyces cerevisiae Rap1 protein is determined by its binding to DNA.

International audienceRap1 is an essential DNA-binding factor from the yeast Saccharomyces cerevisiae involved in transcription and telomere maintenance. Its binding to DNA targets Rap1 at particular loci, and may optimize its ability to form functional macromolecular assemblies. It is a modular protein, rich in large potentially unfolded regions, and comprising BRCT, Myb and RCT well-structured domains. Here, we present the architectures of Rap1 and a Rap1/DNA complex, built through a step-by-step integration of small angle X-ray scattering, X-ray crystallography and nuclear magnetic resonance data. Our results reveal Rap1 structural adjustment upon DNA binding that involves a specific orientation of the C-terminal (RCT) domain with regard to the DNA binding domain (DBD). Crystal structure of DBD in complex with a long DNA identifies an essential wrapping loop, which constrains the orientation of the RCT and affects Rap1 affinity to DNA. Based on our structural information, we propose a model for Rap1 assembly at telomere