Menbrane skeleton in Paramecium tetraurelia : characterization of a novel multigenic family and analysis by GFP and RNAi approaches
Squelette membranaire chez Paramecium tetraurelia : caractérisation d'une nouvelle famille multigénique et analyse par les approches GFP et RNAi
Résumé
The cortex of most ciliated protozoa encloses a membrane skeleton including the epiplasm, a sub-membranous layer extending underneath the cytoplasmic face of the inner alveolar membrane. In Paramecium, epiplasmic scales are centered around ciliary units and are composed of a complex set of proteins named epiplasmins. In the first part of this study, using the sequence of two epiplasmins (EPI-1 and EPI-2), we have contributed to the annotation of Paramecium tetraurelia macronuclear genome and identified 39 additional sequences. Two orthologous sequences were found in the Tetrahymena thermophila genome. Structural analysis of the 43 sequences indicates that the hallmark of this new multigenic family is a 79 amino acids domain flanked by two Q, P and V-rich stretches of sequence that are much more variable in amino-acid composition. A phylogenetic analysis confirmed the existence of 3 sub-families (on a total of 4) that were previously determined on the basis of distinct biochemical properties. The expression of GFP-tagged epiplasmin 1 showed significant labelling of epiplasmic scales and oral structures as well. The second part concerns the functional analysis of epiplasmins using an RNA interference approach. Following the ingestion of bacteria producing double-stranded RNA homologous to the EPI-1 and/or EPI-2 coding sequences, we obtained a phenotype named "boomerang" characterized by repeated blockages of cytokinesis leading to monstrous organisms which are finally lysed after 72 hours of induction. The analysis of induced cells by fluorescence microscopy revealed multiple abnormalities in cortical morphogenesis and karyokinesis processes despite the normal progression of some mechanisms like oral apparatus duplication. Finally, the production of a novel antibody raised against 2 peptides (1 and 2) included in epiplasmins 1 and 2 sequences, and decorating the epiplasm of Paramecium, allowed us to evidence the co-suppression of a sub-family of epiplasmins following the "boomerang" phenotype induction. The epitope of the monoclonal antibody (CTS-32) which labels the overall family of epiplasmins is not contained in peptides 1 and 2.
Chez la paramécie, l'épiplasme se présente sous forme d'écailles indépendantes disposées autour de chaque appareil ciliaire et composées d'une multitude de protéines appelées épiplasmines. Dans une première partie, nous avons contribué à l'annotation du génome macronucléaire de Paramecium tetraurelia à partir de la séquence de 2 épiplasmines (EPI-1 et EPI-2) et identifié 39 gènes supplémentaires. L'analyse des séquences indique que la signature de cette nouvelle famille est un domaine de 79 acides aminés. L'expression de l'épiplasmine 1 couplée à la GFP confirme la localisation de cette protéine au niveau des écailles épiplasmiques. La deuxième partie porte sur l'analyse fonctionnelle d'épiplasmines à partir d'une approche ARN interférence. Nous avons obtenu un phénotype caractérisé par un blocage répété de la cytocinèse conduisant à des organimes monstrueux qui sont finalement lysés après 72 h d'induction. L'analyse en microscopie à fluorescence a révélé l'absence de sillon de division.