Francesca Farina et al. (2007) International Worm Meeting "The Caenorhabditis elegans mitochondrial ADP/ATP translocator ANT-1.1 function is required for embryonic development and interacts with the ras signaling pathway."

Emmanuel Culetto, Norman Breuil, Adriana Alberti, Francesca Farina, Moise Pinto

[International Worm Meeting, 2007]

The Adenine Nucleotide Transporter (ANT) mediates the exchange of mitochondrial ATP with the cytosolic ADP. Mutations in human ANT1 are associated with autosomal dominant progressive external ophtalmoplegia (adPEO), a mitochondrial disorder characterized by progressive muscle weakness and multiple deletions of mitochondrial DNA (mtDNA). Several mechanisms have been proposed to explain the pathogenesis of adPEO, but it is still remains unclear how mutations in the ANT1 lead to mtDNA deletions. The study of ANT proteins in the C. elegans could aid in understanding adPOE. The C. elegans genome encodes four candidates ant paralogs respectively named ant-1.1, ant-1.2, ant-1.3 and ant-1.4. ant-1.1 is mainly localized in the mitochondria of most neurons, pharynx, hypodermis and muscle cells, whereas other three ANT proteins are expressed in a more discrete way in a few nerve cells, muscle cells and the intestine. Moreover, only the disruption of ant-1.1 gene function, through either an internal deletion or RNA interference, results in a developmental phenotype. The ant-1.1(RNAi) animals have smaller body size, slight developmental delay, daf-16 independent lifespan extension, defects in axon guidance and sterility. The mitochondria network in ant-1.1(RNAi) worms is disorganised, consistent with alterations in the mitochondrial function. To investigate the cause of sterility, we analysed both gonad formation and germline differentiation. Most of the ant-1.1(RNAi) animals showed abnormal gonad morphogenesis but some of them exhibited a complete well-shaped gonad. Therefore, we addressed the possibility of some defects in the oogenesis. The oocytes of ant-1.1(RNAi) worms were mostly blocked at the pachytene stage with only few detectable mature oocytes. The ant-1.1(RNAi) sterility could be suppressed by ras gain-of function allele let-60(gf). Moreover, the multivulva phenotype of let-60(gf) but not lin-1(n176) is suppressed by ant-1.1 inactivation. The ant-1.1(RNAi) showed increase resistance to oxidative stress induced by Paraquat, likely because of a basal low level of reactive oxygen species (ROS). These results suggest that ant-1.1 affects the ras signaling pathway by lowering ROS levels. Whatever the mechanism, our experiments provides a model system to study the pathogenesis linked to ANT1 dysfunction and demonstrate the possibility to modulate some aspects of ant-1.1(RNAi) mutant phenotype trough suppressor screens.

Norman Breuil et al. (2007) International Worm Meeting "Modeling mitochondrial heteroplasmy in the nematode Caenorhabditis elegans."

Norman Breuil, Emmanuel Culetto, Laure Rifault, Francesca Farina, Moise Pinto

[International Worm Meeting, 2007]

The mitochondrion plays a critical role in the cellular energy supply and performs other essential role in a variety of metabolic function. The mitochondrion possesses its own genome (mtDNA) coding for 13 polypeptides in the mitochondrial respiratory chain. Typically individuals harbour a single sequence variant of mtDNA. However, germline and somatic mutations can lead to the coexistence of a mixture of mutant and normal mtDNA. This state is defined as heteroplasmy. Some human mitochondrial diseases are often characterized by the coexistence of normal and mutated mtDNA molecules within the same cells. In effect, the disease-associated symptoms are clinically revealed once the mitochondrial mutated version overreach a certain threshold of heteroplasmy. We decided to address the molecular mechanisms controlling heteroplasmy and mtDNA maintenance using the model Caenorhabditis elegans. The C. elegans mtDNA is very similar to the human one, encoding for 12 proteins, is maternally inherited and the existence of heteroplasmic state have been demonstrated (Tsang and Lemire, 2002). We propose to generate heteroplasmic mutants that could be easily detected by drug resistance phenotype, then, to modify the genetic background of these worms by RNAi screening to assess whether there is any modification in the heteroplasmic status that could be detected by direct visual inspection. We aim to isolate animals harbouring point mutations in the mtDNA conferring a selective advantage to the worm: resistance to oligomycin. Actually, in yeast and human cells, point mutations in the mitochondrial gene coding for ATPase 6 subunit lead to oligomycin resistance. Therefore, we hypothesized that C. elegans mutant harbouring mtDNA mutation(s) in this gene could be recovered during the isolation of mutants resistant to the drug. We have shown that oligomycin effectively affect mitochondrial morphology and restricted C. elegans development and survival. In a forward genetic analysis we have screened 3000 F2 hermaphrodites for EMS induced mutations that allow worms to develop in presence of oligomycin. We have isolated 15 oligomycin resistant worms (oli-1 to oli-15) and started to study whether these resistance loci are maternally inherited. Genetic crosses have permitted to show that oli-1 is nuclear and recessive. We identified that a point mutation in the nuclear gene encoding ATP-9 subunit of the mitochondrial ATPsynthase complex is responsible for the oligomycin resistance. For 6 oli mutants, the genetic crosses indicate that the inheritance is nuclear dominant or semi-dominant, whereas oli-7 seems to show a cytoplasmic inheritance. We will present data showing further characterization of the latter mutant.

James K. Phelan et al. (2006) European Worm Meeting "Characterization of ced-8 and ced-8 Interacting Genes"

Michael O. Hengartner, Kelvin Wong, James K. Phelan, Hans H. Jung, Laurent Falquet, Sergio M. Pinto

[European Worm Meeting, 2006]

James K. Phelan1, Kelvin Wong1, Sergio M. Pinto, Laurent Falquet3, Hans H. Jung4 and Michael O. Hengartner1. Mutations in ced-8 were first identified in a screen for genes required for the timely engulfment of developmental cell corpses (Ellis, Jacobson, & Horvitz, 1991). Subsequent work clarified that the persistent corpses were the result of a delay in their generation, rather than a failure of engulfment (Stanfield & Horvitz, 2000). With ten predicted hydrophobic regions, CED-8 is anticipated to be a transmembrane protein and has been proposed to be a transporter. The function of ced-8, however, remains undetermined. Our goals are to clarify the role of ced-8 in cell corpse generation and clearance, and to utilize the mutation as a tool to identify additional genes with which ced-8 interacts. We are in the process of mapping the spatiotemporal expression pattern of ced-8 during the C. elegans lifecycle, and of experimentally determining the membrane topology of CED-8. Potential candidate interacting genes are being tested by double mutant and RNAi analysis. The resulting data will allow educated hypotheses to be formulated as to the function(s) of CED-8. In addition, ced-8 is now known to be the sole C. elegans representative of a gene family with multiple members in mammals. The founding member of the gene family, XK, is mutated in McLeod syndrome, a neuroacanthocytosis disease in which a neuromuscular degeneration is found in association with altered red blood cell morphology. We are attempting to rescue the ced-8 mutant phenotype by expression of representative mammalian cDNAs in order to ascertain which, if any, represent functional orthologs of CED-8.