Contribution des GTPases Ral à l'oncogenèse humaine (recherche et étude de nouveaux partenaires fonctionnels par approche génétique)

Les petites protéines G Ral sont des acteurs essentiels dans la transduction du signal par les proto-oncogènes Ras, dans des conditions physiologiques et pathologiques comme le cancer. Ce travail a pour objectif la recherche et l'étude de nouveaux partenaires fonctionnels de ces protéines par une approche génétique utilisant Drosophila melanogaster. La drosophile possède un seul gène Ral dont la voie de signalisation est analogue à celle des mammifères. Une diminution de la signalisation par Ral conduit à un processus apoptotique temps- et cellule- spécifique se traduisant par un phénotype de " perte de soies mécano-sensorielles ". Un crible génétique par surexpression a identifié des aggravateurs et des suppresseurs de ce phénotype. Les résultats du crible ont été croisés avec les données des cartes d'interactions protéine-protéine et recoupés selon leur appartenance aux groupes d'ontologie et l'existence d'orthologues humains. Ces nouveaux partenaires fonctionnels de Ral ont comme principales fonctions biologiques associées: le cycle cellulaire, la migration, l'apoptose et l'identité cellulaire.TOULOUSE3-BU Santé-Centrale (315552105) / SudocSudocFranceF

PGal4 excision reveals the pleiotropic effects of Voila, a Drosophila locus that affects development and courtship behaviour

0016-6723 (Print) Journal Article Research Support, Non-U.S. Gov'tIn Drosophila melanogaster, the PGal4 transposon inserted at the chromosomal site 86E1-2 is associated with the Voila1 allele that causes multiple phenotypes. Homozygous Voila1/1 flies rarely reach adulthood and heterozygous Voila1/+ adult males display strong homosexual courtship behaviour. Both normal behavioural and developmental phenotypes were rescued by remobilizing the PGal4 element. Yet, the rescue of heterosexual courtship and of adult viability did not occur in the same strains, indicating that these defects have different genetic origins. Furthermore, many strains showed a partial rescue of both characters. Molecular analysis revealed that the PGal4 transposon is inserted upstream of the 5'UTR of the prospero gene. The excision strains with no detectable fragment of the PGal4 transposon remaining showed a rescued viability for homozygote adults. Moreover, the developmental period with the highest homozygote lethality was correlated with the size of PGal4 element that remained inserted at the Voila locus. This suggests a relationship between developmental viability and the amount of DNA inserted within the promoter of prospero

The genetic variant Voila causes gustatory defects during Drosophila development

1529-2401 (Electronic) Journal Article Research Support, Non-U.S. Gov'tVoila(1), an enhancer-trap strain in Drosophila melanogaster, expresses GAL4 in most gustatory neurons, both before and after metamorphosis. Voila(1) expression starts at embryonic stage 10. In the periphery, it labels larval gustatory sensilla in the antennomaxillary complex as well as in the pharynx. GAL4 is also expressed in the CNS in a manner that prefigures expression in adult flies. Most Voila(1/1) homozygotes die between second larval instar and early adulthood. Moreover, escaping Voila(1/1) larvae do not show gustatory responses to NaCl and sucrose. The simultaneous rescue of normal larval gustation together with adult viability after removal of the transposable PGAL4 element suggests that both these phenotypes are caused by the same inserted element

Voila, a new Drosophila courtship variant that affects the nervous system: behavioral, neural, and genetic characterization

0270-6474 (Print) Journal Article Research Support, Non-U.S. Gov'tIn Drosophila melanogaster, a specific PGAL4 transposon induces the Voila1 genetic variant and produces multiple phenotypes. Homozygous Voila1/1 flies rarely reach adulthood, whereas heterozygous Voila1/+ adult males show strong bisexual behavior. Males with a single copy of Voila1 driving the feminizing transgene UAS-transformer show very reduced sexual activity but no overall effect on their behavior. Voila1 is specifically expressed in the nervous system. In the CNS, it is expressed mainly in the mushroom bodies and, to a lesser extent, in the antennal lobes. In the peripheral nervous system, GAL4 expression is almost entirely restricted to the gustatory sensilla. Using chromosomal deficiencies, the behavioral alteration was genetically mapped to the same location as the PGAL4 element (86E1-2). The multiple behavioral effects of the Voila genetic variant are discussed in light of its expression in the nervous system and its genetic basis

A Ral Guanine Exchange Factor-Ral Pathway Is Conserved in Drosophila melanogaster and Sheds New Light on the Connectivity of the Ral, Ras, and Rap Pathways

Ras GTPases are central to many physiological and pathological signaling pathways and act via a combination of effectors. In mammals, at least three Ral exchange factors (RalGEFs) contain a Ras association domain and constitute a discrete subgroup of Ras effectors. Despite their ability to bind activated Rap as well as activated Ras, they seem to act downstream of Ras but not downstream of Rap. We have revisited the Ras/Rap-Ral connections in Drosophila melanogaster by using iterative two-hybrid screens with these three GTPases as primary baits and a subsequent genetic approach. We show that (i) the Ral-centered protein network appears to be extremely conserved in human and flies, (ii) in this network, RGL is a functional Drosophila orthologue of RalGEFs, and (iii) the RGL-Ral pathway functionally interacts with both the Ras and Rap pathways. Our data do not support the paradigmatic model where Ral is in the effector pathway of Ras. They reveal a signaling circuitry where Ral is functionally downstream of the Rap GTPase, at odds with the pathways described for mammalian cell lines. Thus, in vivo data show variations in the connectivity of pathways described for cell lines which might display only a subset of the biological possibilities

Applying mechanical forces on Drosophila tissues in vivo using the StretchCo, a 3D-printable device

Summary: Applying mechanical forces to tissues helps to understand morphogenesis and homeostasis. Additionally, recording the dynamics of living tissues under mechanical constraints is needed to explore tissue biomechanics. Here, we present a protocol to 3D-print a StretchCo device and use it to apply uniaxial mechanical stress on the Drosophila pupal dorsal thorax epithelium. We describe steps for 3D printing, polydimethylsiloxane (PDMS) strip cutting, and glue preparation. We detail procedures for PDMS strip mounting, tissue compaction, and live imaging upon force application. For additional details on the use and execution of this protocol, please refer to Cachoux et al. (2023)1 from which the StretchCo machine has been derived. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics

Erratum: Epithelial tricellular junctions act as interphase cell shape sensors to orient mitosis

International audienc

The Ral/Exocyst Effector Complex Counters c-Jun N-Terminal Kinase-Dependent Apoptosis in Drosophila melanogaster

Ral GTPase activity is a crucial cell-autonomous factor supporting tumor initiation and progression. To decipher pathways impacted by Ral, we have generated null and hypomorph alleles of the Drosophila melanogaster Ral gene. Ral null animals were not viable. Reduced Ral expression in cells of the sensory organ lineage had no effect on cell division but led to postmitotic cell-specific apoptosis. Genetic epistasis and immunofluorescence in differentiating sensory organs suggested that Ral activity suppresses c-Jun N-terminal kinase (JNK) activation and induces p38 mitogen-activated protein (MAP) kinase activation. HPK1/GCK-like kinase (HGK), a MAP kinase kinase kinase kinase that can drive JNK activation, was found as an exocyst-associated protein in vivo. The exocyst is a Ral effector, and the epistasis between mutants of Ral and of msn, the fly ortholog of HGK, suggest the functional relevance of an exocyst/HGK interaction. Genetic analysis also showed that the exocyst is required for the execution of Ral function in apoptosis. We conclude that in Drosophila Ral counters apoptotic programs to support cell fate determination by acting as a negative regulator of JNK activity and a positive activator of p38 MAP kinase. We propose that the exocyst complex is Ral executioner in the JNK pathway and that a cascade from Ral to the exocyst to HGK would be a molecular basis of Ral action on JNK