Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain

p62 has been proposed to mark ubiquitinated protein bodies for autophagic degradation. We report that the Drosophila melanogaster p62 orthologue, Ref(2)P, is a regulator of protein aggregation in the adult brain. We demonstrate that Ref(2)P localizes to age-induced protein aggregates as well as to aggregates caused by reduced autophagic or proteasomal activity. A similar localization to protein aggregates is also observed in D. melanogaster models of human neurodegenerative diseases. Although atg8a autophagy mutant flies show accumulation of ubiquitin- and Ref(2)P-positive protein aggregates, this is abrogated in atg8a/ref(2)P double mutants. Both the multimerization and ubiquitin binding domains of Ref(2)P are required for aggregate formation in vivo. Our findings reveal a major role for Ref(2)P in the formation of ubiquitin-positive protein aggregates both under physiological conditions and when normal protein turnover is inhibited

Mécanismes de la réponse aux stress cellulaires

The projects I have chosen to investigate during my career focus on the control of cell decision to survive or die, leading to the study of tissue homeostasis in Drosophila. I tried to understand the mechanisms regulating survival by examining the Bcl-2 family, as well as a Drosophila model of a human neurodegenerative polyglutamine disease -called Spinocerebellar ataxia type 3- that had been previously established. This background allows me to propose a study of the role of mitochondria in the response to ER stress with an emphasis on mammalian ATF5 and its Drosophila orthologue Crc. Indeed, the roles of ATF5 and Crc have been poorly described. Yet, they seem to lie at the crossroad between the mitochondrial stress response, the endoplasmic reticulum unfolded protein response and viral infection response. This project should lead to a better understanding of the network formed by cell stress responses.Les sujets que j’ai choisi d’étudier depuis le début de mon expérience de recherche ont porté sur le contrôle de la décision de survie ou de mort cellulaire et m’ont conduit à caractériser des mécanismes participant à l’homéostasie tissulaire chez la drosophile. J’ai cherché à comprendre les mécanismes de régulation de la décision de survie ou de mort cellulaire en me focalisant sur les membres de la famille Bcl-2, ainsi que sur un modèle d’une maladie neurodégénérative à polyglutamine humaine (l’ataxie spinocérébelleuse de type 3) établi chez la drosophile. Ces travaux m’ont amené à étudier les conséquences du stress du réticulum endoplasmique et les spécificités de réponse en fonction de la localisation des cellules au sien de leur tissu. Ces travaux me permettent de proposer une étude du rôle de la mitochondrie dans la réponse au stress du réticulum endoplasmique avec une attention particulière pour ATF5 en cellules de mammifères et son orthologue Crc chez la drosophile. En effet, ATF5 et Crc sont peu étudiés et semblent pourtant situés à la confluence de la réponse au stress mitochondrial, de la réponse aux protéines mal conformées du réticulum endoplasmique et de la réponse aux infections virales. Ce projet devrait permettre une meilleure compréhension des communications entre les voies de réponse aux stress cellulaires

Life span extension by dietary restriction is reduced but not abolished by loss of both SIR2 and HST2 in Podospora anserina.

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: Théorie, analyse et ingénierie

National audienceCet ouvrage regroupe rappels de cours, exercices et problèmes corrigés de génétique pour comprendre les concepts et mettre en œuvre les principes de l'analyse génétique. Du fait de l'importance de la drosophile dans la recherche fondamentale, cette nouvelle édition entièrement refondue propose une présentation plus complète de cet organisme modèle et des outils génétiques disponibles qui y ont été intégrés

L'apoptose chez la drosophile : conservation et originalité

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Mitochondria, Bcl-2 family proteins and apoptosomes: of worms, flies and men

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The endoplasmic reticulum unfolded protein response varies depending on the affected region of the tissue but independently from the source of stress

International audienceAccumulation of unfolded proteins and calcium dyshomeostasis induces endoplasmic reticulum (ER) stress, which can be resolved by the unfolded protein response (UPR). We have previously reported that activation of the PERK/ATF4 branch of the UPR, by overexpressing Presenilin in part of the vestigial domain of Drosophila wing imaginal discs, induces both a caspase-dependent apoptosis and a Slpr/JNK/Dilp8-dependent developmental delay that allows compensation of cell death in the tissue. Recently, dDad1 depletion in Drosophila in engrailed-expressing cells of wing imaginal discs was also reported to activate the PERK/ATF4 branch but induced Mekk1/JNK-dependent apoptosis. Here, we assessed whether the stressed cell location in the wing imaginal disc could explain these differences in response to chronic ER stress or whether the stress source could be responsible for the signaling discrepancy. To address this question, we overexpressed a Rhodopsin-1 mutant prone to aggregate either in vestigial-or engrailed-expressing cells. We observed similar responses to the Presenilin overexpression in the vestigial domain and to the dDad1 depletion in the engrailed domain. Therefore, the consequences of a PERK/ATF4 branch activation depend on the position of the cell in the Drosophila wing imaginal disc, suggesting interactions of PERK signaling with developmental pathways involved in the determination or maintenance of wing domains

Screening of suppressors of bax-induced cell death identifies glycerophosphate oxidase-1 as a mediator of debcl-induced apoptosis in Drosophila

International audienceMembers of the Bcl-2 family are key elements of the apoptotic machinery. In mammals, this multigenic family contains about twenty members, which either promote or inhibit apoptosis. We have previously shown that the mammalian pro-apoptotic Bcl-2 family member Bax is very efficient in inducing apoptosis in Drosophila, allowing the study of bax-induced cell death in a genetic animal model. We report here the results of the screening of a P[UAS]-element insertion library performed to identify gene products that modify the phenotypes induced by the expression of bax in Drosophila melanogaster. We isolated 17 putative modifiers involved in various function or process: the ubiquitin/proteasome pathway; cell growth, proliferation and death; pathfinding and cell adhesion; secretion and extracellular signaling; metabolism and oxidative stress. Most of these suppressors also inhibit debcl-induced phenotypes, suggesting that the activities of both proteins can be modulated in part by common signaling or metabolic pathways. Among these suppressors, Glycerophosphate oxidase-1 is found to participate in debcl-induced apoptosis by increasing mitochondrial reactive oxygen species accumulation

zVAD-fmk upregulates caspase-9 cleavage and activity in etoposide-induced cell death of mouse embryonic fibroblasts

International audienceKeywords: Caspase-9 zVAD-fmk p53-dependent cell death Mouse embryonic fibroblast Caspases are key effectors of programmed cell death. Down-and up-regulation of their activity are involved in different pathologies. In most cells, zVAD-fmk prevents apoptosis. However, unexpected effects of zVAD-fmk have been characterized in different laboratories, cell models and cell death processes. We have previously shown that zVAD-fmk accelerates p53-dependent apoptosis in rat embryonic fibroblasts. In this study, we pursued our investigations on zVAD-fmk effects and focused our study at the mitochondrial level in mouse embryonic fibro-blasts (MEFs). In both primary and immortalized (by AgT or 3T9 protocol) MEFs, zVAD-fmk increased etoposide-induced loss of ΔΨm. This increase correlated with an increase of the number of apoptotic cells in primary and 3T9 MEFs, but did not in AgT MEFs. In both types of immortalized MEFs, zVAD-fmk regulated neither p53 levels nor transcriptional activities, suggesting that zVAD-fmk acts downstream of p53. In MEFs, zVAD-fmk increased p53-dependent loss of ΔΨm, cytochrome c release and caspase-9 activity. Indeed, zVAD-fmk inhibited effector caspases (caspases-3,-6,-7) as expected but increased caspase-9 cleavage and activity in etoposide-treated MEFs. Q-VD-OPh, another caspase inhibitor, also increased both loss of ΔΨm and caspase-9 cleavage in etoposide-treated MEFs. Invalidation of bax and bak suppressed p53-dependent cell death and zVAD-fmk regulation of this process. Invalidation of caspase-9 did not inhibit mitochondrial membrane depolarization but suppressed zVAD-fmk amplification of this process. Altogether, our data suggest that caspase-9 activity is up-regulated by zVAD-fmk and is involved in an amplification loop of etoposide-induced cell death at the mitochon-drial level in MEFs