Production et élimination des radicaux libres : les tests cellulaires

International audienc

ETUDE DE LA PROTEINE RB DANS L'APOPTOSE INDUITE PAR INACTIVATION DE L'ANTIGENE T DU VIRUS SV40

L'APOPTOSE ET LA SENESCENCE REPLICATIVE SONT DEUX FORMES D'ARRET IRREVERSIBLE DE LA PROLIFERATION CELLULAIRE. L'APOPTOSE EST UNE MORT CELLULAIRE PHYSIOLOGIQUE TANDIS QUE LA SENESCENCE REFLETE LA CAPACITE PROLIFERATIVE LIMITEE DES CELLULES. AFIN D'ETUDIER LE DETERMINISME MOLECULAIRE DE L'ENGAGEMENT DES CELLULES DANS L'APOPTOSE OU LA SENESCENCE, J'AI UTILISE DES FIBROBLASTES DE RAT IMMORTALISES PAR UN MUTANT THERMOSENSIBLE DE L'ANTIGENE T DU VIRUS SV40. A TEMPERATURE RESTRICTIVE, L'ANTIGENE T EST INACTIVE ET LIBERE LA PROTEINE P53 QUI PEUT INDUIRE L'ARRET DE PROLIFERATION ET L'APOPTOSE. LE PHENOTYPE ASSOCIE A LA PERTE DE L'ETAT IMMORTALISE VARIE EN FONCTION DE LA LIGNEE ETUDIEE : LES CELLULES DE LA LIGNEE RETSAF MEURENT PAR APOPTOSE ALORS QUE LES CELLULES DE LA LIGNEE RETSA15 DEVIENNENT SENESCENTES. OUTRE LA PROTEINE P53, L'ANTIGENE T INHIBE LA PROTEINE ONCOSUPPRESSIVE RB. CETTE PROTEINE EST UN REGULATEUR NEGATIF DU FACTEUR DE TRANSCRIPTION E2F-1 QUI FAVORISE L'ENTREE DES CELLULES EN PHASE S DU CYCLE CELLULAIRE AINSI QUE L'EFFET PRO-APOPTOTIQUE DE P53. L'ETUDE COMPARATIVE DES CELLULES RETSAF ET RETSA15 M'A PERMIS D'OBSERVER QUE LA PROTEINE RB EST REGULEE DE FACON DIFFERENTE DANS CES DEUX LIGNEES, A LA FOIS PAR DES PROCESSUS DE PHOSPHORYLATION ET DE CLIVAGE PROTEOLYTIQUE. A TEMPERATURE RESTRICTIVE, L'INACTIVATION DE LA PROTEINE RB PAR PHOSPHORYLATION DANS LES CELLULES RETSAF FAVORISERAIT L'ACTIVITE DU FACTEUR E2F-1, LA PROGRESSION DES CELLULES DANS LE CYCLE CELLULAIRE ET PEUT ETRE L'APOPTOSE. DE FACON SURPRENANTE, LE CLIVAGE DE LA PROTEINE RB PAR DES PROTEASES A CYSTEINE (CASPASES), TELLES QUE LES CASPASES-6 ET -7, FAVORISERAIT LA SURVIE DES CELLULES RETSA15 ET LEUR ARRET DANS LE CYCLE CELLULAIRE. EN ACCORD AVEC CETTE OBSERVATION, J'AI MIS EN EVIDENCE QUE LA PROTEINE BCL-2, UN INHIBITEUR UNIVERSEL DE L'APOPTOSE, FAVORISE LE CLIVAGE DE RB. CETTE PROPRIETE DE BCL-2 POURRAIT PARTICIPER A UNE VOIE D'INHIBITION DE L'APOPTOSE ENCORE JAMAIS DECRITE.PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Tickets for p53 journey among organelles

International audienc

A corset of adhesions during development establishes individual neural stem cell niches and controls adult behaviour

International audienceNeural stem cells (NSCs) reside in a defined cellular microenvironment, the niche, which supports the generation and integration of neuronal lineages. The mechanisms building a sophisticated niche structure around NSCs, and their functional relevance for neurogenesis are yet to be understood. In the Drosophila larval brain, the cortex glia (CG) encase individual NSC lineages, organizing the stem cell population and newborn neurons into a stereotypic structure. We first found that lineage information is dominant over stem cell fate. We then discovered that, in addition to timing, the balance between multiple adhesion complexes supports the individual encasing of NSC lineages. An intra-lineage adhesion through homophilic Neuroglian interactions provides strong binding between cells of a same lineage, while a weaker interaction through Neurexin-IV exists between CG to NSC lineages. Their loss leads to random, aberrant grouping of several NSC lineages together, and to altered axonal projection of newborn neurons. Further, we link the loss of these two adhesion complexes during development to locomotor hyperactivity in the resulting adults. Altogether, our findings identify a corset of adhesions building a neurogenic niche at the scale of individual stem cell and provide the proof-of-principle that mechanisms supporting niche formation during development define adult behaviour

reaper and bax initiate two different apoptotic pathways affecting mitochondria and antagonized by bcl-2 in Drosophila

International audiencebcl-2 was the first regulator of apoptosis shown to be involved in oncogenesis. Subsequent studies in mammals, in the nematode and in Drosophila revealed wide evolutionary conservation of the regulation of apoptosis. Although dbok/debcl, a member of the bcl-2 gene family described in Drosophila, shows pro-apoptotic activities, no anti-apoptotic bcl-2 family gene has been studied in Drosophila. We have previously reported that the human anti-apoptotic gene bcl-2 is functional in Drosophila, suggesting that the fruit fly shares regulatory mechanisms with vertebrates and the nematode, involving anti-apoptotic members of the bcl-2 family. We now report that bcl-2 suppresses rpr-induced apoptosis in Drosophila. Additionally, we have compared features of bax-and rpr-induced apoptosis. Flow cytometry analysis of wing disc cells demonstrate that both killers trigger mitochondrial defects. Interestingly, bcl-2 suppresses both bax-and rpr-induced mitochondrial defects while the caspase-inhibitor p35 is specific to the rpr pathway. Finally, we show that the inhibition of apoptosis by bcl-2 is associated with the down-regulation of rpr expression

The Drosophila retinoblastoma protein, Rbf1, induces a Debcl-and Drp1-dependent mitochondrial apoptosis

International audienceIn accordance with its tumor suppressor role, the retinoblastoma protein pRb can ensure pro-apoptotic functions. Rbf1, the Drosophila homolog of Rb, also displays a pro-apoptotic activity in proliferative cells. We have previously shown that the Rbf1 pro-apoptotic activity depends on its ability to decrease the level of anti-apoptotic proteins such as the Bcl-2 family protein Buffy. Buffy often acts in an opposite manner to Debcl, the other Drosophila Bcl-2-family protein. Both proteins can localize at the mitochondrion, but the way they control apoptosis still remains unclear. Here, we demonstrate that Debcl and the pro-fission gene Drp1 are necessary downstream of Buffy to trigger a mitochondrial fragmentation during Rbf1-induced apoptosis. Interestingly, Rbf1-induced apoptosis leads to a Debcl-and Drp1-dependent reactive oxygen species production, which in turn activates the Jun Kinase pathway to trigger cell death. Moreover, we show that Debcl and Drp1 can interact and that Buffy inhibits this interaction. Notably, Debcl modulates Drp1 mitochondrial localization during apoptosis. These results provide a mechanism by which Drosophila Bcl-2 family proteins can control apoptosis, and shed light on a link between Rbf1 and mitochondrial dynamics in vivo

Differential adhesion during development establishes individual neural stem cell niches and shapes adult behaviour in Drosophila

International audienceNeural stem cells (NSCs) reside in a defined cellular microenvironment, the niche, which supports the generation and integration of newborn neurons. The mechanisms building a sophisticated niche structure around NSCs and their functional relevance for neurogenesis are yet to be understood. In the Drosophila larval brain, the cortex glia (CG) encase individual NSC lineages in membranous chambers, organising the stem cell population and newborn neurons into a stereotypic structure. We first found that CG wrap around lineage-related cells regardless of their identity, showing that lineage information builds CG architecture. We then discovered that a mechanism of temporally controlled differential adhesion using conserved complexes supports the individual encasing of NSC lineages. An intralineage adhesion through homophilic Neuroglian interactions provides strong binding between cells of a same lineage, while a weaker interaction through Neurexin-IV and Wrapper exists between NSC lineages and CG. Loss of Neuroglian results in NSC lineages clumped together and in an altered CG network, while loss of Neurexin-IV/Wrapper generates larger yet defined CG chamber grouping several lineages together. Axonal projections of newborn neurons are also altered in these conditions. Further, we link the loss of these 2 adhesion complexes specifically during development to locomotor hyperactivity in the resulting adults. Altogether, our findings identify a belt of adhesions building a neurogenic niche at the scale of individual stem cell and provide the proof of concept that niche properties during development shape adult behaviour

Intracellular clusterin causes juxtanuclear aggregate formation and mitochondrial alteration.

International audienceClusterin is a puzzling protein upregulated in many diseased tissues, presented as either a survival or a death protein. The role of clusterin might depend on the final maturation and localization of the protein, which can be secreted or reside inside cells, either after in situ synthesis or uptake of extracellular clusterin. We studied the biological effects of intracellular clusterin and observed that clusterin forms containing the alpha-chain region strongly accumulated in an ubiquitinated form in juxtanuclear aggregates meeting the main criterions of aggresomes and leading to profound alterations of the mitochondrial network. The viability of cells transfected by intracellular forms of clusterin was improved by overexpression of Bcl-2, and caspase inhibition was capable of rescuing cells expressing clusterin, which presented an altered mitochondrial permeability. We propose that, although it might be an inherently pro-survival and anti-apoptotic protein expressed by cells under stress in an attempt to protect themselves, clusterin can become highly cytotoxic when accumulated in the intracellular compartment. This activity might reconcile the opposite purported influences of clusterin on cell survival and explain how clusterin can be causally involved in neurodegeneration

TNF-a activates at least two apoptotic signaling cascades

International audienceApoptosis, the process whereby cells activate an intrinsic death program, can be induced in HeLa cells by TNF-a treatment. The aims of the present study were (i) to examine the precise role and the origin of Reactive Oxygen Species (ROS) in the TNF-a-induced programmed cell death, (ii) to characterize and order the morphological and mitochondrial changes associated with this process and (iii) to link these events with the activation of caspases. Analyses were performed on TNF-a-treated cells in the presence of an anti-oxidant, or of a general caspase inhibitor. To assess the role of mitochondria in the cell death signal transduction, these studies were also realized on HeLa-variant cell lines lacking functional mitochondrial respiratory chain. We show that at least two separate signaling cascades, both mediated by Z-VAD-sensitive caspase(s), contribute to the TNF-a-induced apoptosis of HeLa cells. One signaling pathway involves an early mitochondriadependent ROS production, the other being ROSindependent