Role of the Drosophila Pmi and human TMEM11 proteins in mitochondrial fusion/fission. – Mitopim

Notre projet repose sur deux points forts. Nous disposons d'un lignée de drosophile portant une délétion propre de notre gène d'intérêt. Son étude permet une étude physiologique des conséquences associées à une perte de fonction de ce gène. Par ailleurs, nous disposons de nombreuses lignées de drosophiles chez lesquelles les différents composants et domaines d la mitochondrie sont marqué avec un fluorochrome particulier. Grâce à la qualité des plateformes de microscopie dont nous disposons dans l'institut, nous pouvons suivre «live« les mécanismes de fission fusion des mitochondries et analyser les conséquences de la mutation PMI sur ces processus.

Principales avancées réalisées :
- Nous avons démontré que PMI et son homologue humain TMEM11 codent pour des protéines de la membrane interne des mitochondries et détermine la forme tubulaire de ces organelles.
- Nous avons démontré que PMI déterminent la forme des mitochondries par un mécanisme qui ne dépend pas des processus canoniques de fusion et de fission
- Nous avons démontré que PMI est nécessaire au fonctionnement de la chaine respiratoire des mitochondries. En son absence la production d’ATP est inhibée alors que la production de ROS augmente, expliquant les désordres neurologiques et la durée de vie réduites de drosophile mutante pour PMI.
- Nous nous attachons actuellement à démontrer que PMI est une protéine essentielle à la biogenèse des crêtes mitochondriale. Dans notre modèle de travail PMI déterminerait la longueur des crêtes mitochondriales et ainsi le diamètre des mitochondries expliquant pourquoi en l’absence de PMI les mitochondries perdent leur forme tubulaire et deviennent sphérique

Poursuivre les expériences proposées dans l'ANR

Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways. Rival T, Macchi M, Arnauné-Pelloquin L, Poidevin M, Maillet F, Richard F, Fatmi A, Belenguer P, Royet J. EMBO Rep. 2011 Mar;12(3):223-30.

Our laboratory is working on innate immune defense using Drosophila as a model system. Over the years, we have been unravelling the mechanisms by which Drosophila detect invading microbes. This led us to the first molecular identification and phenotypic characterization of a pattern recognition receptor in invertebrates, the Peptidoglycan Recognition Protein-SA. Further works by us and others, has revealed that PGRPs are key bacterial receptors and signaling modulators in both invertebrates and vertebrates immune response. Recently, we have been studying PGRP-LD. The molecular organization of the PGRP-LD locus is quite atypical. Like only some 20 other loci in the Drosophila genome, PGRP-LD mRNA is transcribed from a bi-cistronic locus that also codes for an uncharacterized protein named Pmi. We have generated Pmi-PGRP-LD double mutants by homologous recombination. Although Pmi-PGRP-LD mutant flies are hatching into phenotypically normal adults, they display a specific neurological phenotype within few days. In normal conditions, they exhibit reduced activity and their locomotion speed is decreased. Under stress conditions, when wild-type flies escape by flying away, mutants are left paralyzed with leg convulsions. This neurological phenotype known as « bang sensitive », reflects the inability of the mutants to mount a proper escape behavior that relies on precise activation of locomotor neuronal circuits. Mutants that display similar phenotypes (sesB, tko…) have been shown to carry mutations in genes coding for mitochondrial proteins. Consistently, we have shown that Pmi-PGRP-LD mutant cells contain giant mitochondria resembling those seen in mitochondria fission mutant drp-1. Importantly, we could show that ectopic expression of Pmi, but not of PGRP-LD, is sufficient, in vivo, to completely rescue these phenotypes. This demonstrates that the inactivation of Pmi and not of PGRP-LD is responsible for the enlarged mitochondria phenotype and the « bang sensitivity». Our results show that Pmi is a novel regulator of mitochondrial morphology. Mitochondrial shape and size are dependent on a balance between two opposed cellular mechanisms: fission and fusion. Both mechanisms implicate a set of Dynamin-related proteins that are evolutionary conserved from yeast to human. The phenotype observed in Pmi mutants suggests that the normal function of Pmi is either to inhibit fusion or to promote fission of mitochondria. Pmi could achieve this function by directly regulating core components of the fission/fusion machinery. Alternatively, it could control mitochondrial shaping via a totally independent mechanism. Our preliminary results indicate that, unexpectedly, Pmi influences mitochondria shape by a mechanism independent from the well-characterized fission/fusion pathways. The main focus of this work is to characterize the role of Pmi and its human ortholog (TMEM11) in mitochondria and to unravel the consequences of its inactivation on the cellular physiology. Since all Drosophila proteins encoded by a bi-cistronic loci that have been characterized so far, are implicated in a common biological process, It is likely that PGRP-LD and Pmi act together to regulate mitochondrial shape. Although this aspect does not constitute the main core of the proposal, we are proposing experiments aimed at studying the putative link between PGRP-LD and mitochondria and therefore between mitochondria function and immune response. The reaserch field studying the links between mitochondria and bacterial immune response has very recently emerged in vertebrates and is totally new in invertebrates.Given the remarkable evolutionary conservation of most fundamental cellular processes, we are confident that our studies on Pmi will be of significant relevance to an in-depth understanding of cellular antimicrobial defense strategies not only in Drosophila but also in other metazoan including humans