Characterization of actors and mechanisms of mitochondrial fusion

Les mitochondries sont des organites dynamiques qui fusionnent et se divisent continuellement. Cette dynamique est requise pour la biogenèse mitochondriale, la fonction et la dégradation. Les relations entre les OXPHOS, la dynamique et les mécanismes assurant la modulation de la dynamique restent largement inconnus. Nous avons étudié grâce à un essai de fusion in vivo, les relations entre la fusion et les OxPhos dans des cellules de levure portant des mutations ponctuelles dans le gène mitochondrial ATP6 qui sont associés à des maladies chez l’homme. Nous montrons que les défauts des OxPhos provoquent des défauts de fusion de la membrane interne mitochondriale mais pas de la membrane externe. L'inhibition sélective de la fusion de la membrane interne peut être mimée par les ionophores qui dissipent le potentiel de membrane interne, mais pas par des inhibiteurs des phosphorylations oxydatives. Nous montrons une inhibition dominante de fusion qui pourrait être un mécanisme d'exclusion des mitochondries dysfonctionnelles du réseau mitochondrial pour les adresser à la mitophagie. Ces résultats indiquent que les défauts de fusion pourraient contribuer à la pathologie des maladies provoquées par des mutations de l'ADNmt. De plus ces résultats impliquent que dans des cellules, l'inhibition de la fusion dominante pourrait permettre l'exclusion des mitochondries dysfonctionnelles du réseau mitochondrial. La fusion mitochondriale implique de nombreuses protéines de la superfamille des dynamines. Si ces protéines ont été identifiées, les mécanismes moléculaires permettant la fusion restent indéterminés. Dans le but de comprendre ces mécanismes, nous avons choisi de caractériser les protéines Mitofusine 1 et 2, essentielles à la fusion des membranes externes mitochondriales. Ces protéines sont composées de deux domaines coiled-coil et un domaine N-terminal GTPase et des domaines hydrophobes prédits pour être des segments transmembranaires. Après la détermination des activités GTPase des mitofusines, nous avons reconstitué les mitofusines ou des fragments des mitofusines dans des liposomes afin d'étudier leur capacité à fusionner ces liposomes. Les mitofusines, permettent la fusion des liposomes contenant des cardiolipides. Étonnamment, ces événements sont indépendants de la présence du GTP mais nécessitent Mg2+ dans la solution. En utilisant la microscopie électronique, nous montrons que les mitofusines 1 et 2 induisent une déformation des liposomes. Cette capacité permettant de créer localement des régions très courbes (et donc fusogènes) ouvre un nouvel angle pour comprendre les mécanismes moléculaires de la fusion mitochondriale.Mitochondria are dynamic organelles that continuously fuse and divide. This dynamic is required for mitochondrial biogenesis, function and degradation. The cross-talk between OXPHOS and dynamics and the mechanisms ensuring modulation of dynamics remain largely unraveled. We have investigated the relationship between fusion and OXPHOS in yeast cells carrying point mutations in the mitochondrial ATP6 gene that are associated to human diseases. We show that OXPHOS defects provoke severe defects of inner membrane, but not outer membrane fusion. Selective inhibition of inner membrane fusion can be recapitulated by ionophores that dissipate the inner membrane potential, but not by inhibitors of OXPHOS. We show a dominant inhibition of fusion that further provides a mechanism for the exclusion of defective mitochondria from the functional mitochondrial network, a pre-requisite for their selective targeting to mitophagy. These results suggest that defects of fusion could contribute to the pathology of diseases caused by mtDNA mutations. Moreover, these results imply that in cells, inhibition of dominant fusion could allow the exclusion of dysfunctional mitochondria mitochondrial network. Mitochondrial fusion involves many proteins of the superfamily of dynamin. If these proteins have been identified, the molecular mechanisms of fusion remain undetermined. In order to understand these mechanisms, we choose to characterize Mitofusin 1 and 2 proteins, essential for outer mitochondrial membrane fusion. These transmembrane proteins are consisting of two coiled-coil domains and one N-terminal GTPase domain. We have characterized GTPase activity of Mitofusin and reconstituted Mitofusins or fragments of Mitofusins into liposomes to study their capacity to fuse these liposomes. Full-length mitofusins can fuse liposomes containing cardiolipins. Surprisingly, these fusion events are independent of GTP but require Mg2+ in the buffer. Using electron microscopy, we show that mitofusin 1 and 2 induce local deformation of liposomes. This capacity of mitofusins to locally create highly curved (and thus fusogenic) membrane regions opens a new angle to understand the molecular mechanisms of mitochondrial fusion

Characterization of actors and mechanisms of mitochondrial fusion

Les mitochondries sont des organites dynamiques qui fusionnent et se divisent continuellement. Cette dynamique est requise pour la biogenèse mitochondriale, la fonction et la dégradation. Les relations entre les OXPHOS, la dynamique et les mécanismes assurant la modulation de la dynamique restent largement inconnus. Nous avons étudié grâce à un essai de fusion in vivo, les relations entre la fusion et les OxPhos dans des cellules de levure portant des mutations ponctuelles dans le gène mitochondrial ATP6 qui sont associés à des maladies chez l’homme. Nous montrons que les défauts des OxPhos provoquent des défauts de fusion de la membrane interne mitochondriale mais pas de la membrane externe. L'inhibition sélective de la fusion de la membrane interne peut être mimée par les ionophores qui dissipent le potentiel de membrane interne, mais pas par des inhibiteurs des phosphorylations oxydatives. Nous montrons une inhibition dominante de fusion qui pourrait être un mécanisme d'exclusion des mitochondries dysfonctionnelles du réseau mitochondrial pour les adresser à la mitophagie. Ces résultats indiquent que les défauts de fusion pourraient contribuer à la pathologie des maladies provoquées par des mutations de l'ADNmt. De plus ces résultats impliquent que dans des cellules, l'inhibition de la fusion dominante pourrait permettre l'exclusion des mitochondries dysfonctionnelles du réseau mitochondrial. La fusion mitochondriale implique de nombreuses protéines de la superfamille des dynamines. Si ces protéines ont été identifiées, les mécanismes moléculaires permettant la fusion restent indéterminés. Dans le but de comprendre ces mécanismes, nous avons choisi de caractériser les protéines Mitofusine 1 et 2, essentielles à la fusion des membranes externes mitochondriales. Ces protéines sont composées de deux domaines coiled-coil et un domaine N-terminal GTPase et des domaines hydrophobes prédits pour être des segments transmembranaires. Après la détermination des activités GTPase des mitofusines, nous avons reconstitué les mitofusines ou des fragments des mitofusines dans des liposomes afin d'étudier leur capacité à fusionner ces liposomes. Les mitofusines, permettent la fusion des liposomes contenant des cardiolipides. Étonnamment, ces événements sont indépendants de la présence du GTP mais nécessitent Mg2+ dans la solution. En utilisant la microscopie électronique, nous montrons que les mitofusines 1 et 2 induisent une déformation des liposomes. Cette capacité permettant de créer localement des régions très courbes (et donc fusogènes) ouvre un nouvel angle pour comprendre les mécanismes moléculaires de la fusion mitochondriale.Mitochondria are dynamic organelles that continuously fuse and divide. This dynamic is required for mitochondrial biogenesis, function and degradation. The cross-talk between OXPHOS and dynamics and the mechanisms ensuring modulation of dynamics remain largely unraveled. We have investigated the relationship between fusion and OXPHOS in yeast cells carrying point mutations in the mitochondrial ATP6 gene that are associated to human diseases. We show that OXPHOS defects provoke severe defects of inner membrane, but not outer membrane fusion. Selective inhibition of inner membrane fusion can be recapitulated by ionophores that dissipate the inner membrane potential, but not by inhibitors of OXPHOS. We show a dominant inhibition of fusion that further provides a mechanism for the exclusion of defective mitochondria from the functional mitochondrial network, a pre-requisite for their selective targeting to mitophagy. These results suggest that defects of fusion could contribute to the pathology of diseases caused by mtDNA mutations. Moreover, these results imply that in cells, inhibition of dominant fusion could allow the exclusion of dysfunctional mitochondria mitochondrial network. Mitochondrial fusion involves many proteins of the superfamily of dynamin. If these proteins have been identified, the molecular mechanisms of fusion remain undetermined. In order to understand these mechanisms, we choose to characterize Mitofusin 1 and 2 proteins, essential for outer mitochondrial membrane fusion. These transmembrane proteins are consisting of two coiled-coil domains and one N-terminal GTPase domain. We have characterized GTPase activity of Mitofusin and reconstituted Mitofusins or fragments of Mitofusins into liposomes to study their capacity to fuse these liposomes. Full-length mitofusins can fuse liposomes containing cardiolipins. Surprisingly, these fusion events are independent of GTP but require Mg2+ in the buffer. Using electron microscopy, we show that mitofusin 1 and 2 induce local deformation of liposomes. This capacity of mitofusins to locally create highly curved (and thus fusogenic) membrane regions opens a new angle to understand the molecular mechanisms of mitochondrial fusion

Dynamique et morphologie mitochondriales

Les mitochondries sont des organites dynamiques qui se déplacent, se divisent et fusionnent continuellement. L’équilibre fusion-fission détermine si elles forment, dans la cellule, des filaments interconnectés ou apparaissent comme une collection de structures ponctiformes indépendantes. Les machineries de fusion et fission sont conservées des levures aux mammifères et comprennent trois GTPases de la famille des dynamines : Dnm1/DRP1 (nomenclature levure/ homme pour dynamin-related protein), impliquée dans la fission, et Fzo1/MFN (mitofusine) et Mgm1/OPA1 (optic atrophy 1), requises pour la fusion. Alors que l’identification et la caractérisation des acteurs de la dynamique mitochondriale, de leur mécanisme d’action, de leurs fonctions et de leur régulation continuent à être l’objet de nombreuses recherches, la pertinence de ce processus est attestée par son rôle dans le fonctionnement mitochondrial, la survie cellulaire, le développement embryonnaire et son implication dans des maladies neurologiques

Ezrin activation by LOK phosphorylation involves a PIP2-dependent wedge mechanism

How cells specify morphologically distinct plasma membrane domains is poorly understood. Prior work has shown that restriction of microvilli to the apical aspect of epithelial cells requires the localized activation of the membrane-F-actin linking protein ezrin. Using an in vitro system, we now define a multi-step process whereby the kinase LOK specifically phosphorylates ezrin to activate it. Binding of PIP2 to ezrin induces a conformational change permitting the insertion of the LOK C-terminal domain to wedge apart the membrane and F-actin-binding domains of ezrin. The N-terminal LOK kinase domain can then access a site 40 residues distal from the consensus sequence that collectively direct phosphorylation of the appropriate threonine residue. We suggest that this elaborate mechanism ensures that ezrin is only phosphorylated at the plasma membrane, and with high specificity by the apically localized kinase LOK. DOI: http://dx.doi.org/10.7554/eLife.22759.00

Escherichia coli and colorectal carcinogenesis: interactions with intestinal mucosa and role of Ceacam6 overexpression

International audienc

The heptad repeat domain 1 of Mitofusin has membrane destabilization function in mitochondrial fusion

International audienceMitochondria are double-membrane-bound organelles that constantly change shape through membrane fusion and fission. Outer mitochondrial membrane fusion is controlled by Mitofusin, whose molecular architecture consists of an N-terminal GTPase domain, a first heptad repeat domain (HR1), two transmembrane domains, and a second heptad repeat domain (HR2). The mode of action of Mitofusin and the specific roles played by each of these functional domains in mitochondrial fusion are not fully understood. Here, using a combination of in situ and in vitro fusion assays, we show that HR1 induces membrane fusion and possesses a conserved amphipathic helix that folds upon interaction with the lipid bilayer surface. Our results strongly suggest that HR1 facilitates membrane fusion by destabilizing the lipid bilayer structure, notably in membrane regions presenting lipid packing defects. This mechanism for fusion is thus distinct from that described for the heptad repeat domains of SNARE and viral proteins, which assemble as membrane-bridging complexes, triggering close membrane apposition and fusion, and is more closely related to that of the C-terminal amphipathic tail of the Atlastin protein

LdFlabarin, a new BAR domain membrane protein of Leishmania flagellum.

International audienceDuring the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein

Morphological control enables nanometer-scale dissection of cell-cell signaling complexes

International audienceProtein micropatterning enables robust control of cell positioning on electron-microscopy substrates for cryogenic electron tomography (cryo-ET). However, the combination of regulated cell boundaries and the underlying electron-microscopy substrate (EM-grids) provides a poorly understood microenvironment for cell biology. Because substrate stiffness and morphology affect cellular behavior, we devised protocols to characterize the nanometer-scale details of the protein micropatterns on EM-grids by combining cryo-ET, atomic force microscopy, and scanning electron microscopy. Measuring force displacement characteristics of holey carbon EM-grids, we found that their effective spring constant is similar to physiological values expected from skin tissues. Despite their apparent smoothness at light-microscopy resolution, spatial boundaries of the protein micropatterns are irregular at nanometer scale. Our protein micropatterning workflow provides the means to steer both positioning and morphology of cell doublets to determine nanometer details of punctate adherens junctions. Our workflow serves as the foundation for studying the fundamental structural changes governing cell-cell signaling

Characterization of heterogeneity in nanodisc samples using Feret signatures

International audienceNanodiscs have become a popular tool in structure determination of membrane proteins using cryogenic electron microscopy and single particle analysis. However, the structure determination of small membrane proteins remains challenging. When the embedded protein is in the same size range as the nanodisc, the nanodisc can significantly contribute to the alignment and classification during the structure determination process. In those cases, it is crucial to minimize the heterogeneity in the nanodisc preparations to assure maximum accuracy in the classification and alignment steps of single particle analysis. Here, we introduce a new in-silico method for the characterization of nanodisc samples that is based on analyzing the Feret diameter distribution of their particle projection as imaged in the electron microscope. We validated the method with comprehensive simulation studies and show that Feret signatures can detect subtle differences in nanodisc morphologies and composition that might otherwise go unnoticed. We used the method to identify a specific biochemical nanodisc preparation with low size variations, allowing us to obtain a structure of the 23-kDa single-span membrane protein Bcl-xL while embedded in a nanodisc. Feret signature analysis can steer experimental data collection strategies, allowing more efficient use of high-end data collection hardware, as well as image analysis investments in studies where nanodiscs significantly contribute to the total volume of the full molecular species

Mitochondrial DNA Mutations Provoke Dominant Inhibition of Mitochondrial Inner Membrane Fusion

<div><p>Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA). We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS) due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial <em>ATP6</em> gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP) or to maternally inherited Leigh Syndrome (MILS) in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from the network of functional, fusogenic mitochondria.</p> </div