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Publications Départements Biologie Cellulaire


  • I. Altinoglu, C. J. Merrifield, et Y. Yamaichi, « Single molecule super-resolution imaging of bacterial cell pole proteins with high-throughput quantitative analysis pipeline », Scientific Reports, vol. 9, nᵒ 1, p. 6680, avr. 2019.
    Résumé : Bacteria show sophisticated control of their cellular organization, and many bacteria deploy different polar landmark proteins to organize the cell pole. Super-resolution microscopy, such as Photo-Activated Localization Microscopy (PALM), provides the nanoscale localization of molecules and is crucial for better understanding of organization and dynamics in single-molecule. However, analytical tools are not fully available yet, in particular for bacterial cell biology. For example, quantitative and statistical analyses of subcellular localization with multiple cells from multiple fields of view are lacking. Furthermore, brightfield images are not sufficient to get accurate contours of small and low contrast bacterial cells, compared to subpixel presentation of target molecules. Here we describe a novel analytic tool for PALM which integrates precisely drawn cell outlines, of either inner membrane or periplasm, labelled by PALM-compatible fluorescent protein fusions, with molecule data for >10,000 molecules from >100 cells by fitting each cell into an oval arc. In the vibrioid bacterium Vibrio cholerae, the polar anchor HubP constitutes a big polar complex which includes multiple proteins involved in chemotaxis and the flagellum. With this pipeline, HubP is shown to be slightly skewed towards the inner curvature side of the cell, while its interaction partners showed rather loose polar localization.
    Mots-clés : BIOCELL, DBG, EQYY.

  • E. M. Bayer, T. Calì, F. Giordano, A. Hamacher-Brady, et L. Pellegrini, « EMBO Workshop: Membrane Contact Sites in Health and Disease », Contact, vol. 2, p. 2515256419825931, janv. 2019.

  • E. de Langre, O. Penalver, P. Hémon, J. - M. Frachisse, M. _B. Bogeat-Triboulot, J. - M. Frachisse, M. - B. Bogeat-Triboulot, B. Niez, E. Badel, et B. Moulia, « Nondestructive and Fast Vibration Phenotyping of Plants », Plant Phenomics, vol. 2019, 2019.
    Résumé : The frequencies of free oscillations of plants, or plant parts, depend on their geometries, stiffnesses, and masses. Besides direct biomechanical interest, free frequencies also provide insights into plant properties that can usually only be measured destructively or with low-throughput techniques (e.g., change in mass, tissue density, or stiffness over development or with stresses). We propose here a new high-throughput method based on the noncontact measurements of the free frequencies of the standing plant. The plant is excited by short air pulses (typically 100 ms). The resulting motion is recorded by a high speed video camera (100 fps) and processed using fast space and time correlation algorithms. In less than a minute the mechanical behavior of the plant is tested over several directions. The performance and versatility of this method has been tested in three contrasted species: tobacco (Nicotiana benthamian), wheat (Triticum aestivum L.), and poplar (Populus sp.), for a total of more than 4000 data points. In tobacco we show that water stress decreased the free frequency by 15%. In wheat we could detect variations of less than 1 g in the mass of spikes. In poplar we could measure frequencies of both the whole stem and leaves. The work provides insight into new potential directions for development of phenotyping.
    Mots-clés : BIOCELL, MINION.

  • M. Esposito, S. Hermann-Le Denmat, et A. Delahodde, « Contribution of ERMES subunits to mature peroxisome abundance », PloS One, vol. 14, nᵒ 3, p. e0214287, 2019.
    Résumé : Eukaryotic organelles share different components and establish physical contacts to communicate throughout the cell. One of the best-recognized examples of such interplay is the metabolic cooperation and crosstalk between mitochondria and peroxisomes, both organelles being functionally and physically connected and linked to the endoplasmic reticulum (ER). In Saccharomyces cerevisiae, mitochondria are linked to the ER by the ERMES complex that facilitates inter-organelle calcium and phospholipid exchanges. Recently, peroxisome-mitochondria contact sites (PerMit) have been reported and among Permit tethers, one component of the ERMES complex (Mdm34) was shown to interact with the peroxin Pex11, suggesting that the ERMES complex or part of it may be involved in two membrane contact sites (ER-mitochondria and peroxisome- mitochondria). This opens the possibility of exchanges between these three membrane compartments. Here, we investigated in details the role of each ERMES subunit on peroxisome abundance. First, we confirmed previous studies from other groups showing that absence of Mdm10 or Mdm12 leads to an increased number of mature peroxisomes. Secondly, we showed that this is not simply due to respiratory function defect, mitochondrial DNA (mtDNA) loss or mitochondrial network alteration. Finally, we present evidence that the contribution of ERMES subunits Mdm10 and Mdm12 to peroxisome number involves two different mechanisms.
    Mots-clés : BIOCELL, biogenesis, complex, dna, endoplasmic-reticulum, er, FDMITO, inheritance, mitochondrial morphology, outer-membrane protein, smp domains, yeast.

  • S. Gervason, D. Larkem, A. B. Mansour, T. Botzanowski, C. S. Müller, L. Pecqueur, G. Le Pavec, A. Delaunay-Moisan, O. Brun, J. Agramunt, A. Grandas, M. Fontecave, V. Schünemann, S. Cianférani, C. Sizun, M. B. Tolédano, et B. D'Autréaux, « Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide-processing functions of ferredoxin-2 and frataxin », Nature Communications, vol. 10, nᵒ 1, p. 3566, août 2019.
    Résumé : Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies.
    Mots-clés : BIOCELL, SOC.
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  • M. Guichard, J. - M. Allain, M. Wolfe Bianchi, et J. - M. Frachisse, « Root Hair Sizer: an algorithm for high throughput recovery of different root hair and root developmental parameters », Plant Methods, vol. 15, nᵒ 1, p. 104, 2019.
    Résumé : Background: The root is an important organ for water and nutrient uptake, and soil anchorage. It is equipped with root hairs (RHs) which are elongated structures increasing the exchange surface with the soil. RHs are also studied as a model for plant cellular development, as they represent a single cell with specific and highly regulated polarized elongation. For these reasons, it is useful to be able to accurately quantify RH length employing standardized procedures. Methods commonly employed rely on manual steps and are therefore time consuming and prone to errors, restricting analysis to a short segment of the root tip. Few partially automated methods have been reported to increase measurement efficiency. However, none of the reported methods allow an accurate and standardized definition of the position along the root for RH length measurement, making data comparison difficult. Results: We developed an image analysis algorithm that semi-automatically detects RHs and measures their length along the whole differentiation zone of roots. This method, implemented as a simple automated script in ImageJ/Fiji software that we termed Root Hair Sizer, slides a rectangular window along a binarized and straightened image of root tips to estimate the maximal RH length in a given measuring interval. This measure is not affected by heavily bent RHs and any bald spots. RH length data along the root are then modelled with a sigmoidal curve, generating several biologically significant parameters such as RH length, positioning of the root differentiation zone and, under certain conditions, RH growth rate. Conclusions: Image analysis with Root Hair Sizer and subsequent sigmoidal modelling of RH length data provide a simple and efficient way to characterize RH growth in different conditions, equally suitable to small and large scale phenotyping experiments.
    Mots-clés : BIOCELL, Cell elongation, Image analysis, Medicago truncatula, MINION, Phenotyping, Root, Root hair.
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  • A. M. Hartley, N. Lukoyanova, Y. Zhang, A. Cabrera-Orefice, S. Arnold, B. Meunier, N. Pinotsis, et A. Maréchal, « Structure of yeast cytochrome c oxidase in a supercomplex with cytochrome bc1 », Nature Structural & Molecular Biology, vol. 26, nᵒ 1, p. 78-83, janv. 2019.
    Résumé : Cytochrome c oxidase (complex IV, CIV) is known in mammals to exist independently or in association with other respiratory proteins to form supercomplexes (SCs). In Saccharomyces cerevisiae, CIV is found solely in an SC with cytochrome bc1 (complex III, CIII). Here, we present the cryogenic electron microscopy (cryo-EM) structure of S. cerevisiae CIV in a III2IV2 SC at 3.3 Å resolution. While overall similarity to mammalian homologs is high, we found notable differences in the supernumerary subunits Cox26 and Cox13; the latter exhibits a unique arrangement that precludes CIV dimerization as seen in bovine. A conformational shift in the matrix domain of Cox5A-involved in allosteric inhibition by ATP-may arise from its association with CIII. The CIII-CIV arrangement highlights a conserved interaction interface of CIII, albeit one occupied by complex I in mammalian respirasomes. We discuss our findings in the context of the potential impact of SC formation on CIV regulation.
    Mots-clés : architecture, BIOCELL, BIOMIT, isoforms, kinetics, mechanisms, mitochondrial complex, phosphorylation, purification, respiration, subunit, system.

  • C. Jenzer, E. Simionato, C. Largeau, V. Scarcelli, C. Lefebvre, et R. Legouis, « Autophagy mediates phosphatidylserine exposure and phagosome degradation during apoptosis through specific functions of GABARAP/LGG-1 and LC3/LGG-2 », Autophagy, vol. 15, nᵒ 2, p. 228-241, févr. 2019.
    Résumé : Phagocytosis and macroautophagy/autophagy are 2 processes involved in lysosome-mediated clearance of extracellular and intracellular components, respectively. Recent studies have identified the recruitment of the autophagic protein LC3 during phagocytosis of apoptotic corpses in what is now called LC3-associated phagocytosis (LAP). LAP is a distinct process from autophagy but it relies on some members of the autophagy pathway to allow efficient degradation of the phagocytosed cargo. We investigated whether both LC3/LGG-2 and GABARAP/LGG-1 are involved in phagocytosis of apoptotic corpses during embryonic development of Caenorhabditis elegans. We discovered that both LGG-1 and LGG-2 are involved in the correct elimination of apoptotic corpses, but that they have different functions. lgg-1 and lgg-2 mutants present a delay in phagocytosis of apoptotic cells but genetic analyses indicate that LGG-1 and LGG-2 act upstream and downstream of the engulfment pathways, respectively. Moreover, LGG-1 and LGG-2 display different cellular localizations with enrichment in apoptotic corpses and phagocytic cells, respectively. For both LGG-1 and LGG-2, subcellular localization is vesicular and dependent on UNC-51/ULK1, BEC-1/BECN1 and the lipidation machinery, indicating that their functions during phagocytosis of apoptotic corpses mainly rely on autophagy. Finally, we show that LGG-1 is involved in the exposure of the 'eat-me signal' phosphatidylserine at the surface of the apoptotic cell to allow its recognition by the phagocytic cell, whereas LGG-2 is involved in later steps of phagocytosis to allow efficient cell corpse clearance by mediating the maturation/degradation of the phagosome.
    Mots-clés : Apoptosis, BIOCELL, Caenorhabditis elegans, LAP process, LAP process, OTOFAG, VPS-39.

  • S. Jiang, C. Koolmeister, J. Misic, S. Siira, I. Kühl, E. S. Ramos, M. Miranda, M. Jiang, V. Posse, O. Lytovchenko, I. Atanassov, F. A. Schober, R. Wibom, K. Hultenby, D. Milenkovic, C. M. Gustafsson, A. Filipovska, et N. - G. Larsson, « TEFM regulates both transcription elongation and RNA processing in mitochondria », EMBO reports, vol. 20, nᵒ 6, p. e48101, avr. 2019.
    Résumé : Regulation of replication and expression of mitochondrial DNA (mtDNA) is essential for cellular energy conversion via oxidative phosphorylation. The mitochondrial transcription elongation factor (TEFM) has been proposed to regulate the switch between transcription termination for replication primer formation and processive, near genome-length transcription for mtDNA gene expression. Here, we report that Tefm is essential for mouse embryogenesis and that levels of promoter-distal mitochondrial transcripts are drastically reduced in conditional Tefm-knockout hearts. In contrast, the promoter-proximal transcripts are much increased in Tefm knockout mice, but they mostly terminate before the region where the switch from transcription to replication occurs, and consequently, de novo mtDNA replication is profoundly reduced. Unexpectedly, deep sequencing of RNA from Tefm knockouts revealed accumulation of unprocessed transcripts in addition to defective transcription elongation. Furthermore, a proximity-labeling (BioID) assay showed that TEFM interacts with multiple RNA processing factors. Our data demonstrate that TEFM acts as a general transcription elongation factor, necessary for both gene transcription and replication primer formation, and loss of TEFM affects RNA processing in mammalian mitochondria.
    Mots-clés : BIOCELL, BIOMIT, dna-replication, granules, hybrid, identification, maintenance, mtDNA replication, polymerase, ribosomal-protein l12, RNA processing, site, stability, transcription elongation, translation.

  • K. Keatley, S. Stromei-Cleroux, T. Wiltshire, N. Rajala, G. Burton, W. V. Holt, D. T. J. Littlewood, A. G. Briscoe, J. Jung, K. Ashkan, S. J. Heales, G. J. Pilkington, B. Meunier, J. E. McGeehan, I. P. Hargreaves, et R. E. McGeehan, « Integrated Approach Reveals Role of Mitochondrial Germ-Line Mutation F18L in Respiratory Chain, Oxidative Alterations, Drug Sensitivity, and Patient Prognosis in Glioblastoma », International Journal of Molecular Sciences, vol. 20, nᵒ 13, juill. 2019.
    Résumé : Glioblastoma is the most common and malignant primary brain tumour in adults, with a dismal prognosis. This is partly due to considerable inter- and intra-tumour heterogeneity. Changes in the cellular energy-producing mitochondrial respiratory chain complex (MRC) activities are a hallmark of glioblastoma relative to the normal brain, and associate with differential survival outcomes. Targeting MRC complexes with drugs can also facilitate anti-glioblastoma activity. Whether mutations in the mitochondrial DNA (mtDNA) that encode several components of the MRC contribute to these phenomena remains underexplored. We identified a germ-line mtDNA mutation (m. 14798T > C), enriched in glioblastoma relative to healthy controls, that causes an amino acid substitution F18L within the core mtDNA-encoded cytochrome b subunit of MRC complex III. F18L is predicted to alter corresponding complex III activity, and sensitivity to complex III-targeting drugs. This could in turn alter reactive oxygen species (ROS) production, cell behaviour and, consequently, patient outcomes. Here we show that, despite a heterogeneous mitochondrial background in adult glioblastoma patient biopsy-derived cell cultures, the F18L substitution associates with alterations in individual MRC complex activities, in particular a 75% increase in MRC complex II_III activity, and a 34% reduction in CoQ10, the natural substrate for MRC complex III, levels. Downstream characterisation of an F18L-carrier revealed an 87% increase in intra-cellular ROS, an altered cellular distribution of mitochondrial-specific ROS, and a 64% increased sensitivity to clomipramine, a repurposed MRC complex III-targeting drug. In patients, F18L-carriers that received the current standard of care treatment had a poorer prognosis than non-carriers (373 days vs. 415 days, respectively). Single germ-line mitochondrial mutations could predispose individuals to differential prognoses, and sensitivity to mitochondrial targeted drugs. Thus, F18L, which is present in blood could serve as a useful non-invasive biomarker for the stratification of patients into prognostically relevant groups, one of which requires a lower dose of clomipramine to achieve clinical effect, thus minimising side-effects.
    Mots-clés : BIOCELL, BIOMIT, clomipramine, CoQ10, glioblastoma, mitochondria, mtDNA, mutation, OXPHOS, prognosis, ROS.
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  • C. Largeau, E. Culetto, et R. Legouis, « Subcellular Localization of ESCRT-II in the Nematode C. elegans by Correlative Light Electron Microscopy », Methods in Molecular Biology (Clifton, N.J.), vol. 1998, p. 49-61, 2019.
    Résumé : In this chapter, we report a protocol to perform correlative light electron microscopy (CLEM) on adult Caenorhabditis elegans. We use a specific fixation protocol, which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins and, subsequently, with transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows determining the subcellular localization of the fluorescent protein.We used CLEM to characterize the subcellular localization of the C. elegans ESCRT-II component VPS-36. VPS-36 protein localization in C. elegans muscle cell is strongly correlated with the sarcoplasmic reticulum network. Together with genetic evidences, the CLEM data support a role for ESCRT-II proteins in sarcoplasmic reticulum membrane shaping.
    Mots-clés : BIOCELL, Electron microscopy, ESCRT-II, Freeze substitution, GMA resin, Green fluorescent protein, High pressure freezing, Muscle, OTOFAG, Sarcoplasmic reticulum, VPS-36.

  • C. Largeau et R. Legouis, « Correlative Light and Electron Microscopy to Analyze LC3 Proteins in Caenorhabditis elegans Embryo », Methods in Molecular Biology (Clifton, N.J.), vol. 1880, p. 281-293, 2019.
    Résumé : In this chapter, we present a protocol to perform correlative light and electron microscopy (CLEM) on Caenorhabditis elegans embryos. We use a specific fixation method which preserves both the GFP fluorescence and the structural integrity of the samples. Thin sections are first analyzed by light microscopy to detect GFP-tagged proteins, then by transmission electron microscopy (TEM) to characterize the ultrastructural anatomy of cells. The superimposition of light and electron images allows to determine the subcellular localization of the fluorescent protein. We have used this method to characterize the roles of autophagy in the phagocytosis of apoptotic cells in C. elegans embryos. We analyzed in apoptotic cell and phagocytic cell the localization of the two homologs of LC3/GABARAP proteins, namely, LGG-1 and LGG-2.
    Mots-clés : BIOCELL, Freeze substitution, GMA resin, Green fluorescent protein, High-pressure freezing, LC3-associated phagocytosis, LGG-1, LGG-2, OTOFAG.

  • R. Le Bars, M. W. Bianchi, et C. Lefebvre, « Three-Dimensional Surface Rendering of ESCRT Proteins Microscopy Data Using UCSF Chimera Software », Methods in Molecular Biology (Clifton, N.J.), vol. 1998, p. 149-161, 2019.
    Résumé : Visualization of subcellular localization of ESCRT proteins and their interactions with different cellular compartments are critical to understand their function. This approach requires the generation of an important amount of 3D fluorescence microscopy data that is not always easy to visualize and apprehend.We describe a step-by-step protocol for 3D surface rendering of confocal microscopy acquisitions using the free software UCSF-Chimera, generating snapshots and animations to facilitate analysis and presentation of subcellular localization data.
    Mots-clés : 3D animation, 3D fluorescence confocal microscopy, BIOCELL, ESCRT, MINION, OTOFAG, PF, PHOT, Surface rendering, UCSF Chimera.

  • A. Malkamäki, B. Meunier, M. Reidelbach, P. R. Rich, et V. Sharma, « The H channel is not a proton transfer path in yeast cytochrome c oxidase », Biochimica Et Biophysica Acta. Bioenergetics, vol. 1860, nᵒ 9, p. 717-723, juill. 2019.
    Résumé : Cytochrome c oxidases (CcOs) in the respiratory chains of mitochondria and bacteria are primary consumers of molecular oxygen, converting it to water with the concomitant pumping of protons across the membrane to establish a proton electrochemical gradient. Despite a relatively well understood proton pumping mechanism of bacterial CcOs, the role of the H channel in mitochondrial forms of CcO remains debated. Here, we used site-directed mutagenesis to modify a central residue of the lower span of the H channel, Q413, in the genetically tractable yeast Saccharomyces cerevisiae. Exchange of Q413 to several different amino acids showed no effect on rates and efficiencies of respiratory cell growth, and redox potential measurements indicated minimal electrostatic interaction between the 413 locus and the nearest redox active component heme a. These findings clearly exclude a primary role of this section of the H channel in proton pumping in yeast CcO. In agreement with the experimental data, atomistic molecular dynamics simulations and continuum electrostatic calculations on wildtype and mutant yeast CcOs highlight potential bottlenecks in proton transfer through this route. Our data highlight the preference for neutral residues in the 413 locus, precluding sufficient hydration for formation of a proton conducting wire.
    Mots-clés : BIOCELL, BIOMIT, Cell respiration, Electron transfer, MD simulations, Mitochondria, Proton pumping.

  • J. Morizet, G. Ducourthial, W. Supatto, A. Boutillon, R. Legouis, M. - C. Schanne-Klein, C. Stringari, et E. Beaurepaire, « High-speed polarization-resolved third-harmonic microscopy », Optica, vol. 6, nᵒ 3, p. 385-388, mars 2019.
    Résumé : Polarization-resolved third-harmonic generation (P-THG) is a sensitive probe of material anisotropy and molecular ordering. Despite its promises, this property has little been used in biological tissues due to the lack of measurement schemes compatible with dynamic samples. We report here on the development of a fast P-THG microscope where excitation polarization is switched between line scans using an electro-optic modulator, providing temporal resolution in the 10 ms range for the polarimetric measurement. We demonstrate novel applications enabled by this approach, associated with Fourier-based analysis: probing molecular order in deforming lipid structures undergoing phase transition; revealing crystallinity of flowing particles in the zebrafish embryo's inner ear; and detecting birefringence in vivo. These results establish that P-THG is ideally suited for probing lipid organization and mineralization in dynamic biological environments. (C) 2019 Optical Society of America under the terms of the OSA Open Access Publishing Agreement
    Mots-clés : BIOCELL, cells, modulated 2nd-harmonic generation, OTOFAG.

  • P. Mounkoro, T. Michel, R. Benhachemi, G. Surpateanu, B. I. Iorga, N. Fisher, et B. Meunier, « Mitochondrial complex III Qi -site inhibitor resistance mutations found in laboratory selected mutants and field isolates », Pest Management Science, vol. 75, nᵒ 8, p. 2107-2114, août 2019.
    Résumé : BACKGROUND: Complex III inhibitors targeting the Qi -site have been known for decades; some are used or being developed as antimicrobial compounds. Target site resistance mutations have been reported in laboratory-selected mutants and in field isolates. Here, we present a brief overview of mutations found in laboratory-selected resistant mutants. We also provide a study of mutations observed in field isolates of Plasmopara viticola, in particular the ametoctradin resistance substitution, S34L that we analysed in the yeast model. RESULTS: A survey of laboratory mutants showed that resistance could be caused by a large number of substitutions in the Qi -site. Four residues seemed key in term of resistance: N31, G37, L198 and K228. Using yeast, we analysed the effect of the ametoctradin resistance substitution S34L reported in field isolates of P. viticola. We showed that S34L caused a high level of resistance combined with a loss of complex III activity and growth competence. CONCLUSION: Use of single site Qi -site inhibitors is expected to result in the selection of resistant mutants. However, if the substitution is associated with a fitness penalty, as may be the case with S34L, resistance development might not be an insuperable obstacle, although careful monitoring is required. © 2018 Society of Chemical Industry.
    Mots-clés : bc1 complex, BIOCELL, BIOMIT, fungicides, QiI, resistance, target site mutation, yeast model.

  • P. Mounkoro, T. Michel, S. Blandin, M. - P. Golinelli-Cohen, E. Davioud-Charvet, et B. Meunier, « Investigating the mode of action of the redox-active antimalarial drug plasmodione using the yeast model », Free Radical Biology & Medicine, vol. 141, p. 269-278, juin 2019.
    Résumé : Malaria is caused by protozoan parasites and remains a major public health issue in subtropical areas. Plasmodione (3-[4-(trifluoromethyl)benzyl]-menadione) is a novel early lead compound displaying fast-acting antimalarial activity. Treatment with this redox active compound disrupts the redox balance of parasite-infected red blood cells. In vitro, the benzoyl analogue of plasmodione can act as a subversive substrate of the parasite flavoprotein NADPH-dependent glutathione reductase, initiating a redox cycling process producing ROS. Whether this is also true in vivo remains to be investigated. Here, we used the yeast model to investigate the mode of action of plasmodione and uncover enzymes and pathways involved in its activity. We showed that plasmodione is a potent inhibitor of yeast respiratory growth, that in drug-treated cells, the ROS-sensitive aconitase was impaired and that cells with a lower oxidative stress defence were highly sensitive to the drug, indicating that plasmodione may act via an oxidative stress. We found that the mitochondrial respiratory chain flavoprotein NADH-dehydrogenases play a key role in plasmodione activity. Plasmodione and metabolites act as substrates of these enzymes, the reaction resulting in ROS production. This in turn would damage ROS-sensitive enzymes leading to growth arrest. Our data further suggest that plasmodione is a pro-drug whose activity is mainly mediated by its benzhydrol and benzoyl metabolites. Our results in yeast are coherent with existing data obtained in vitro and in Plasmodium falciparum, and provide additional hypotheses that should be investigated in parasites.
    Mots-clés : Antimalarial drug, BIOCELL, BIOMIT, Drug mode of action, Mitochondrial respiratory chain, Oxidative stress, Yeast model.

  • M. Perez, Y. Guerringue, B. Ranty, C. Pouzet, A. Jauneau, E. Robe, C. Mazars, J. P. Galaud, et D. Aldon, « Specific TCP transcription factors interact with and stabilize PRR2 within different nuclear sub-domains », Plant Science: An International Journal of Experimental Plant Biology, vol. 287, p. 110197, oct. 2019.
    Résumé : Plants possess a large set of transcription factors both involved in the control of plant development or in plant stress responses coordination. We previously identified PRR2, a Pseudo-Response Regulator, as a plant-specific CML-interacting partner. We reported that PRR2 acts as a positive actor of plant defense by regulating the production of antimicrobial compounds. Here, we report new data on the interaction between PRR2 and transcription factors belonging to the Teosinte branched Cycloidea and PCF (TCP) family. TCPs have been described to be involved in plant development and immunity. We evaluated the ability of PRR2 to interact with seven TCPs representative of the different subclades of the family. PRR2 is able to interact with TCP13, TCP15, TCP19 and TCP20 in yeast two-hybrid system and in planta interactions were validated for TCP19 and TCP20. Transient expression in tobacco highlighted that PRR2 protein is more easily detected when co-expressed with TCP19 or TC20. This stabilization is associated with a specific sub-nuclear localization of the complex in Cajal bodies or in nuclear speckles according to the interaction of PRR2 with TCP19 or TCP20 respectively. The interaction between PRR2 and TCP19 or TCP20 would contribute to the biological function in specific nuclear compartments.
    Mots-clés : Arabidopsis thaliana, BIOCELL, FRET-FLIM, MINION, Nuclear localization, Protein stabilization, Protein-protein interaction, Pseudo-response regulator, TCP, Transcription factor.

  • Y. Pillon, H. Randriambanona, D. Alphonso Gonzales, P. P. Lowry II, T. Jaffré, et S. Merlot, « Parallel ecological filtering of ultramafic soils in three distant island floras », Journal of Biogeography, 2019.

  • M. Pottier, J. Dumont, C. Masclaux-Daubresse, et S. Thomine, « Autophagy is essential for optimal translocation of iron to seeds in Arabidopsis », Journal of Experimental Botany, vol. 70, nᵒ 3, p. 859-869, févr. 2019.
    Résumé : Autophagy is an essential recycling mechanism making micronutrients available in vegetative organs for subsequent reallocation to seeds
    Mots-clés : BIOCELL, MINION.
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  • E. S. Ramos, E. Motori, C. Brüser, I. Kühl, A. Yeroslaviz, B. Ruzzenente, J. H. K. Kauppila, J. D. Busch, K. Hultenby, B. H. Habermann, S. Jakobs, N. - G. Larsson, et A. Mourier, « Mitochondrial fusion is required for regulation of mitochondrial DNA replication », PLOS Genetics, vol. 15, nᵒ 6, p. e1008085, 2019.
    Résumé : Mitochondrial dynamics is an essential physiological process controlling mitochondrial content mixing and mobility to ensure proper function and localization of mitochondria at intracellular sites of high-energy demand. Intriguingly, for yet unknown reasons, severe impairment of mitochondrial fusion drastically affects mtDNA copy number. To decipher the link between mitochondrial dynamics and mtDNA maintenance, we studied mouse embryonic fibroblasts (MEFs) and mouse cardiomyocytes with disruption of mitochondrial fusion. Super-resolution microscopy revealed that loss of outer mitochondrial membrane (OMM) fusion, but not inner mitochondrial membrane (IMM) fusion, leads to nucleoid clustering. Remarkably, fluorescence in situ hybridization (FISH), bromouridine labeling in MEFs and assessment of mitochondrial transcription in tissue homogenates revealed that abolished OMM fusion does not affect transcription. Furthermore, the profound mtDNA depletion in mouse hearts lacking OMM fusion is not caused by defective integrity or increased mutagenesis of mtDNA, but instead we show that mitochondrial fusion is necessary to maintain the stoichiometry of the protein components of the mtDNA replisome. OMM fusion is necessary for proliferating MEFs to recover from mtDNA depletion and for the marked increase of mtDNA copy number during postnatal heart development. Our findings thus link OMM fusion to replication and distribution of mtDNA.
    Mots-clés : BIOCELL, BIOMIT, Confocal microscopy, DNA replication, Heart, Membrane fusion, Mitochondria, Mitochondrial DNA, Point mutation, Southern blot.
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  • L. Scorrano, M. A. De Matteis, S. Emr, F. Giordano, G. Hajnóczky, B. Kornmann, L. L. Lackner, T. P. Levine, L. Pellegrini, K. Reinisch, R. Rizzuto, T. Simmen, H. Stenmark, C. Ungermann, et M. Schuldiner, « Coming together to define membrane contact sites », Nature Communications, vol. 10, nᵒ 1, p. 1287, mars 2019.
    Résumé : Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research.
    Mots-clés : BIOCELL, COAST, er-mitochondria, high-capacity, imaging reveals, lipid-synthesis, mitochondria-associated membranes, plasma-membrane, proteomic analysis, rat-liver, rough endoplasmic-reticulum, saccharomyces-cerevisiae.

  • C. H. Sellem, A. Humbert, et A. Sainsard-Chanet, « Mutations in the phosphatase domain of the 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase result in the transcriptional activation of the alternative oxidase and gluconeogenic pathways in Podospora anserina », Fungal Genetics and Biology, vol. 130, p. 1-10, sept. 2019.
    Résumé : By screening suppressors of a respiratory mutant lacking a functional cytochrome pathway in the filamentous fungus Podospora anserina, we isolated a mutation located in the phosphatase domain of the bi-functional enzyme 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2). We show that the inactivation of the phosphatase but not of the kinase domain is responsible for the suppressor effect that results from the activation of the RSEs transcription factors that control expression of AOX, an alternative oxidase able to bypass the mitochondria cytochrome pathway of respiration. Remarkably, activation of the RSEs also stimulates the expression of the gluconeogenic enzymes, fructose-1,6 bi-phosphatase (FBPase-1) and phosphoenolpyruvate carboxykinase (PCK-1). We thus reveal in P. anserina an apparently paradoxical situation where the inactivation of the phosphatase domain of PFK-2/FBPase-2, supposed to stimulate glycolysis, is correlated with the transcriptional induction of the gluconeogenic enzymes. Phylogenic analysis revealed the presence of multiple presumed PFK-2/FBPase-2 isoforms in all the species of tested Ascomycetes.
    Mots-clés : Alternative oxidase regulation, BIOCELL, FDMITO, Glycolysis and gluconeogenesis, PFK-2/FBPase-2, Podospora anserina.

  • P. Silar, J. - M. Dauget, V. Gautier, P. Grognet, M. Chablat, S. Hermann-Le Denmat, A. Couloux, P. Wincker, et R. Debuchy, « A gene graveyard in the genome of the fungus Podospora comata », Molecular Genetics and Genomics, vol. 294, nᵒ 1, p. 177-190, févr. 2019.
    Résumé : Mechanisms involved in fine adaptation of fungi to their environment include differential gene regulation associated with single nucleotide polymorphisms and indels (including transposons), horizontal gene transfer, gene copy amplification, as well as pseudogenization and gene loss. The two Podospora genome sequences examined here emphasize the role of pseudogenization and gene loss, which have rarely been documented in fungi. Podospora comata is a species closely related to Podospora anserina, a fungus used as model in several laboratories. Comparison of the genome of P. comata with that of P. anserina, whose genome is available for over 10 years, should yield interesting data related to the modalities of genome evolution between these two closely related fungal species that thrive in the same types of biotopes, i.e., herbivore dung. Here, we present the genome sequence of the mat+isolate of the P. comata reference strain T. Comparison with the genome of the mat+isolate of P. anserina strain S confirms that P. anserina and P. comata are likely two different species that rarely interbreed in nature. Despite having a 94-99% of nucleotide identity in the syntenic regions of their genomes, the two species differ by nearly 10% of their gene contents. Comparison of the species-specific gene sets uncovered genes that could be responsible for the known physiological differences between the two species. Finally, we identified 428 and 811 pseudogenes (3.8 and 7.2% of the genes) in P. anserina and P. comata, respectively. Presence of high numbers of pseudogenes supports the notion that difference in gene contents is due to gene loss rather than horizontal gene transfers. We propose that the high frequency of pseudogenization leading to gene loss in P. anserina and P. comata accompanies specialization of these two fungi. Gene loss may be more prevalent during the evolution of other fungi than usually thought.
    Mots-clés : anserina, BIOCELL, biodiversity, DBG, DSMC, EDC, FDMITO, Lasiosphaeriaceae, MRP, neanderthal, Podospora anserina, Podospora comata, Pseudogene, pseudogenes, sequence, Sordariales, Speciation.

  • G. Vigani, Á. Solti, S. Thomine, et K. Philippar, « Essential and Detrimental - an Update on Intracellular Iron Trafficking and Homeostasis », Plant & Cell Physiology, vol. 60, nᵒ 7, p. 1420-1439, juill. 2019.
    Résumé : Chloroplasts, mitochondria, and vacuoles represent characteristic organelles of the plant cell, with a predominant function in cellular metabolism. Chloroplasts are the site of photosynthesis and therefore basic and essential for photoautotrophic growth of plants. Mitochondria produce energy during respiration and vacuoles act as internal waste and storage compartments. Moreover, chloroplasts and mitochondria are sites for the biosynthesis of various compounds of primary and secondary metabolism. For photosynthesis and energy generation, the internal membranes of chloroplasts and mitochondria are equipped with electron transport chains. To perform proper electron transfer and several biosynthetic functions, both organelles contain transition metals and here iron is by far the most abundant. Although iron is thus essential for plant growth and development, it becomes toxic when present in excess and/or in its free, ionic form. The harmful effect of the latter is caused by the generation of oxidative stress. As a consequence, iron transport and homeostasis have to be tightly controlled during plant growth and development. In addition to the corresponding transport and homeostasis proteins, the vacuole plays an important role as an intracellular iron storage and release compartment at certain developmental stages. In this review, we will summarize current knowledge on iron transport and homeostasis in chloroplasts, mitochondria, and vacuoles. In addition, we aim to integrate the physiological impact of intracellular iron homeostasis on cellular and developmental processes.
    Mots-clés : BIOCELL, chloroplast, iron homeostasis, iron transport, MINION, mitochondrion, vacuole.

  • M. - K. Zahoor, M. Poidevin, C. Lecerf, D. Garrido, et J. Montagne, « A Drosophila genetic screen for suppressors of S6kinase-dependent growth identifies the F-box subunit Archipelago/FBXW7 », Molecular genetics and genomics: MGG, vol. 294, nᵒ 3, p. 573-582, juin 2019.
    Résumé : This study was designed to identify novel negative regulators of the Drosophila S6kinase (dS6K). S6K is a downstream effector of the growth-regulatory complex mTORC1 (mechanistic-Target-of-Rapamycin complex 1). Nutrients activate mTORC1, which in turn induces the phosphorylation of S6K to promote cell growth, whereas fasting represses mTORC1 activity. Here, we screened 11,000 RNA-interfering (RNAi) lines and retained those that enhanced a dS6K-dependent growth phenotype. Since RNAi induces gene knockdown, enhanced tissue growth supports the idea that the targeted gene acts as a growth suppressor. To validate the resulting candidate genes, we monitored dS6K phosphorylation and protein levels in double-stranded RNAi-treated S2 cells. We identified novel dS6K negative regulators, including gene products implicated in basal cellular functions, suggesting that feedback inputs modulate mTORC1/dS6K signaling. We also identified Archipelago (Ago), the Drosophila homologue of FBXW7, which is an E3-ubiquitin-ligase subunit that loads ubiquitin units onto target substrates for proteasome-mediated degradation. Despite a previous report showing an interaction between Ago/FBXW7 and dS6K in a yeast two-hybrid assay and the presence of an Ago/FBXW7-consensus motif in the dS6K polypeptide, we could not see a direct interaction in immunoprecipitation assay. Nevertheless, we observed that loss-of-ago/fbxw7 in larvae resulted in an increase in dS6K protein levels, but no change in the levels of phosphorylated dS6K or dS6K transcripts, suggesting that Ago/FBXW7 indirectly controls dS6K translation or stability. Through the identification of novel negative regulators of the downstream target, dS6K, our study may help deciphering the underlying mechanisms driving deregulations of mTORC1, which underlies several human diseases.
    Mots-clés : activation, Ago, Ago/FBXW7, binding pocket, BIOCELL, cellular growth, fbxw7, Genetic screen, mammalian target, METABO, mtor, p70 s6 kinase, phosphorylation, protein, Protein degradation, S2 cells, Signaling, tumor-suppressor, ubiquitin ligases.


  • T. Avin-Wittenberg, F. Baluška, P. V. Bozhkov, P. H. Elander, A. R. Fernie, G. Galili, A. Hassan, D. Hofius, E. Isono, R. Le Bars, C. Masclaux-Daubresse, E. A. Minina, H. Peled-Zehavi, N. S. Coll, L. M. Sandalio, B. Satiat-Jeunemaitre, A. Sirko, P. S. Testillano, et H. Batoko, « Autophagy-related approaches for improving nutrient use efficiency and crop yield protection », Journal of Experimental Botany, vol. 69, nᵒ 6, p. 1335-1353, mars 2018.
    Mots-clés : BIOCELL, CYTO, DYNBSJ, PF, PHOT.

  • T. Avin-Wittenberg, F. Baluška, P. V. Bozhkov, P. H. Elander, A. R. Fernie, G. Galili, A. Hassan, D. Hofius, E. Isono, R. Le Bars, C. Masclaux-Daubresse, E. A. Minina, H. Peled-Zehavi, N. S. Coll, L. M. Sandalio, B. Satiat-Jeunemaitre, A. Sirko, P. S. Testillano, et H. Batoko, « Corrigendum: Autophagy-related approaches for improving nutrient use efficiency and crop yield protection », Journal of Experimental Botany, vol. 69, nᵒ 12, p. 3173, mai 2018.
    Mots-clés : BIOCELL, CYTO, DYNBSJ, PF, PHOT.

  • E. L. Bastow, V. S. Garcia de la Torre, A. E. Maclean, R. T. Green, S. Merlot, S. Thomine, et J. Balk, « Vacuolar iron stores gated by NRAMP3 and NRAMP4 are the primary source of iron in germinating seeds », Plant Physiology, vol. 177, nᵒ 3, p. 1267-1276, mai 2018.
    Résumé : During seed germination, iron (Fe) stored in vacuoles is exported by the redundant NRAMP3 and NRAMP4 transporter proteins. A double nramp3 nramp4 mutant is unable to mobilize Fe stores and does not develop in the absence of external Fe. We used RNA sequencing to compare gene expression in nramp3 nramp4 and wild type during germination and early seedling development. Even though sufficient Fe was supplied, the Fe-responsive transcription factors bHLH38, 39, 100 and 101 and their downstream targets FRO2 and IRT1 mediating Fe uptake were strongly upregulated in the nramp3 nramp4 mutant. Activation of the Fe deficiency response was confirmed by increased ferric chelate reductase activity in the mutant. At early stages, genes important for chloroplast redox control (FSD1, SAPX), Fe homeostasis (FER1, SUFB) and chlorophyll metabolism (HEMA1, NYC1) were downregulated, indicating limited Fe availability in plastids. In contrast, expression of FRO3, encoding a ferric reductase involved in Fe import into the mitochondria, was maintained and Fe-dependent enzymes in the mitochondria were unaffected in nramp3 nramp4. Together these data show that a failure to mobilize Fe stores during germination triggered Fe deficiency responses and strongly affected plastids but not mitochondria.
    Mots-clés : BIOCELL, MINION.

  • D. Carmona-Gutierrez, M. A. Bauer, A. Zimmermann, A. Aguilera, N. Austriaco, K. Ayscough, R. Balzan, S. Bar-Nun, A. Barrientos, P. Belenky, M. Blondel, R. J. Braun, M. Breitenbach, W. C. Burhans, S. Buettner, D. Cavalieri, M. Chang, K. F. Cooper, M. Côrte-Real, V. Costa, C. Cullin, I. Dawes, J. Dengjel, M. B. Dickman, T. Eisenberg, B. Fahrenkrog, N. Fasel, K. - U. Froehlich, A. Gargouri, S. Giannattasio, P. Goffrini, C. W. Gourlay, C. M. Grant, M. T. Greenwood, N. Guaragnella, T. Heger, J. Heinisch, E. Herker, J. M. Herrmann, S. Hofer, A. Jiménez-Ruiz, H. Jungwirth, K. Kainz, D. P. Kontoyiannis, P. Ludovico, S. Manon, E. Martegani, C. Mazzoni, L. A. Megeney, C. Meisinger, J. Nielsen, T. Nystroem, H. D. Osiewacz, T. F. Outeiro, H. - O. Park, T. Pendl, D. Petranovic, S. Picot, P. Polčic, T. Powers, M. Ramsdale, M. Rinnerthaler, P. Rockenfeller, C. Ruckenstuhl, R. Schaffrath, M. Segovia, F. F. Severin, A. Sharon, S. J. Sigrist, C. Sommer-Ruck, M. J. Sousa, J. M. Thevelein, K. Thevissen, V. Titorenko, M. B. Toledano, M. Tuite, F. - N. Voegtle, B. Westermann, J. Winderickx, S. Wissing, S. Woelfl, Z. J. Zhang, R. Y. Zhao, B. Zhou, L. Galluzzi, G. Kroemer, et F. Madeo, « Guidelines and recommendations on yeast cell death nomenclature », Microbial Cell, vol. 5, nᵒ 1, p. 4-31, janv. 2018.

  • E. H. Dawson, T. P. M. Bailly, J. Dos Santos, C. Moreno, M. Devilliers, B. Maroni, C. Sueur, A. Casali, B. Ujvari, F. Thomas, J. Montagne, et F. Mery, « Social environment mediates cancer progression in Drosophila », Nature Communications, vol. 9, nᵒ 1, p. 3574, sept. 2018.
    Résumé : The influence of oncogenic phenomena on the ecology and evolution of animal species is becoming an important research topic. Similar to host-pathogen interactions, cancer negatively affects host fitness, which should lead to the selection of host control mechanisms, including behavioral traits that best minimize the proliferation of malignant cells. Social behavior is suggested to influence tumor progression. While the ecological benefits of sociality in gregarious species are widely acknowledged, only limited data are available on the role of the social environment on cancer progression. Here, we exposed adult Drosophila, with colorectal-like tumors, to different social environments. We show how subtle variations in social structure have dramatic effects on the progression of tumor growth. Finally, we reveal that flies can discriminate between individuals at different stages of tumor development and selectively choose their social environment accordingly. Our study demonstrates the reciprocal links between cancer and social interactions and how sociality may impact health and fitness in animals and its potential implications for disease ecology.
    Mots-clés : BIOCELL, METABO.

  • T. Di Mattia, L. P. Wilhelm, S. Ikhlef, C. Wendling, D. Spehner, Y. Nominé, F. Giordano, C. Mathelin, G. Drin, C. Tomasetto, et F. Alpy, « Identification of MOSPD2, a novel scaffold for endoplasmic reticulum membrane contact sites », EMBO reports, vol. 19, nᵒ 7, juill. 2018.
    Résumé : Membrane contact sites are cellular structures that mediate interorganelle exchange and communication. The two major tether proteins of the endoplasmic reticulum (ER), VAP-A and VAP-B, interact with proteins from other organelles that possess a small VAP-interacting motif, named FFAT [two phenylalanines (FF) in an acidic track (AT)]. In this study, using an unbiased proteomic approach, we identify a novel ER tether named motile sperm domain-containing protein 2 (MOSPD2). We show that MOSPD2 possesses a Major Sperm Protein (MSP) domain which binds FFAT motifs and consequently allows membrane tethering in vitro MOSPD2 is an ER-anchored protein, and it interacts with several FFAT-containing tether proteins from endosomes, mitochondria, or Golgi. Consequently, MOSPD2 and these organelle-bound proteins mediate the formation of contact sites between the ER and endosomes, mitochondria, or Golgi. Thus, we characterized here MOSPD2, a novel tethering component related to VAP proteins, bridging the ER with a variety of distinct organelles.
    Mots-clés : BIOCELL, COAST, endoplasmic reticulum, ER–organelle contact, FFAT motif, membrane contact site, VAP proteins.

  • A. Dreinert, A. Wolf, T. Mentzel, B. Meunier, et M. Fehr, « The cytochrome bc1 complex inhibitor Ametoctradin has an unusual binding mode », Biochimica et Biophysica Acta (BBA) - Bioenergetics, vol. 1859, nᵒ 8, p. 567-576, avr. 2018.
    Résumé : Ametoctradin is an agricultural fungicide that selectively inhibits the cytochrome bc1 complex of oomycetes. Previous spectrophotometric studies using the purified cytochrome bc1 complex from Pythium sp. showed that Ametoctradin binds to the Qo-site of the enzyme. However, as modeling studies suggested a binding mode like that of the substrate ubiquinol, the possibility for a dual Qo- and Qi-site binding mode was left open. In this work, binding studies and enzyme assays with mitochondrial membrane preparations from Pythium sp. and an S. cerevisiae strain with a modified Qi-site were used to investigate further the binding mode of Ametoctradin. The results obtained argue that the compound could bind to both the Qo- and Qi-sites of the cytochrome bc1 complex and that its position or binding pose in the Qi-site differs from that of Cyazofamid and Amisulbrom, the two Qi-site-targeting, anti-oomycetes compounds. Furthermore, the data support the argument that Ametoctradin prefers binding to the reduced cytochrome bc1 complex. Thus, Ametoctradin has an unusual binding mode and further studies with this compound may offer the opportunity to better understand the catalytic cycle of the cytochrome bc1 complex.
    Mots-clés : Ametoctradin, Amisulbrom, BIOCELL, BIOMIT, Cyazofamid, Cytochrome bc(1) complex, Initium, Oomycetes, Respiration inhibitor, Respiratory complex III.

  • G. Dubeaux, J. Neveu, E. Zelazny, et G. Vert, « Metal Sensing by the IRT1 Transporter-Receptor Orchestrates Its Own Degradation and Plant Metal Nutrition », Molecular Cell, vol. 69, nᵒ 6, p. 953-964.e5, mars 2018.
    Résumé : Plant roots forage the soil for iron, the concentration of which can be dramatically lower than those needed for growth. Soil iron uptake uses the broad metal spectrum IRT1 transporter that also transports zinc, manganese, cobalt, and cadmium. Sophisticated iron-dependent transcriptional regulatory mechanisms allow plants to tightly control the abundance of IRT1, ensuring optimal absorption of iron. Here, we uncover that IRT1 acts as a transporter and receptor (transceptor), directly sensing excess of its non-iron metal substrates in the cytoplasm, to regulate its own degradation. Direct metal binding to a histidine-rich stretch in IRT1 triggers its phosphorylation by the CIPK23 kinase and facilitates the subsequent recruitment of the IDF1 E3 ligase. CIPK23-driven phosphorylation and IDF1-mediated lysine-63 polyubiquitination are jointly required for efficient endosomal sorting and vacuolar degradation of IRT1. Thus, IRT1 directly senses elevated non-iron metal concentrations and integrates multiple substrate-dependent regulations to optimize iron uptake and protect plants from highly reactive metals.
    Mots-clés : Arabidopsis, BIOCELL, degradation, metal homeostasis, phosphorylation, plant, receptor, sensing, transceptor, transporter, UBINET, ubiquitin.

  • R. Enrique Gomez, J. Joubès, N. Valentin, H. Batoko, B. Satiat-Jeunemaître, et A. Bernard, « Lipids in membrane dynamics during autophagy in plants », Journal of Experimental Botany, vol. 69, nᵒ 6, p. 1287-1299, mars 2018.
    Résumé : Autophagy is a critical pathway for plant adaptation to stress. Macroautophagy relies on the biogenesis of a specialized membrane named the phagophore that maturates into a double membrane vesicle. Proteins and lipids act synergistically to promote membrane structure and functions, yet research on autophagy has mostly focused on autophagy-related proteins while knowledge of supporting lipids in the formation of autophagic membranes remains scarce. This review expands on studies in plants with examples from other organisms to present and discuss our current understanding of lipids in membrane dynamics associated with the autophagy pathway in plants.
    Mots-clés : Arabidopsis, autophagy, BIOCELL, DYNBSJ, homeostasis, lipids, membrane, Plant.

  • M. R. Fassad, A. Shoemark, P. le Borgne, F. Koll, M. Patel, M. Dixon, J. Hayward, C. Richardson, E. Frost, L. Jenkins, T. Cullup, E. M. K. Chung, M. Lemullois, A. Aubusson-Fleury, C. Hogg, D. R. Mitchell, A. - M. Tassin, et H. M. Mitchison, « C11orf70 Mutations Disrupting the Intraflagellar Transport-Dependent Assembly of Multiple Axonemal Dyneins Cause Primary Ciliary Dyskinesia », American Journal of Human Genetics, vol. 102, nᵒ 5, p. 956-972, mai 2018.
    Résumé : Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomized left-right body asymmetry. PCD is mostly caused by mutations affecting the core axoneme structure of motile cilia that is essential for movement. Genes that cause PCD when mutated include a group that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified two unrelated families (three affected children) with mutations in the uncharacterized C11orf70 gene (official gene name CFAP300). The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Tagged C11orf70 in Paramecium and Chlamydomonas localizes mainly in the cytoplasm with a small amount in the ciliary component. IFT139/TTC21B (IFT-A protein) and FLA10 (IFT kinesin) depletion experiments show that its transport within cilia is IFT dependent. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD.
    Mots-clés : BIOCELL, BIOCIL, dynein, intraflagellar transport, Kartagener syndrome, mutation, Paramecium, primary ciliary dyskinesia.

  • M. R. Fassad, A. Shoemark, M. Legendre, R. A. Hirst, F. Koll, P. le Borgne, B. Louis, F. Daudvohra, M. P. Patel, L. Thomas, M. Dixon, T. Burgoyne, J. Hayes, A. G. Nicholson, T. Cullup, L. Jenkins, S. B. Carr, P. Aurora, M. Lemullois, A. Aubusson-Fleury, J. - F. Papon, C. O'Callaghan, S. Amselem, C. Hogg, E. Escudier, A. - M. Tassin, et H. M. Mitchison, « Mutations in Outer Dynein Arm Heavy Chain DNAH9 Cause Motile Cilia Defects and Situs Inversus », American Journal of Human Genetics, vol. 103, nᵒ 6, p. 984-994, déc. 2018.
    Résumé : Motile cilia move body fluids and gametes and the beating of cilia lining the airway epithelial surfaces ensures that they are kept clear and protected from inhaled pathogens and consequent respiratory infections. Dynein motor proteins provide mechanical force for cilia beating. Dynein mutations are a common cause of primary ciliary dyskinesia (PCD), an inherited condition characterized by deficient mucociliary clearance and chronic respiratory disease coupled with laterality disturbances and subfertility. Using next-generation sequencing, we detected mutations in the ciliary outer dynein arm (ODA) heavy chain gene DNAH9 in individuals from PCD clinics with situs inversus and in one case male infertility. DNAH9 and its partner heavy chain DNAH5 localize to type 2 ODAs of the distal cilium and in DNAH9-mutated nasal respiratory epithelial cilia we found a loss of DNAH9/DNAH5-containing type 2 ODAs that was restricted to the distal cilia region. This confers a reduced beating frequency with a subtle beating pattern defect affecting the motility of the distal cilia portion. 3D electron tomography ultrastructural studies confirmed regional loss of ODAs from the distal cilium, manifesting as either loss of whole ODA or partial loss of ODA volume. Paramecium DNAH9 knockdown confirms an evolutionarily conserved function for DNAH9 in cilia motility and ODA stability. We find that DNAH9 is widely expressed in the airways, despite DNAH9 mutations appearing to confer symptoms restricted to the upper respiratory tract. In summary, DNAH9 mutations reduce cilia function but some respiratory mucociliary clearance potential may be retained, widening the PCD disease spectrum.
    Mots-clés : BIOCELL, BIOCIL, diagnosis, DNAH9, dynein, dyskinesia, genes, genetics, motile cilia, mutation, primary ciliary dyskinesia, situs inversus, tomography.

  • F. Giordano, « Non-vesicular lipid trafficking at the endoplasmic reticulum-mitochondria interface », Biochemical Society Transactions, vol. 46, nᵒ 2, p. 437-452, 2018.
    Résumé : Mitochondria are highly dynamic organelles involved in various cellular processes such as energy production, regulation of calcium homeostasis, lipid trafficking, and apoptosis. To fulfill all these functions and preserve their morphology and dynamic behavior, mitochondria need to maintain a defined protein and lipid composition in both their membranes. The maintenance of mitochondrial membrane identity requires a selective and regulated transport of specific lipids from/to the endoplasmic reticulum (ER) and across the mitochondria outer and inner membranes. Since they are not integrated in the classical vesicular trafficking routes, mitochondria exchange lipids with the ER at sites of close apposition called membrane contact sites. Deregulation of such transport activities results in several pathologies including cancer and neurodegenerative disorders. However, we are just starting to understand the function of ER-mitochondria contact sites in lipid transport, what are the proteins involved and how they are regulated. In this review, we summarize recent insights into lipid transport pathways at the ER-mitochondria interface and discuss the implication of recently identified lipid transfer proteins in these processes.
    Mots-clés : Animals, BIOCELL, Biological Transport, Calcium, COAST, Endoplasmic Reticulum, Homeostasis, Humans, Lipid Metabolism, Mitochondria.

  • Y. Guerringue, S. Thomine, et J. - M. Frachisse, « Sensing and transducing forces in plants with MSL10 and DEK1 mechanosensors », FEBS letters, vol. 592, nᵒ 12, p. 1968-1979, mai 2018.
    Résumé : Mechanosensitive (MS) channels behave as microprobes that transduce mechanical tension into electric and ion signals. The plasma membrane anion-permeable channel AtMSL10 belongs to the first family of MS channels (MscS-LIKE) that has been characterized in Arabidopsis thaliana. In the same membrane, a rapidly activated calcium MS channel activity (RMA) associated with the presence of the DEFECTIVE KERNEL1 (AtDEK1) protein has been recently described. In this Review, based on the comparison of the specific properties of AtMSL10 and RMA, we put forward hypotheses on the mechanism of activation of these two channels, their respective roles in signalling and also raise the question of the molecular identity of RMA. Finally, we propose functions for these two channels within the context of plant mechanotransduction.
    Mots-clés : BIOCELL, mechanosensitive channel, mechanotransduction, MINION, plant.

  • P. Junková, M. Daněk, D. Kocourková, J. Brouzdová, K. Kroumanová, E. Zelazny, M. Janda, R. Hynek, J. Martinec, et O. Valentová, « Mapping of Plasma Membrane Proteins Interacting With Arabidopsis thaliana Flotillin 2 », Frontiers in Plant Science, vol. 9, p. 991, 2018.
    Résumé : Arabidopsis flotillin 2 (At5g25260) belongs to the group of plant flotillins, which are not well characterized. In contrast, metazoan flotillins are well known as plasma membrane proteins associated with membrane microdomains that act as a signaling hub. The similarity of plant and metazoan flotillins, whose functions most likely consist of affecting other proteins via protein-protein interactions, determines the necessity of detecting their interacting partners in plants. Nevertheless, identifying the proteins that form complexes on the plasma membrane is a challenging task due to their low abundance and hydrophobic character. Here we present an approach for mapping Arabidopsis thaliana flotillin 2 plasma membrane interactors, based on the immunoaffinity purification of crosslinked and enriched plasma membrane proteins with mass spectrometry detection. Using this approach, 61 proteins were enriched in the AtFlot-GFP plasma membrane fraction, and 19 of them were proposed to be flotillin 2 interaction partners. Among our proposed partners of Flot2, proteins playing a role in the plant response to various biotic and abiotic stresses were detected. Additionally, the use of the split-ubiquitin yeast system helped us to confirm that plasma-membrane ATPase 1, early-responsive to dehydration stress protein 4, syntaxin-71, harpin-induced protein-like 3, hypersensitive-induced response protein 2 and two aquaporin isoforms interact with flotillin 2 directly. Based on the results of our study and the reported properties of Flot2 interactors, we propose that Flot2 complexes may be involved in plant-pathogen interactions, water transport and intracellular trafficking.
    Mots-clés : Arabidopsis flotillin 2, BIOCELL, immunopurification, intracellular trafficking, mass spectrometry, plant–pathogen interaction, protein–protein interactions, split-ubiquitin yeast system, UBINET, water transport.

  • A. Krieger-Liszkay et S. Thomine, « Importing Manganese into the Chloroplast: Many Membranes to Cross », Molecular Plant, vol. 11, nᵒ 9, p. 1109-1111, août 2018.
    Mots-clés : arabidopsis, B3S, BIOCELL, cells, homeostasis, MINION, MROP, photosynthesis, photosystem-ii, protein.

  • C. Lefebvre, R. Legouis, et E. Culetto, « ESCRT and autophagies: Endosomal functions and beyond », Seminars in Cell & Developmental Biology, vol. 74, p. 21-28, 2018.
    Résumé : ESCRT (endosomal sorting complex required for transport) machinery has been initially identified for its role during endocytosis, which allows membrane proteins and lipids to be degraded in the lysosome. ESCRT function is required to form intraluminal vesicles permitting internalization of cytosolic components or membrane embedded cargoes and promoting endosome maturation. ESCRT machinery also contributes to multiple key cell mechanisms in which it reshapes membranes. In addition, ESCRT actively participates in different types of autophagy processes for degrading cytosolic components, such as endosomal microautophagy and macroautophagy. During macroautophagy, ESCRT promotes formation of multivesicular bodies, which can fuse with autophagosomes to generate amphisomes. This latter fusion probably brings to autophagosomes key membrane molecules necessary for the subsequent fusion with lysosomes. Interestingly, during macroautophagy, ESCRT proteins could be involved in non-canonical functions such as vesicle tethering or phagophore membrane sealing. Additionally, ESCRT subunits could directly interact with key autophagy related proteins to build a closer connection between endocytosis and autophagy pathways.
    Mots-clés : Amphisome, Animals, ATG, Autophagosome, Autophagy, BIOCELL, Endosomal Sorting Complexes Required for Transport, Endosome, Endosomes, ESCRT, Humans, OTOFAG.

  • A. Maréchal, A. M. Hartley, T. P. Warelow, B. Meunier, et P. R. Rich, « Comparison of redox and ligand binding behaviour of yeast and bovine cytochrome c oxidases using FTIR spectroscopy », Biochimica et Biophysica Acta (BBA) - Bioenergetics, mai 2018.
    Résumé : Redox and CO photolysis FTIR spectra of yeast cytochrome c oxidase WT and mutants are compared to those from bovine and P. denitrificans CcOs in order to establish common functional features. All display changes that can be assigned to their E242 (bovine numbering) equivalent and to weakly H-bonded water molecules. The additional redox-sensitive band reported at 1736 cm-1 in bovine CcO and previously assigned to D51 is absent from yeast CcO and couldn't be restored by introduction of a D residue at the equivalent position of the yeast protein. Redox spectra of yeast CcO also show much smaller changes in the amide I region, which may relate to structural differences in the region around D51 and the subunit I/II interface.
    Mots-clés : BIOCELL, BIOMIT, Carboxyl groups, Cytochrome c oxidase, Infrared spectroscopy, Mitochondria, Oxidoreduction, Site-directed mutagenesis.

  • J. Marion, R. Le Bars, L. Besse, H. Batoko, et B. Satiat-Jeunemaitre, « Multiscale and Multimodal Approaches to Study Autophagy in Model Plants », Cells, vol. 7, nᵒ 1, janv. 2018.
    Résumé : Autophagy is a catabolic process used by eukaryotic cells to maintain or restore cellular and organismal homeostasis. A better understanding of autophagy in plant biology could lead to an improvement of the recycling processes of plant cells and thus contribute, for example, towards reducing the negative ecological consequences of nitrogen-based fertilizers in agriculture. It may also help to optimize plant adaptation to adverse biotic and abiotic conditions through appropriate plant breeding or genetic engineering to incorporate useful traits in relation to this catabolic pathway. In this review, we describe useful protocols for studying autophagy in the plant cell, taking into account some specificities of the plant model.
    Mots-clés : Arabidopsis, autophagosome, Autophagy, autophagy assays, BIOCELL, CYTO, DYNBSJ, methods, PHOT, plant cells, Tobacco.

  • K. Mishev, Q. Lu, B. Denoo, F. Peurois, W. Dejonghe, J. Hullaert, R. De Rycke, S. Boeren, M. Bretou, S. De Munck, I. Sharma, K. Goodman, K. Kalinowska, V. Storme, L. S. L. Nguyen, A. Drozdzecki, S. Martins, W. Nerinckx, D. Audenaert, G. Vert, A. Madder, M. S. Otegui, E. Isono, S. N. Savvides, W. Annaert, S. De Vries, J. Cherfils, J. Winne, et E. Russinova, « Nonselective Chemical Inhibition of Sec7 Domain-Containing ARF GTPase Exchange Factors », Plant Cell, vol. 30, nᵒ 10, p. 2573-2593, oct. 2018.
    Résumé : Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans-Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA.
    Mots-clés : arabidopsis-thaliana, BIOCELL, brefeldin-a, endomembrane trafficking, golgi network/early endosome, plant-cells, plasma-membrane, protein complexes, receptor kinase bri1, small molecules, UBINET, wide analysis.

  • M. Mishra, H. Jiang, H. A. Chawsheen, M. Gerard, M. B. Toledano, et Q. Wei, « Nrf2-activated expression of sulfiredoxin contributes to urethane-induced lung tumorigenesis », Cancer Letters, vol. 432, p. 216-226, sept. 2018.
    Résumé : Lung cancer is the leading cause of cancer death worldwide. Cigarette smoking and exposure to chemical carcinogens are among the risk factors of lung tumorigenesis. In this study, we found that cigarette smoke condensate and urethane significantly stimulated the expression of sulfiredoxin (Srx) at the transcript and protein levels in cultured normal lung epithelial cells, and such stimulation was mediated through the activation of nuclear related factor 2 (Nrf2). To study the role of Srx in lung cancer development in vivo, mice with Srx wildtype, heterozygous or knockout genotype were subjected to the same protocol of urethane treatment to induce lung tumors. By comparing tumor multiplicity and volume between groups of mice with different genotype, we found that Srx knockout mice had a significantly lower number and smaller size of lung tumors. Mechanistically, we demonstrated that loss of Srx led to a decrease of tumor cell proliferation as well as an increase of tumor cell apoptosis. These data suggest that Srx may have an oncogenic role that contributes to the development of lung cancer in smokers or urethane-exposed human subjects.
    Mots-clés : Antioxidant, BIOCELL, Cell growth and proliferation, DBG, Oxidative stress, Peroxiredoxins, REMOD, Signal transduction, SOC.

  • D. Naquin, C. Panozzo, G. Dujardin, E. van Dijk, Y. D'Aubenton-Carafa, et C. Thermes, « Complete Sequence of the Intronless Mitochondrial Genome of the Saccharomyces cerevisiae Strain CW252 », Microbiology Resource Announcements, vol. 6, nᵒ 17, p. UNSP e00219-18, avr. 2018.
    Résumé : The mitochondrial genomes of Saccharomyces cerevisiae strains contain up to 13 introns. An intronless recombinant genome introduced into the nuclear background of S. cerevisiae strain W303 gave the S. cerevisiae CW252 strain, which is used to model mitochondrial respiratory pathologies. The complete sequence of this mitochondrial genome was obtained using a hybrid assembling methodology.
    Mots-clés : BIOCELL, BIOMIT, NGS, PF.

  • S. Rahuel-Clermont et M. B. Toledano, « Parsing protein sulfinic acid switches », Nature Chemical Biology, vol. 14, nᵒ 11, p. 991-993, nov. 2018.
    Mots-clés : BIOCELL, cysteine, peroxiredoxin, SOC, sulfiredoxin.

  • N. Romero-Barrios et G. Vert, « Proteasome-independent functions of lysine-63 polyubiquitination in plants », The New Phytologist, vol. 217, nᵒ 3, p. 995-1011, févr. 2018.
    Résumé : Contents Summary 995 I. Introduction 995 II. The plant Ub machinery 996 III. From Ub to Ub linkage types in plants 997 IV. Increasing analytical resolution for K63 polyUb in plants 998 V. How to build K63 polyUb chains? 998 VI. Cellular roles of K63 polyUb in plants 999 VII. Physiological roles of K63 polyUb in plants 1004 VIII. Future perspectives: towards the next level of the Ub code 1006 Acknowledgements 1006 References 1007 SUMMARY: Ubiquitination is a post-translational modification essential for the regulation of eukaryotic proteins, having an impact on protein fate, function, localization or activity. What originally appeared to be a simple system to regulate protein turnover by the 26S proteasome is now known to be the most intricate regulatory process cells have evolved. Ubiquitin can be arranged in countless chain assemblies, triggering various cellular outcomes. Polyubiquitin chains using lysine-63 from ubiquitin represent the second most abundant type of ubiquitin modification. Recent studies have exposed their common function in proteasome-independent functions in non-plant model organisms. The existence of lysine-63 polyubiquitination in plants is, however, only just emerging. In this review, we discuss the recent advances on the characterization of ubiquitin chains and the molecular mechanisms driving the formation of lysine-63-linked ubiquitin modifications. We provide an overview of the roles associated with lysine-63 polyubiquitination in plant cells in the light of what is known in non-plant models. Finally, we review the crucial roles of lysine-63 polyubiquitin-dependent processes in plant growth, development and responses to environmental conditions.
    Mots-clés : Autophagy, BIOCELL, UBINET.

  • L. Shi, F. Koll, O. Arnaiz, et J. Cohen, « The Ciliary Protein IFT57 in the Macronucleus of Paramecium », The Journal of Eukaryotic Microbiology, vol. 65, nᵒ 1, p. 12-27, janv. 2018.
    Résumé : The intraflagellar transport IFT57 protein is essential for ciliary growth and maintenance. Also known as HIPPI, human IFT57 can be translocated to the nucleus via a molecular partner of the Huntingtin, Hip1, inducing gene expression changes. In Paramecium tetraurelia, we identified four IFT57 genes forming two subfamilies IFT57A/B and IFT57C/D arising from whole genome duplications. The depletion of proteins of the two subfamilies induced ciliary defects and IFT57A and IFT57C localized in basal bodies and cilia. We observed that IFT57A, but not IFT57C, is also present in the macronucleus and able to traffic toward the developing anlage during autogamy. Analysis of chimeric IFT57A-IFT57C-GFP-tagged proteins allowed us to identify a region of IFT57A necessary for nuclear localization. We studied the localization of the unique IFT57 protein of Paramecium caudatum, a species, which diverged from P. tetraurelia before the whole genome duplications. The P. caudatumIFT57C protein was excluded from the nucleus. We also analyzed whether the overexpression of IFT57A in Paramecium could affect gene transcription as the human protein does in HeLa cells. The expression of some genes was indeed affected by overexpression of IFT57A, but the set of affected genes poorly overlaps the set of genes affected in human cells.
    Mots-clés : ANGE, BIOCELL, BIOCIL, cilia, DBG, HIPPI, IFT57 /HIPPI, intraflagellar transport, intraflagellar transport (IFT), Macronucleus, MICMAC, Paramecium.

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