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Neuhaus, Jean-Marc

Nom
Neuhaus, Jean-Marc
Affiliation principale
Fonction
Professeur ordinaire
Email
jean-marc.neuhaus@unine.ch
Identifiants
https://libra.unine.ch/handle/123456789/300

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  • Publication
    Accès libre
    The functions of RMR proteins in the "Physcomitrella patens" secretory pathway
    (2017)
    Fahr, Noémie
    ;
    Neuhaus, Jean-Marc 
    Chez les plantes, les vacuoles occupent un grand nombre de fonctions, allant du maintien de la pression de turgescence et de la rigidité cellulaire en passant par le stockage ou la dégradation de diverses molécules. Deux types de vacuoles ayant un pH distinct peuvent coexister au sein d’une même cellule. Les vacuoles acides sont considérées comme étant des homologues aux lysosomes présents dans les cellules animales, tandis que les vacuoles neutres sont impliquées dans le stockage de protéines et de métabolites secondaires. L’adressage des protéines à la vacuole lytique a été largement étudié et les récepteurs vacuolaires impliqués, les VSRs, sont des protéines bien caractérisées. À l’opposé, les connaissances sur l’adressage des protéines à la vacuole neutre ou de stockage sont moindres. Les protéines RMR sont très probablement les récepteurs vacuolaires impliqués, bien que la délétion des cinq gènes RMR chez Physcomitrella patens n’ait conduit à aucun phénotype visible. Ce travail a donc pour objectif l’élucidation du rôle des protéines RMR chez la mousse.
    Dans le premier chapitre de cette thèse nous avons regroupé les principales données concernant le système sécrétoire et endomembranaire chez les plantes, et nous avons également documenté comment les protéines sont adressées aux vacuoles.
    Dans le second chapitre, nous avons étudié le système sécrétoire de la mousse en développant une bibliothèque de marqueurs fluorescents. Différents mécanismes cellulaires semblent conservés entre les mousses et les plantes à fleurs.
    Dans le troisième chapitre, nous nous sommes intéressés à la caractérisation des simples et quintuple knock-out mutants RMR. Nous avons finalement obtenu un phénotype de tri vacuolaire: un défaut d’adressage est observé avec le marqueur fluorescent Citrine-Card dans les simples, triple et quintuple KO mutants. Le signal fluorescent a été détecté dans le réticulum endoplasmique chez les mutants, tandis que la fluorescence est observée dans la vacuole centrale chez le WT. Cela montre que l’adressage à la vacuole d’une protéine comportant ce ctVSD est dépendant des RMRs.
    Dans la dernière partie de ce travail, nous avons identifié des partenaires interagissant très probablement avec la partie cytosolique de PpRMR2 par des analyses de GST pull-down et de spectrométrie de masse., Plant vacuoles play a wide range of functions within the cell, from the maintenance of turgor pressure and rigidity, to the storage or degradation of various molecules. Two types of vacuoles with distinct pH can coexist in the same single cell. Acidic vacuoles can be considered as homologues to animal lysosomes while neutral vacuoles are involved in proteins and secondary metabolites storage. Targeting of proteins to the lytic vacuole has been extensively studied and the vacuolar receptors involved, the VSRs, are now well characterized in higher plants. However, less is known about the traffic of proteins to the neutral/storage vacuole. RMR proteins are thought to be vacuolar receptors for the neutral/storage vacuole. However, the complete deletion of the five RMR genes in Physcomitrella patens did not lead to any developmental phenotype. This work aimed to investigate the role of RMR proteins in the moss.
    In the first chapter, we review the plant secretory pathway system and how proteins are targeted to vacuoles.
    In the second chapter, we studied the moss secretory pathway by developing a fluorescent reporter library. Several mechanisms seem to be conserved between the moss and the flowering plants.
    In the third chapter, we focused on the characterization of the single and quintuple knock-out RMR mutants. We finally obtained a trafficking phenotype: the fluorescent reporter Citrine-Card was mistargeted in the single, triple and quintuple KO mutants. Fluorescent signal was detected in endoplasmic reticulum in the mutants, while it was observed in the central vacuole in WT. Trafficking to vacuole of a protein carrying this ctVSD was RMR-dependent.
    In the last part of this thesis, we identified some putative binding partners of the cytosolic part of PpRMR2 by GST pull-down assay and mass spectrometry analysis.
  • Publication
    Accès libre
    Study of moss vacuoles and functional characterization of the putative vacuolar receptors: the RMR proteins
    (2012)
    Ayachi, Sanaa
    ;
    Neuhaus, Jean-Marc 
    The vacuolar system of plants is a key element of plant growth and development, it fulfils many other functions. Plant cell can have more than two different vacuolar sorting systems: the lytic and the (seed) protein storage or vegetative storage vacuoles. Soluble vacuolar proteins are sorted through the secretory pathway to these vacuoles by three different routes, depending on different types of Vacuolar Sorting Determinants (VSD) and involving several types of receptors and vesicles. The AtRMR proteins has been identified in cellular structures associated with the seed storage vacuole pathway (Jiang et al. 2000). Based on its localisation and homology to a known vacuolar receptor, it has been hypothesised to be a receptor protein for the C-terminal type of VSD (ct-VSD) involved in sorting to the storage vacuole. The genome of Physcomitrella patens contains five genes coding for RMR proteins.
    My work hypothesis is that the vacuolar system of higher plants has evolved from simple ancestors, which might have been preserved in lower plants. This evolution is reflected in the gene families involved in vacuole biogenesis. In a first part, we established the moss P. patens as a model system for the study of the secretory pathways. In a second part, we performed a comparative study of the plant-specific aspects of the vacuolar system. And finally in a third part, we tried to establish the functional role of PpRMR genes by the analysis of the complete RMR deletion mutants. Several strategies were considered to investigate a putative disorder due to RMRs loss of function. So far, no phenotype was detected in the mutants. Nevertheless the absence of the RMR family gene seems not to be necessary for moss development.
  • Publication
    Accès libre
    The knock-out of ARP3a gene affects F-actin cytoskeleton organization altering cellular tip growth, morphology and development in moss Physcomitrella patens
    (2008)
    Finka, Andrija
    ;
    Saidi, Younousse
    ;
    Goloubinoff, Pierre
    ;
    Neuhaus, Jean-Marc 
    ;
    Zrÿd, Jean-Pierre
    ;
    Schaefer, Didier G.
    The seven subunit Arp2/3 complex is a highly conserved nucleation factor of actin microfilaments. We have isolated the genomic sequence encoding a putative Arp3a protein of the moss Physcomitrella patens. The disruption of this ARP3A gene by allele replacement has generated loss-of-function mutants displaying a complex developmental phenotype. The loss-of function of ARP3A gene results in shortened, almost cubic chloronemal cells displaying affected tip growth and lacking differentiation to caulonemal cells. In moss arp3a mutants, buds differentiate directly from chloronemata to form stunted leafy shoots having differentiated leaves similar to wild type. Yet, rhizoids never differentiate from stem epidermal cells. To characterize the F-actin organization in the arp3a-mutated cells, we disrupted ARP3A gene in the previously described HGT1 strain expressing conditionally the GFP-talin marker. In vivo observation of the F-actin cytoskeleton during P. patens development demonstrated that loss-of-function of Arp3a is associated with the disappearance of specific F-actin cortical structures associated with the establishment of localized cellular growth domains. Finally, we show that constitutive expression of the P. patens Arp3a and its Arabidopsis thaliana orthologs efficiently complement the mutated phenotype indicating a high degree of evolutionary conservation of the Arp3 function in land plants.