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1. Characterization of the epidermal-dermal junction in hiPSC-derived skin organoids

Author

Ramovs, Veronika, Janssen, Hans, Fuentes, Ignacia, Pitaval, Amandine, Rachidi, Walid, Chuva de Sousa Lopes, Susana, Freund, Christian, Gidrol, Xavier, Mummery, Christine, Raymond, Karine, Bertacchi Griffi, Nathalie, Leiden University Medical Center (LUMC), Netherlands Cancer Institute (NKI), Antoni van Leeuwenhoek Hospital, Universidad del Desarrollo, Biomicrotechnologie et génomique fonctionnelle (BIOMICS), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Ghent University Hospital

Subjects
collagen, [SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], epidermal-dermal junction, hiPSC-derived hair-bearing skin organoids
Abstract

Human induced pluripotent stem cell (hiPSC)-derived hair-bearing skin organoids offer exciting new possibilities for modeling diseases like epidermolysis bullosa (EB). These inherited diseases affect 1 in 30,000 people worldwide and result from perturbed expression and/or structure of components of the epidermal-dermal junction (EDJ). To establish whether hiPSC-derived skin organoids might be able to capture salient features of EB, it is thus important to characterize their EDJ. Here, we report successful generation of hair-bearing skin organoids from two hiPSC lines that exhibited fully stratified interfollicular epidermis. Using immunofluorescence and electron microscopy, we showed that basal keratinocytes in organoids adhere to laminin-332 and type IV collagen-rich basement membrane via type I hemidesmosomes and integrin β1-based adhesion complexes. Importantly, we demonstrated that EDJs in organoids are almost devoid of type VII collagen, a fibril that mediates anchorage of the epidermis to dermis. This should be considered when using skin organoids for EB modeling., Les îlots de Langerhans isolés de donneurs en état de mort encéphalique constituent actuellement la seule source de cellules pour la transplantation de patients atteints de diabète de type 1. Cette approche thérapeutique reste cependant compromise par la rareté des donneurs et par certains aspects techniques. L’utilisation de sources alternatives de cellules productrices d’insuline est donc un enjeu tant thérapeutique que pour la recherche pharmacologique. Plusieurs équipes dans le monde, dont la nôtre, développent des modèles de culture cellulaire en 3D, les Langerhanoïdes , qui sont physiologiquement proches des îlots pancréatiques humains. Dans cette revue, nous décrivons les récentes avancées mimant la niche pancréatique (matrice extracellulaire, vascularisation, microfluidique), permettant ainsi d’accroître la fonctionnalité de ces Langerhanoïdes .

Published
2022

2. Les Langerhanoïdes, des organoïdes d’îlots pancréatiques

Author

Anastasia Papoz, Flora Clément, Camille Laporte, Emily Tubbs, Xavier Gidrol, Amandine Pitaval, Biomicrotechnologie et génomique fonctionnelle (BIOMICS), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Département d'Architectures, Conception et Logiciels Embarqués-LETI (DACLE-LETI), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Laboratory of Fundamental and Applied Bioenergetics = Laboratoire de bioénergétique fondamentale et appliquée (LBFA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Bertacchi Griffi, Nathalie, Centre d'Etude et de Recherche Travail Organisation Pouvoir (CERTOP), Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), and Sciences, EDP

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], General Medicine, General Biochemistry, Genetics and Molecular Biology
Abstract

The extension of islet transplantation to a wider number of Type 1 diabetic patients is compromised by the scarcity of donors, the reduced ex vivo survival of pancreatic islets and the use of immunosuppressive treatments. Islets of Langerhans isolated from brain-dead donors are currently the only cell source for transplantation. Thus, it is crucial to find an alternative and an abundant source of functional insulin secreting cells not only for clinical use but also for the development of research dedicated to the screening of drugs and to the development of new therapeutic targets. Several groups around the world, including ours, develop 3D culture models as Langerhanoids that closely mimick human pancreatic islets physiology. In this review, we describe recent advances to mimic the pancreatic niche (extracellular matrix, vascularization, microfluidics) allowing better functionality of Langerhanoids., Les îlots de Langerhans isolés de donneurs en état de mort encéphalique constituent actuellement la seule source de cellules pour la transplantation de patients atteints de diabète de type 1. Cette approche thérapeutique reste cependant compromise par la rareté des donneurs et par certains aspects techniques. L’utilisation de sources alternatives de cellules productrices d’insuline est donc un enjeu tant thérapeutique que pour la recherche pharmacologique. Plusieurs équipes dans le monde, dont la nôtre, développent des modèles de culture cellulaire en 3D, les Langerhanoïdes , qui sont physiologiquement proches des îlots pancréatiques humains. Dans cette revue, nous décrivons les récentes avancées mimant la niche pancréatique (matrice extracellulaire, vascularisation, microfluidique), permettant ainsi d’accroître la fonctionnalité de ces Langerhanoïdes .

Published
2022

3. Ultrasensitive Quantification of Recombinant Proteins Using AAA-MS

Author

Sylvie Kieffer-Jaquinod, Mathilde Louwagie, Virginie Brun, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Bertacchi Griffi, Nathalie

Subjects
0301 basic medicine, chemistry.chemical_classification, Chromatography, Absolute quantification, [SDV]Life Sciences [q-bio], 010401 analytical chemistry, Mass spectrometry, 01 natural sciences, 0104 chemical sciences, law.invention, Amino acid, [SDV] Life Sciences [q-bio], 03 medical and health sciences, Hydrolysis, Amino acid analysis, 030104 developmental biology, chemistry, law, Recombinant DNA, Target protein, ComputingMilieux_MISCELLANEOUS
Abstract

Recombinant proteins are essential components of therapeutic, biotechnological, food, and household products. In some cases, recombinant proteins must be purified and their quantity and/or concentration precisely determined. In this chapter, we describe a protocol for the quantification of purified recombinant proteins. The protocol is based on a microwave-assisted acidic hydrolysis of the target protein followed by high-resolution mass spectrometry (HRMS) analysis of the hydrolytic products. Absolute quantification is obtained by adding controlled amounts of labeled amino acids that serve as standards.

Published
2019

4. Single-particle mass spectrometry with arrays of frequency-addressed nanomechanical resonators

Author

Sebastien Hentz, Thomas Alava, Laurent Duraffourg, Ariel Brenac, Shawn Fostner, Guillaume Jourdan, Marc Sansa, Eric Colinet, Michael L. Roukes, Marc Gely, Eric Sage, Martial Defoort, Akshay Naik, Christophe Masselon, R. Morel, Laboratoire d'Electronique et des Technologies de l'Information (CEA-LETI), Université Grenoble Alpes (UGA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes (UGA), UBT - Ultra-basses températures, Institut Néel (NEEL), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), SPINtronique et TEchnologie des Composants (SPINTEC), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Département Intégration Hétérogène sur Silicium (DIHS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), California Institute of Technology (CALTECH), Équipe NanoBioSystèmes (LAAS-NBS), Laboratoire d'analyse et d'architecture des systèmes [Toulouse] (LAAS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées, CEA, Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Département Nanotec (D2NT), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), and Bertacchi Griffi, Nathalie

Subjects
Physics - Instrumentation and Detectors, Materials science, Science, [SDV]Life Sciences [q-bio], General Physics and Astronomy, FOS: Physical sciences, 02 engineering and technology, Mass spectrometry, 01 natural sciences, Article, General Biochemistry, Genetics and Molecular Biology, Mass spectrometry imaging, Resonator, [PHYS.PHYS.PHYS-INS-DET]Physics [physics]/Physics [physics]/Instrumentation and Detectors [physics.ins-det], lcsh:Science, ComputingMilieux_MISCELLANEOUS, Nanoelectromechanical systems, Multidisciplinary, business.industry, 010401 analytical chemistry, Detector, technology, industry, and agriculture, General Chemistry, Instrumentation and Detectors (physics.ins-det), 021001 nanoscience & nanotechnology, 0104 chemical sciences, [SDV] Life Sciences [q-bio], Mass spectrum, Optoelectronics, lcsh:Q, 0210 nano-technology, business, Order of magnitude, Beam (structure)
Abstract

One of the main challenges to overcome to perform nanomechanical mass spectrometry analysis in a practical time frame stems from the size mismatch between the analyte beam and the small nanomechanical detector area. We report here the demonstration of mass spectrometry with arrays of 20 multiplexed nanomechanical resonators; each resonator is designed with a distinct resonance frequency which becomes its individual address. Mass spectra of metallic aggregates in the MDa range are acquired with more than one order of magnitude improvement in analysis time compared to individual resonators. A 20 NEMS array is probed in 150 ms with the same mass limit of detection as a single resonator. Spectra acquired with a conventional time-of-flight mass spectrometer in the same system show excellent agreement. We also demonstrate how mass spectrometry imaging at the single-particle level becomes possible by mapping a 4-cm-particle beam in the MDa range and above., Nano-electro-mechanical system-based mass spectrometry holds promise for detecting supramolecular assemblies at large molecular weights, but its efficiency is too poor to be practical. Sage et al. overcome this problem using a nanomechanical resonator array, which significantly decreases detection time.

Published
2018

5. PIWI Slicing and EXD1 Drive Biogenesis of Nuclear piRNAs from Cytosolic Targets of the Mouse piRNA Pathway

Author

Marie-Odile Fauvarque, David Homolka, Ramesh S. Pillai, Zhaolin Yang, Radha Raman Pandey, K.M. Chen, Andrew A. McCarthy, Ravi Sachidanandam, Bruno Kotska Rodino Janeiro, Michael Reuter, Laboratoire européen de biologie moléculaire - European Molecular Biology Laboratory (EMBL Grenoble), European Molecular Biology Laboratory [Grenoble] (EMBL), Department of Molecular Biology [Geneva, Switzerland], Université de Genève = University of Geneva (UNIGE), Unit for Virus Host-Cell Interactions [Grenoble] (UVHCI), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS), Cold Spring Harbor Laboratory, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Bertacchi Griffi, Nathalie, University of Geneva [Switzerland], Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Université Joseph Fourier - Grenoble 1 (UJF), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, Exonucleases, Male, Models, Molecular, [SDV]Life Sciences [q-bio], Tdrd12, RNA-binding protein, Retrotransposon, piRNA, Cytosol, RNA interference, Drosophila Proteins, RNA Processing, Post-Transcriptional, RNA, Small Interfering, ComputingMilieux_MISCELLANEOUS, Genetics, RNA-Binding Proteins, Argonaute, Cell biology, [SDV] Life Sciences [q-bio], Drosophila melanogaster, Argonaute Proteins, Female, RNA Interference, endocrine system, Molecular Sequence Data, Piwi-interacting RNA, Mice, Transgenic, Inchworming, Biology, Article, 03 medical and health sciences, Slicer, RasiRNA, Animals, Protein Interaction Domains and Motifs, Exd1, Amino Acid Sequence, Molecular Biology, Cell Nucleus, PIWI, urogenital system, Cell Biology, Bombyx, 030104 developmental biology, Gene Expression Regulation, Cytoplasm, Multiprotein Complexes, Ping-pong, Carrier Proteins, Biogenesis
Abstract

Summary PIWI-interacting RNAs (piRNAs) guide PIWI proteins to suppress transposons in the cytoplasm and nucleus of animal germ cells, but how silencing in the two compartments is coordinated is not known. Here we demonstrate that endonucleolytic slicing of a transcript by the cytosolic mouse PIWI protein MILI acts as a trigger to initiate its further 5′→3′ processing into non-overlapping fragments. These fragments accumulate as new piRNAs within both cytosolic MILI and the nuclear MIWI2. We also identify Exonuclease domain-containing 1 (EXD1) as a partner of the MIWI2 piRNA biogenesis factor TDRD12. EXD1 homodimers are inactive as a nuclease but function as an RNA adaptor within a PET (PIWI-EXD1-Tdrd12) complex. Loss of Exd1 reduces sequences generated by MILI slicing, impacts biogenesis of MIWI2 piRNAs, and de-represses LINE1 retrotransposons. Thus, piRNA biogenesis triggered by PIWI slicing, and promoted by EXD1, ensures that the same guides instruct PIWI proteins in the nucleus and cytoplasm., Graphical Abstract, Highlights • MILI slicing triggers entry of target transcript into piRNA processing • Slicing generates non-overlapping, contiguous primary piRNAs in MILI and MIWI2 • EXD1 is a component of a PET (PIWI-EXD1-TDRD12) complex • RNA-binding protein EXD1 is critical for MIWI2 piRNA biogenesis, Yang et al. show how cytoplasmic slicing of a target transcript by a PIWI endonuclease triggers its conversion into piRNAs that are loaded into both cytosolic and nuclear PIWI proteins, ensuring coordinated silencing in the two compartments. The inactive nuclease EXD1 functions as an RNA-binding in this process.

Published
2016
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6. Calcium influx mediates the chemoattractant-induced translocation of the arrestin-related protein AdcC in Dictyostelium

Author

Christian Delphin, Agnès Journet, Adeline Cieren, Laurence Aubry, Lauriane Mas, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), [GIN] Grenoble Institut des Neurosciences (GIN), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Genetics and Chemogenomics (GenChem), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Bertacchi Griffi, Nathalie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, biology, [SDV]Life Sciences [q-bio], Signal transducing adaptor protein, chemical and pharmacologic phenomena, Chemotaxis, Cell Biology, biology.organism_classification, Dictyostelium, Dictyostelium discoideum, Cell biology, [SDV] Life Sciences [q-bio], 03 medical and health sciences, 030104 developmental biology, Membrane protein, Arrestin, ComputingMilieux_MISCELLANEOUS, Calcium signaling, C2 domain
Abstract

Arrestins are key adaptor proteins that control the fate of cell-surface membrane proteins and modulate downstream signaling cascades. The Dictyostelium discoideum genome encodes six arrestin-related proteins, harboring additional modules besides the arrestin domain. Here, we studied AdcB and AdcC, two homologs that contain C2 and SAM domains. We showed that AdcC - in contrast to AdcB - responds to various stimuli (such as the chemoattractants cAMP and folate) known to induce an increase in cytosolic calcium by transiently translocating to the plasma membrane, and that calcium is a direct regulator of AdcC localization. This response requires the calcium-dependent membrane-targeting C2 domain and the double SAM domain involved in AdcC oligomerization, revealing a mode of membrane targeting and regulation unique among members of the arrestin clan. AdcB shares several biochemical properties with AdcC, including in vitro binding to anionic lipids in a calcium-dependent manner and auto-assembly as large homo-oligomers. AdcB can interact with AdcC; however, its intracellular localization is insensitive to calcium. Therefore, despite their high degree of homology and common characteristics, AdcB and AdcC are likely to fulfill distinct functions in amoebae.

Published
2018

7. Deubiquitinating enzymes related to autophagy : new therapeutic opportunities?

Author

Jacomin, Anne-Claire, Taillebourg, Emmanuel, Fauvarque, Marie-Odile, Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Bertacchi Griffi, Nathalie

Subjects
0301 basic medicine, autophagy, [SDV]Life Sciences [q-bio], Cellular homeostasis, Aggrephagy, Review, DUB, Deubiquitinating enzyme, 03 medical and health sciences, Chaperone-mediated autophagy, Ubiquitin, Mitophagy, ubiquitin, aggrephagy, chaperone-mediated autophagy, lcsh:QH301-705.5, small-molecule, ComputingMilieux_MISCELLANEOUS, biology, deubiquitinating enzymes, Autophagy, ubiquitin proteases, General Medicine, QP, 3. Good health, Cell biology, inhibitor, [SDV] Life Sciences [q-bio], 030104 developmental biology, mitophagy, lcsh:Biology (General), biology.protein, Deubiquitination
Abstract

Autophagy is an evolutionary conserved catabolic process that allows for the degradation of intracellular components by lysosomes. This process can be triggered by nutrient deprivation, microbial infections or other challenges to promote cell survival under these stressed conditions. However, basal levels of autophagy are also crucial for the maintenance of proper cellular homeostasis by ensuring the selective removal of protein aggregates and dysfunctional organelles. A tight regulation of this process is essential for cellular survival and organismal health. Indeed, deregulation of autophagy is associated with a broad range of pathologies such as neuronal degeneration, inflammatory diseases, and cancer progression. Ubiquitination and deubiquitination of autophagy substrates, as well as components of the autophagic machinery, are critical regulatory mechanisms of autophagy. Here, we review the main evidence implicating deubiquitinating enzymes (DUBs) in the regulation of autophagy. We also discuss how they may constitute new therapeutic opportunities in the treatment of pathologies such as cancers, neurodegenerative diseases or infections.

Published
2018

8. Combined inhibition of PI3K and Src kinases demonstrates synergistic therapeutic efficacy in clear-cell renal carcinoma

Author

Roelants, Caroline, Giacosa, Sofia, Pillet, Catherine, Bussat, Rémi, Champelovier, Pierre, Bastien, Olivier, Guyon, Laurent, Arnoux, Valentin, Cochet, Claude, Filhol, Odile, Invasion mechanisms in angiogenesis and cancer (IMAC), Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Département d'anatomie et cythologie pathologique, CHU Grenoble-Hôpital Michallon, Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Grenoble Alpes - UFR Médecine (UGA UFRM), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Grenoble Alpes (UGA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Bertacchi Griffi, Nathalie

Subjects
[SDV] Life Sciences [q-bio], 3D culture, [SDV]Life Sciences [q-bio], kidney cancer, protein kinase, synthetic lethality, targeted combinational therapy, ComputingMilieux_MISCELLANEOUS, Research Paper
Abstract

Potent inhibitors of PI3K (GDC-0941) and Src (Saracatinib) exhibit as individual agents, excellent oral anticancer activity in preclinical models and have entered phase II clinical trials in various cancers. We found that PI3K and Src kinases are dysregulated in clear cell renal carcinomas (ccRCCs), an aggressive disease without effective targeted therapies. In this study we addressed this challenge by testing GDC-0941 and Saracatinib as either single agents or in combination in ccRCC cell lines, as well as in mouse and PDX models. Our findings demonstrate that combined inhibition of PI3K and Src impedes cell growth and invasion and induces cell death of renal carcinoma cells providing preclinical evidence for a pairwise combination of these anticancer drugs as a rational strategy to improve renal cancer treatment.

Published
2018

9. The Rim Pathway Mediates Antifungal Tolerance in Candida albicans through Newly Identified Rim101 Transcriptional Targets, Including Hsp90 and Ipt1

Author

Morgane Champleboux, Danièle Maubon, Cécile Garnaud, Yan Wang, Encar García-Oliver, Sébastien Bailly, M. Cornet, Quentin Despinasse, Jérôme Govin, Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Adaptation et pathogénie des micro-organismes [Grenoble] (LAPM), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes - UFR Pharmacie (UGA UFRP), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Hypoxie : Physiopathologie Respiratoire et Cardiovasculaire (HP2 ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), ANR-11-PDOC-0011,EpiGam,Epigénétique et dynamique chromatinienne des gamètes : modèle 'spores de levure' et protéomique(2011), European Project: 304003,EC:FP7:PEOPLE,FP7-PEOPLE-2011-CIG,EPIGAM2(2012), Bertacchi Griffi, Nathalie, Retour Post-Doctorants - Epigénétique et dynamique chromatinienne des gamètes : modèle 'spores de levure' et protéomique - - EpiGam2011 - ANR-11-PDOC-0011 - RPDOC - VALID, Epigenetics and chromatin dynamics in gametes : using yeast spores and advanced proteomics - EPIGAM2 - - EC:FP7:PEOPLE2012-03-01 - 2016-02-29 - 304003 - VALID, Thérapeutique Recombinante Expérimentale (TIMC-IMAG-TheREx), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Etude de la dynamique des protéomes (EDyP ), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Department of Electrical and Computer Engineering [Minneapolis] (ECE), University of Minnesota [Twin Cities] (UMN), University of Minnesota System-University of Minnesota System, Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), and Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, Azoles, Antifungal Agents, Echinocandin, [SDV]Life Sciences [q-bio], Mutant, Antifungal drug, Microbiology, Fungal Proteins, 03 medical and health sciences, Echinocandins, Candida albicans, medicine, Pharmacology (medical), HSP90 Heat-Shock Proteins, Transcription factor, ComputingMilieux_MISCELLANEOUS, Pharmacology, chemistry.chemical_classification, biology, biology.organism_classification, Corpus albicans, [SDV] Life Sciences [q-bio], 030104 developmental biology, Infectious Diseases, chemistry, Susceptibility, Azole, medicine.drug, Signal Transduction
Abstract

Invasive candidiasis (IC) is a major cause of morbidity and mortality despite antifungal treatment. Azoles and echinocandins are used as first-line therapies for IC. However, their efficacy is limited by yeast tolerance and the emergence of acquired resistance. Tolerance is a reversible stage created due to the yeast's capacity to counter antifungal drug exposure, leading to persistent growth. For Candida albicans , multiple stress signaling pathways have been shown to contribute to this adaptation. Among them, the pH-responsive Rim pathway, through its transcription factor Rim101p, was shown to mediate azole and echinocandin tolerance. The Rim pathway is fungus specific, is conserved among the members of the fungal kingdom, and plays a key role in pathogenesis and virulence. The present study aimed at confirming the role of Rim101p and investigating the implication of the other Rim proteins in antifungal tolerance in C. albicans , as well as the mechanisms underlying it. Time-kill curve experiments and colony formation tests showed that genetic inhibition of all the Rim factors enhances echinocandin and azole antifungal activity. Through RNA sequencing analysis of a rim101 −/− mutant, a strain constitutively overexpressing RIM101 , and control strains, we discovered novel Rim-dependent genes involved in tolerance, including HSP90 , encoding a major molecular chaperone, and IPT1 , involved in sphingolipid biosynthesis. Rim mutants were also hypersensitive to pharmacological inhibition of Hsp90. Taken together, these data suggest that Rim101 acts upstream of Hsp90 and that targeting the Rim pathway in combination with existing antifungal drugs may represent a promising antifungal strategy to indirectly but specifically target Hsp90 in yeasts.

Published
2018

10. BMP9, but not BMP10, acts as a quiescence factor on tumor growth, vessel normalization and metastasis in a mouse model of breast cancer

Author

Marie, Ouarné, Claire, Bouvard, Gabriela, Boneva, Christine, Mallet, Johnny, Ribeiro, Agnès, Desroches-Castan, Emmanuelle, Soleilhac, Emmanuelle, Tillet, Olivier, Peyruchaud, Sabine, Bailly, Bertacchi Griffi, Nathalie, Biologie du Cancer et de l'Infection (BCI ), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Physiopathologie, diagnostic et traitements des maladies osseuses / Pathophysiology, Diagnosis & Treatments of Bone Diseases (LYOS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Lyon, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Mecanismes et Traitements des Metastases Osseuses des Tumeurs Solides, and M.O. was supported by the PhD program of CEA/DRF (IRTELIS). This work was supported by INSERM (Institut National de la Santé et de la Recherche Médicale, U1036), CEA (Commissariat à l’Energie Atomique et aux Energies Alternatives, DRF/BIG/BCI), UGA (University Grenoble-Alpes, BCI), the Ligues Départementales contre le Cancer de la Loire et de la Savoie, the association Maladie de Rendu-Osler (AMRO-HHT France), the Association pour la Recherche sur le Cancer (ARC), the Fondation Recherche Médicale (FRM) and the Labex GRAL. We thank the Fondation Maladies Rares (FMR) for the generation of Bmp10 lox/lox mice.

Subjects
Activin Receptors, Type II, [SDV]Life Sciences [q-bio], Breast Neoplasms, Mammary Neoplasms, Animal, BMP9, lcsh:RC254-282, Tumor angiogenesis, Metastasis, Mice, Growth Differentiation Factor 2, Animals, Humans, Neoplasm Metastasis, Mice, Knockout, Research, Endothelial Cells, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, E0771, Mammary tumor, BMP10, [SDV] Life Sciences [q-bio], Disease Models, Animal, Bone Morphogenetic Proteins, Female, Endothelium, Vascular, Activin Receptors, Type I, Signal Transduction
Abstract

Background Angiogenesis has become an attractive target for cancer therapy. However, despite the initial success of anti-VEGF (Vascular endothelial growth factor) therapies, the overall survival appears only modestly improved and resistance to therapy often develops. Other anti-angiogenic targets are thus urgently needed. The predominant expression of the type I BMP (bone morphogenetic protein) receptor ALK1 (activin receptor-like kinase 1) in endothelial cells makes it an attractive target, and phase I/II trials are currently being conducted. ALK1 binds with strong affinity to two ligands that belong to the TGF-ß family, BMP9 and BMP10. In the present work, we addressed their specific roles in tumor angiogenesis, cancer development and metastasis in a mammary cancer model. Methods For this, we used knockout (KO) mice for BMP9 (constitutive Gdf2-deficient), for BMP10 (inducible Bmp10-deficient) and double KO mice (Gdf2 and Bmp10) in a syngeneic immunocompetent orthotopic mouse model of spontaneous metastatic breast cancer (E0771). Results Our studies demonstrate a specific role for BMP9 in the E0771 mammary carcinoma model. Gdf2 deletion increased tumor growth while inhibiting vessel maturation and tumor perfusion. Gdf2 deletion also increased the number and the mean size of lung metastases. On the other hand, Bmp10 deletion did not significantly affect the E0771 mammary model and the double deletion (Gdf2 and Bmp10) did not lead to a stronger phenotype than the single Gdf2 deletion. Conclusions Altogether, our data show that in a tumor environment BMP9 and BMP10 play different roles and thus blocking their shared receptor ALK1 is maybe not appropriate. Indeed, BMP9, but not BMP10, acts as a quiescence factor on tumor growth, lung metastasis and vessel normalization. Our results also support that activating rather than blocking the BMP9 pathway could be a new strategy for tumor vessel normalization in order to treat breast cancer. Electronic supplementary material The online version of this article (10.1186/s13046-018-0885-1) contains supplementary material, which is available to authorized users.

Published
2018

11. A meiotic XPF–ERCC1-like complex recognizes joint molecule recombination intermediates to promote crossover formation

Author

Raphaelle Laureau, Lepakshi Ranjha, Petr Cejka, Jessica Andreani, Ambra Fernandez-Vega, Yohann Couté, Raphael Guerois, Arnaud de Muyt, Valérie Borde, Daniel Holoch, Claire Ramus, Raphaël Margueron, Alexandra Pyatnitskaya, Jérôme Govin, Elodie Girard, Sorbonne Université - Faculté de Médecine (SU FM), Sorbonne Université (SU), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Assemblage moléculaire et intégrité du génome (AMIG), Département Biochimie, Biophysique et Biologie Structurale (B3S), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Universität Zürich [Zürich] = University of Zurich (UZH), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Dynamique de l'information génétique : bases fondamentales et cancer (DIG CANCER), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Grenoble Alpes - École supérieure du professorat et de l'éducation - Académie de Grenoble (UGA ESPE Grenoble), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Génétique et Biologie du Développement, Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Dynamique de l'information génétique : bases fondamentales et cancer (DIGBFC), Bertacchi Griffi, Nathalie, Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)

Subjects
0301 basic medicine, Saccharomyces cerevisiae Proteins, Chromosomal Proteins, Non-Histone, [SDV]Life Sciences [q-bio], Crossover, XPF–ERCC1, homologous recombination, Biology, 03 medical and health sciences, chemistry.chemical_compound, Protein Domains, Meiosis, Chromosome (genetic algorithm), Genetics, meiosis, DNA Breaks, Double-Stranded, Crossing Over, Genetic, Synaptonemal complex, Zip2–Zip4–Spo16 complex, ComputingMilieux_MISCELLANEOUS, Nuclease, crossover, Endodeoxyribonucleases, DNA, Cell biology, DNA-Binding Proteins, [SDV] Life Sciences [q-bio], 030104 developmental biology, chemistry, biology.protein, Chromosomes, Fungal, Homologous recombination, Microtubule-Associated Proteins, Recombination, Research Paper, Developmental Biology
Abstract

Meiotic crossover formation requires the stabilization of early recombination intermediates by a set of proteins and occurs within the environment of the chromosome axis, a structure important for the regulation of meiotic recombination events. The molecular mechanisms underlying and connecting crossover recombination and axis localization are elusive. Here, we identified the ZZS (Zip2–Zip4–Spo16) complex, required for crossover formation, which carries two distinct activities: one provided by Zip4, which acts as hub through physical interactions with components of the chromosome axis and the crossover machinery, and the other carried by Zip2 and Spo16, which preferentially bind branched DNA molecules in vitro. We found that Zip2 and Spo16 share structural similarities to the structure-specific XPF–ERCC1 nuclease, although it lacks endonuclease activity. The XPF domain of Zip2 is required for crossover formation, suggesting that, together with Spo16, it has a noncatalytic DNA recognition function. Our results suggest that the ZZS complex shepherds recombination intermediates toward crossovers as a dynamic structural module that connects recombination events to the chromosome axis. The identification of the ZZS complex improves our understanding of the various activities required for crossover implementation and is likely applicable to other organisms, including mammals., Genes & Development, 32 (3-4), ISSN:0890-9369, ISSN:1549-5477

Published
2018
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12. Evidence for rRNA 2′-O-methylation plasticity: Control of intrinsic translational capabilities of human ribosomes

Author

Virginie Marchand, Yohann Couté, Yuri Motorin, Jenny Erales, Virginie Marcel, Pierre Bertin, Marat Yusupov, Agnès Baudin-Baillieu, Jean-Jacques Diaz, Florian Laforêts, Gabriel Therizols, Annie Adrait, Frédéric Catez, Olivier Namy, Melanie Meyer, Sandra Gillot, Stephane Belin, Baptiste Panthu, Sandra E. Ghayad, Théophile Ohlmann, Maxime Garcia, Centre de Recherche en Cancérologie de Lyon ( CRCL ), Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Bioingénierie Moléculaire, Cellulaire et Thérapeutique ( BMCT ), Université de Lorraine ( UL ) -Centre National de la Recherche Scientifique ( CNRS ), Centre International de Recherche en Infectiologie ( CIRI ), École normale supérieure - Lyon ( ENS Lyon ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), École normale supérieure - Lyon ( ENS Lyon ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Laboratoire de Biologie à Grande Échelle ( BGE - UMR S1038 ), Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université Grenoble Alpes [Saint Martin d'Hères]-Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Institut de Biosciences et Biotechnologies de Grenoble ( BIG ), Université de Grenoble-Alpes-Commissariat à l'Énergie Atomique et aux Énergies Alternatives (CEA) - Grenoble-Centre National de la Recherche Scientifique ( CNRS ), Institut de Génétique et de Biologie Moléculaire et Cellulaire ( IGBMC ), Université de Strasbourg ( UNISTRA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de génétique et microbiologie [Orsay] ( IGM ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Ingénierie Moléculaire et Physiopathologie Articulaire ( IMoPA ), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut des Sciences Pharmaceutiques et Biologiques [Lyon] (ISPB), Bioingénierie Moléculaire, Cellulaire et Thérapeutique (BMCT), Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Contrôle traductionnel des ARNm eucaryotes et viraux – Translational control of Eukaryotic and Viral RNAs, Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Ingénierie Moléculaire et Physiopathologie Articulaire (IMoPA), Centre International de Recherche en Infectiologie - UMR (CIRI), Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université de Lorraine (UL), Bertacchi Griffi, Nathalie, École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)

Subjects
0301 basic medicine, Chromosomal Proteins, Non-Histone, [SDV]Life Sciences [q-bio], 5.8S ribosomal RNA, [ SDV.BBM.BM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Biology, Methylation, RNA epigenetics, 03 medical and health sciences, 23S ribosomal RNA, Humans, Ribosome profiling, ComputingMilieux_MISCELLANEOUS, Multidisciplinary, [ SDV.BC ] Life Sciences [q-bio]/Cellular Biology, fibrillarin, RRNA 2'-O-methylation, translational control, 2′-O-methylation, Translation (biology), Ribosomal RNA, Biological Sciences, Molecular biology, Cell biology, [SDV] Life Sciences [q-bio], Internal ribosome entry site, 030104 developmental biology, Gene Expression Regulation, RNA, Ribosomal, Gene Knockdown Techniques, Protein Biosynthesis, Eukaryotic Ribosome, ribosomal RNA, HeLa Cells
Abstract

Ribosomal RNAs (rRNAs) are main effectors of messenger RNA (mRNA) decoding, peptide-bond formation, and ribosome dynamics during translation. Ribose 2′-O-methylation (2′-O-Me) is the most abundant rRNA chemical modification, and displays a complex pattern in rRNA. 2′-O-Me was shown to be essential for accurate and efficient protein synthesis in eukaryotic cells. However, whether rRNA 2′-O-Me is an adjustable feature of the human ribosome and a means of regulating ribosome function remains to be determined. Here we challenged rRNA 2′-O-Me globally by inhibiting the rRNA methyl-transferase fibrillarin in human cells. Using RiboMethSeq, a nonbiased quantitative mapping of 2′-O-Me, we identified a repertoire of 2′-O-Me sites subjected to variation and demonstrate that functional domains of ribosomes are targets of 2′-O-Me plasticity. Using the cricket paralysis virus internal ribosome entry site element, coupled to in vitro translation, we show that the intrinsic capability of ribosomes to translate mRNAs is modulated through a 2′-O-Me pattern and not by nonribosomal actors of the translational machinery. Our data establish rRNA 2′-O-Me plasticity as a mechanism providing functional specificity to human ribosomes.

Published
2017

13. Experimental investigation of the evaporation of droplets in diamagnetic levitation

Author

Semenov, Sergey, Haguet, Vincent, Jeandey, Christian, Antoni, Mickaël, Department of Chemistry, Lomonosov Moscow State University, Lomonosov Moscow State University (MSU), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Matériaux divisés, interfaces, réactivité, électrochimie (MADIREL), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Institut de Chimie du CNRS (INC), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Aix Marseille Université (AMU)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Bertacchi Griffi, Nathalie

Subjects
[PHYS]Physics [physics], [PHYS] Physics [physics]
Abstract

International audience; The time evolution of levitating pure water and water-ethanol droplets is investigated. Droplets are trapped in stable diamagnetic levitation in the magnetic well produced by a cylindrical permanent magnet. Radius of evaporating droplets and their temperature differences with air are monitored over time using a thermographic camera. Experimental setup A cylindrical permanent magnet made by 8 NdFeB sectors with radial magnetization and covered by a pole piece (Figure 1(a)) is used to trap diamagnetic droplets in levitation. A screwed brass base maintains all the NdFeB sectors together. In addition, a cylindrical hole of 1.6 mm diameter is realized along the symmetry axis of this set up. The gap between the sectors and the pole piece is adjusted in order to produce a potential well where diamagnetic levitation can be achieved [1,2]. Droplets are generated by a piezoelectric jetting device. It consists of a tiny glass tube covered with a piezoelectric ceramic (Figure 1(b)). A capillary transports the liquid solution from a 15 mL test tube to the glass tube. The applied potential in the piezoelectric ceramic is computer-controlled. As the ceramic shrinks, pressure builds up in the glass tube and a jet of microdroplets (i.e. droplets with a radius of few tens of micrometers) is ejected from the tube nozzle (Figure 1(b)). The ejection energy depends upon the applied potential and therefore determines the properties of the jet. The main difficulty in this procedure consists in the requirement for microdroplets to fall into the magnetic well. As the jet is extremely sensitive to disturbances of environment, it tends to behave randomly. A confinement facility is used to limit this drawback. Once microdroplets are trapped in the magnetic well, they spontaneously coalesce after few seconds and form larger droplets. When millimeter-sized droplets are produced (i.e., with a radius larger than 200 m), the jetting is stopped. Figure 1. (a) Top view of the permanent magnet. (b) Experimental setup made of a piezoelectric nozzle, the magnet and a thermographic camera. The microdroplet jet from the piezoelectric nozzle to the magnetic levitation site is schematically plotted with a full blue line. A thermographic camera placed above the magnetic levitation site captures infrared (IR) radiation emitted by the droplet in the spectral wavelength range from 7 m to 10 m. It records a field of view (FOV) of ~2.0 mm x 1.6 mm and allows measuring the droplet size as well as the intensity of emitted IR radiation over time. Images are grabbed on a 160 x 128 matrix of sensors and post treated with the ImageJ software. The spatial resolution is about 12.8 m. Thermography measurements depend on the IR emissivity and observation angles. Accessing to the temperature field in the full FOV is therefore not straightforward (although sensitivity of the camera is less than 20 mK), especially for ethanol that is transparent in the considered spectral range. Used liquid solutions comprise pure water and water/ethanol mixtures (20%, 40%, 60% and 80%, 96% v/v). Water is deionized. In order to achieve a better reproducibility, experiments were carried out several times for each solution. Room temperature T and humidity H are 203 °C and 305%, respectively.

Published
2017

14. Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries

Author

Pandey, Radha Raman, Homolka, David, Chen, Kuan-Ming, Sachidanandam, Ravi, Fauvarque, Marie-Odile, Pillai, Ramesh, Miska, Eric, Department of Molecular Biology [Geneva, Switzerland], University of Geneva [Switzerland], Cold Spring Harbor Laboratory, Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université de Genève = University of Geneva (UNIGE), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Department of Oncological Sciences [New York, NY, USA], Icahn School of Medicine at Mount Sinai [New York] (MSSM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), This work was supported by the Swiss National Science Foundation: ERC Transfer Grant (GermMethylation), Project Grant (Origin-of-pi), and funding from the NCCR RNA & Disease to RSP. Funding is also acknowledged from the National Institutes of Health (1R01HD069592, and Bertacchi Griffi, Nathalie

Subjects
0301 basic medicine, Cancer Research, Small RNA, Somatic cell, [SDV]Life Sciences [q-bio], Biochemistry, Germline, Animal Cells, Medicine and Health Sciences, Drosophila Proteins, RNA, Small Interfering, Genetics (clinical), ComputingMilieux_MISCELLANEOUS, Ribonucleoprotein, Genetics, biology, Drosophila Melanogaster, Physics, Animal Models, Enzymes, Precipitation Techniques, [SDV] Life Sciences [q-bio], Insects, Ovaries, Physical sciences, Nucleic acids, Experimental Organism Systems, Nucleic acid thermodynamics, Helicases, Drosophila, RNA annealing, Female, Anatomy, Cellular Types, Drosophila melanogaster, Research Article, endocrine system, Arthropoda, lcsh:QH426-470, Precursor Cells, Annealing (genetics), Biophysics, Piwi-interacting RNA, Biosynthesis, Research and Analysis Methods, 03 medical and health sciences, Model Organisms, Animals, Immunoprecipitation, RasiRNA, Uridine, Molecular Biology, Ecology, Evolution, Behavior and Systematics, urogenital system, Ovary, Organisms, Reproductive System, Biology and Life Sciences, Proteins, Cell Biology, RNA helicases, biology.organism_classification, Invertebrates, lcsh:Genetics, Fertility, Germ Cells, 030104 developmental biology, Enzymology, DNA Transposable Elements, RNA, Biogenesis
Abstract

Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5′→3′ directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5′ ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5′→3′ helicase activity, and mutations that abolish this activity also reduce piRNA processing in vivo. Another somatic piRNA pathway factor Yb, an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery., Author summary PIWI-interacting RNAs (piRNAs) are 24–30 nucleotide (nt) small RNAs that are exclusively expressed in animal germlines and are essential for suppression of transposable elements or ‘jumping genes’. Millions of piRNAs are produced from single-stranded transcripts that arise from large RNA polymerase II transcription units called piRNA clusters. Since they resemble other RNA pol II products like protein-coding mRNAs or non-coding RNAs, how piRNA precursors are selectively recruited to the biogenesis machinery is a big mystery. Here we demonstrate that artificial tethering of specific piRNA biogenesis factors to a reporter transcript is sufficient to identify it as a piRNA precursor in Drosophila ovaries and in an ovarian cell culture model. This results in fragmentation of the transcript into thousands of piRNAs, which are generated as a series of non-overlapping (phased) fragments that are loaded into PIWI proteins. Our study indicates that localization of the tethered factors to cytoplasmic perinuclear granules called nuage/Yb bodies is necessary for processing. Any mutations in the tethered factors that disrupt this sub-cellular localization, reduce processing. We believe that our strategy will allow generation of designer small RNAs to target almost any germline gene, and also provides a valuable tool to dissect the molecular mechanism of piRNA biogenesis.

Published
2017

15. Introducing plasma/serum glycodepletion for the targeted proteomics analysis of cytolysis biomarkers

Author

Guillaume Picard, Yohann Couté, Pauline Maes, Sandrine Donadio-Andréi, Michel Jaquinod, Claire Lacoste, Philippe Ichai, Virginie Brun, Jamila Faivre, Christophe Bruley, Jérôme Garin, Mathilde Louwagie, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM), Physiopathologie et traitement des maladies du foie, Université Paris-Sud - Paris 11 (UP11)-Hôpital Paul Brousse-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Spectrométrie de Masse BioOrganique, Département des Sciences Analytiques, Institut Pluridisciplinaire Hubert Curien (LSMBO-DSA-IPHC), Centre National de la Recherche Scientifique (CNRS), Hotel Technol Morandat, Independent, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Master Information-Communication spécialité Documentation, Université de Poitiers, Institut universitaire de formation des maîtres - Nord-Pas-de-Calais (IUFM Nord-Pas-de-Calais), Université d'Artois (UA), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire d'étude de la dynamique des protéomes (LEDyP), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Pathogénèse et traitement de l'hépatite fulminante et du cancer du foie, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), Développement de la protéomique comme outil d'investigation fonctionelle et d'annotation des génomes, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Bertacchi Griffi, Nathalie, and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
0301 basic medicine, Proteomics, Glycosylation, [SDV]Life Sciences [q-bio], Context (language use), Chemical Fractionation, Analytical Chemistry, Hepatitis, 03 medical and health sciences, Tandem Mass Spectrometry, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Humans, Amino Acid Sequence, Secretory pathway, ComputingMilieux_MISCELLANEOUS, Glycoproteins, chemistry.chemical_classification, Chemistry, Albumin, Proteins, Blood Proteins, Blood proteins, 3. Good health, [SDV] Life Sciences [q-bio], Cytolysis, 030104 developmental biology, Biochemistry, Biomarker (medicine), Glycoprotein, Biomarkers, Chromatography, Liquid
Abstract

A major class of clinical biomarkers is constituted of intracellular proteins which are leaking into the blood following ischemia, exposure to toxic xenobiotics or mechanical aggression. Their ectopic presence in plasma/serum is an indicator of tissue damage and raises a warning signal. These proteins, referred to as cytolysis biomarkers, are generally of cytoplasmic origin and as such, are devoid of glycosylation. In contrast, most plasma/serum proteins originate from the hepatic secretory pathway and are heavily glycosylated (at the exception of albumin). Recent advances in targeted proteomics have supported the parallelized evaluation of new blood biomarkers. However, these analytical methods must be combined with prefractionation strategies that reduce the complexity of plasma/serum matrix. In this article, we present the glycodepletion method, which reverses the hydrazide-based glycocapture concept to remove plasma/serum glycoproteins from plasma/serum matrix and facilitates the detection of cytolysis biomarkers. Glycodepletion was integrated to a targeted proteomics pipeline to evaluate 4 liver cytolysis biomarker candidates in the context of acetaminophen-induced acute hepatitis.

Published
2017

16. Histone Variant H2A.L.2 Guides Transition Protein-Dependent Protamine Assembly in Male Germ Cells

Author

Sophie Barral, Patrick Héry, Jérôme Govin, Matthieu Gérard, Fayçal Boussouar, Sandrine Curtet, Emilie Montellier, Hitoshi Kurumizaka, Denis Puthier, Takashi Urahama, Sophie Rousseaux, Maud de Dieuleveult, Guillaume Charbonnier, François Fenaille, Saadi Khochbin, Hitoshi Shiota, Yuichi Morozumi, Hiroki Tanaka, Thierry Buchou, Nicolas Fernandez-Nunez, Yohann Couté, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Waseda University [Tokyo, Japan], Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et de Technologies de Saclay (IBITECS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Interactions et dynamique des environnements de surface (IDES), Université Paris-Sud - Paris 11 (UP11)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), Technologies avancées pour le génôme et la clinique (TAGC), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Etude du Métabolisme des Médicaments (LEMM), Service de Pharmacologie et Immunoanalyse (SPI), Médicaments et Technologies pour la Santé (MTS), Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Paris-Saclay-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Médicaments et Technologies pour la Santé (MTS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Biologie moléculaire et cellulaire de la différenciation, Université Joseph Fourier - Grenoble 1 (UJF)-Institut Albert Bonniot-Institut National de la Santé et de la Recherche Médicale (INSERM), DBJC-SBMS, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Epigénomique des mammifères (REMOD), Département Biologie des Génomes (DBG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Waseda University, Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Bertacchi Griffi, Nathalie, Institut National de la Santé et de la Recherche Médicale (INSERM)-EFS-CHU Grenoble-Université Joseph Fourier - Grenoble 1 (UJF), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Université Paris-Saclay-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
Male, 0301 basic medicine, NAP1L4, Mice, 129 Strain, Chromosomal Proteins, Non-Histone, Spermiogenesis, [SDV]Life Sciences [q-bio], Biology, Transfection, Genome, Histones, 03 medical and health sciences, 0302 clinical medicine, Chlorocebus aethiops, Databases, Genetic, Animals, Nucleosome, Genetic Predisposition to Disease, Protamines, Spermatogenesis, Molecular Biology, Infertility, Male, Mice, Knockout, Genetics, Transition (genetics), Computational Biology, Gene Expression Regulation, Developmental, Cell Biology, Chromatin Assembly and Disassembly, Spermatozoa, Protamine, Chromatin, Nucleosomes, Cell biology, [SDV] Life Sciences [q-bio], Transformation (genetics), Fertility, Phenotype, 030104 developmental biology, Histone, COS Cells, biology.protein, 030217 neurology & neurosurgery
Abstract

International audience; Histone replacement by transition proteins (TPs) and protamines (Prms) constitutes an essential step for the successful production of functional male gametes, yet nothing is known on the underlying functional interplay between histones, TPs, and Prms. Here, by studying spermatogenesis in the absence of a spermatid-specific histone variant, H2A.L.2, we discover a fundamental mechanism involved in the transformation of nucleosomes into nucleoprotamines. H2A.L.2 is synthesized at the same time as TPs and enables their loading onto the nucleosomes. TPs do not displace histones but rather drive the recruitment and processing of Prms, which are themselves responsible for histone eviction. Altogether, the incorporation of H2A.L.2 initiates and orchestrates a series of successive transitional states that ultimately shift to the fully compacted genome of the mature spermatozoa. Hence, the current view of histone-to-nucleoprotamine transition should be revisited and include an additional step with H2A.L.2 assembly prior to the action of TPs and Prms.

Published
2017

17. CAP-Gly proteins contribute to microtubule-dependent trafficking via interactions with the C-terminal aromatic residue of α-tubulin

Author

Andrieux, Annie, Aubry, Laurence, Boscheron, Cécile, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), [GIN] Grenoble Institut des Neurosciences (GIN), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Bertacchi Griffi, Nathalie

Subjects
p150Glued, [SDV]Life Sciences [q-bio], fungi, Brief Report - Commissioned, Biological Transport, [SDV] Life Sciences [q-bio], microtubules, trafficking, Tubulin, +Tips tyrosination, Animals, Humans, CLIP170, endosome, Microtubule-Associated Proteins, ComputingMilieux_MISCELLANEOUS
Abstract

SUMMARY In mammals, the C-terminal tyrosine residue of α-tubulin is subjected to removal/re-addition cycles resulting in tyrosinated microtubules and detyrosinated Glu-microtubules. CLIP170 and its yeast ortholog (Bik1) interact weakly with Glu-microtubules. Recently, we described a Microtubule- Rho1- and Bik1-dependent mechanism involved in Snc1 routing. Here, we further show a contribution of the yeast p150Glued ortholog (Nip100) in Snc1 trafficking. Both CLIP170 and p150Glued are CAP-Gly-containing proteins that belong to the microtubule +end-tracking protein family (known as +Tips). We discuss the +Tips-dependent role of microtubules in trafficking, the role of CAP-Gly proteins as possible molecular links between microtubules and vesicles, as well as the contribution of the Rho1-GTPase to the regulation of the +Tips repertoire and the partners associated with microtubules.

Published
2017

18. New Insight Into the Pathogenesis of Cerebral Small-Vessel Diseases

Author

Matilde Valeria Ursini, Kristin Müller, Markus Schwaninger, Gilles Courtois, Institute for Experimental and Clinical Pharmacology and Toxicology [Lübeck, Germany], University of Lübeck [Germany], Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institute of Genetics and Biophysics - 'Adriano Buzzati-Traverso' [Naples, Italy] ( IGB-CNR), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Bertacchi Griffi, Nathalie, Universität zu Lübeck = University of Lübeck [Lübeck], and Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
0301 basic medicine, Pathology, medicine.medical_specialty, [SDV]Life Sciences [q-bio], CADASIL, Blood–brain barrier, Leukoencephalopathy, 03 medical and health sciences, 0302 clinical medicine, IKBKG, Medicine, Animals, Humans, Vascular dementia, ComputingMilieux_MISCELLANEOUS, Advanced and Specialized Nursing, transforming growth factor beta, biology, business.industry, Incontinentia pigmenti, Transforming growth factor beta, blood-brain barrier, medicine.disease, 3. Good health, [SDV] Life Sciences [q-bio], 030104 developmental biology, medicine.anatomical_structure, Cerebral Small Vessel Diseases, biology.protein, Neurology (clinical), Alzheimer's disease, Alzheimer disease, Cardiology and Cardiovascular Medicine, business, incontinentia pigmenti, 030217 neurology & neurosurgery
Abstract

Cerebral small-vessel diseases (SVD) are a common cause of vascular dementia and disability in the elderly. Apart from risk containment, little can be done to prevent or to treat SVD. Although most cases occur sporadically, hereditary forms of the disease provide the chance to elucidate the molecular and cellular mechanisms leading to microvascular pathology. Along this line, authoritative reviews on SVD have been published.1-3 Here, we would like to extend current concepts of SVD by highlighting a molecular pathway that seems to be involved in several well-defined SVD, some of which are hereditary. As a new piece of evidence, we will focus on incontinentia pigmenti (IP), which has recently been shown to be associated with microvascular pathology of the brain and retina.4,5 As will be elaborated below, IP is a SVD that does not present in the elderly but rather during infancy. The young age of onset and the nonprogressive microvascular pathology lead to a clinical picture that differs from other SVD. We suggest the term developmental SVD for this form of presentation. IP is caused by mutations in the NEMO gene (NF-kappaB essential modulator), an essential modulator of the transcription factor nuclear factor (NF)-?B.6 NEMO maintains the viability of brain endothelial cells and stabilizes the blood-brain barrier (BBB).5 In NEMO-deficient animals, there is rarefaction of cerebral capillaries and hypoperfusion of the central nervous system (CNS). Disruption of the transforming growth factor (TGF)-?-activated kinase (TAK1) upstream of NEMO produces a similar pathology.5 Interestingly, TGF-? has also been involved in the pathogenesis of cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), and the vascular changes of Alzheimer disease (AD),7-11 suggesting disturbed TGF-? signaling as a common ...

Published
2017

19. DAPAR & ProStaR: software to perform statistical analyses in quantitative discovery proteomics

Author

Wieczorek, Samuel, Combes, Florence, Lazar, Cosmin, Giai Gianetto, Quentin, Gatto, Laurent, Dorffer, Alexia, Hesse, Anne-Marie, Couté, Yohann, Ferro, Myriam, Bruley, Christophe, Burger, Thomas, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Département Image et Traitement Information (ITI), Université européenne de Bretagne - European University of Brittany (UEB)-Télécom Bretagne-Institut Mines-Télécom [Paris] (IMT), Bertacchi Griffi, Nathalie, Gatto, Laurent [0000-0002-1520-2268], and Apollo - University of Cambridge Repository

Subjects
Proteomics, [SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], Gene Expression, Proteins, Peptides, Applications Notes, Software, ComputingMilieux_MISCELLANEOUS
Abstract

UNLABELLED: DAPAR and ProStaR are software tools to perform the statistical analysis of label-free XIC-based quantitative discovery proteomics experiments. DAPAR contains procedures to filter, normalize, impute missing value, aggregate peptide intensities, perform null hypothesis significance tests and select the most likely differentially abundant proteins with a corresponding false discovery rate. ProStaR is a graphical user interface that allows friendly access to the DAPAR functionalities through a web browser. AVAILABILITY AND IMPLEMENTATION: DAPAR and ProStaR are implemented in the R language and are available on the website of the Bioconductor project (http://www.bioconductor.org/). A complete tutorial and a toy dataset are accompanying the packages. CONTACT: samuel.wieczorek@cea.fr, florence.combes@cea.fr, thomas.burger@cea.fr.

Published
2016

20. A functional endosomal pathway is necessary for lysosome biogenesis in Drosophila

Author

Anne-Claire Jacomin, Marie-Odile Fauvarque, Emmanuel Taillebourg, Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Bertacchi Griffi, Nathalie

Subjects
0301 basic medicine, Endosome, Cathepsin L, [SDV]Life Sciences [q-bio], Endosomes, Biology, Endocytosis, Deubiquitinating enzyme, QH301, 03 medical and health sciences, Lysosomal-Associated Membrane Protein 1, Lysosome, Autophagy, medicine, Animals, Endosomal system, ComputingMilieux_MISCELLANEOUS, Lysosomal biogenesis, Autophagosomes, Cell Biology, Cell biology, [SDV] Life Sciences [q-bio], Drosophila melanogaster, 030104 developmental biology, medicine.anatomical_structure, Membrane protein, Biochemistry, biology.protein, Lysosomes, Biogenesis, Research Article
Abstract

Background Lysosomes are the major catabolic compartment within eukaryotic cells, and their biogenesis requires the integration of the biosynthetic and endosomal pathways. Endocytosis and autophagy are the primary inputs of the lysosomal degradation pathway. Endocytosis is specifically needed for the degradation of membrane proteins whereas autophagy is responsible for the degradation of cytoplasmic components. We previously identified the deubiquitinating enzyme UBPY/USP8 as being necessary for lysosomal biogenesis and productive autophagy in Drosophila. Because UBPY/USP8 has been widely described for its function in the endosomal system, we hypothesized that disrupting the endosomal pathway itself may affect the biogenesis of the lysosomes. Results In the present study, we blocked the progression of the endosomal pathway at different levels of maturation of the endosomes by expressing in fat body cells either dsRNAs or dominant negative mutants targeting components of the endosomal machinery: Shibire, Rab4, Rab5, Chmp1 and Rab7. We observed that inhibition of endosomal trafficking at different steps in vivo is systematically associated with defects in lysosome biogenesis, resulting in autophagy flux blockade. Conclusion Our results show that the integrity of the endosomal system is required for lysosome biogenesis and productive autophagy in vivo. Electronic supplementary material The online version of this article (doi:10.1186/s12860-016-0115-7) contains supplementary material, which is available to authorized users.

Published
2016

21. Human Proteome Project Mass Spectrometry Data Interpretation Guidelines 2.1

Author

Gilbert S. Omenn, Jennifer E. Van Eyk, Mark S. Baker, Robert L. Moritz, Christopher M. Overall, Ulrike Kusebauch, William S. Hancock, Henning Hermjakob, Eric W. Deutsch, Lennart Martens, Ruedi Aebersold, Yves Vandenbrouck, Young Ki Paik, Lydie Lane, Susan T. Weintraub, Institute for Systems Biology [Seattle] (ISB), Centre for Blood Research (CBR), University of British Columbia (UBC), Australian Proteome Analysis Facility, Macquarie University, Yonsei Proteome Research Center and Department of Biochemistry, Yonsei University, Département de science des protéines humaines [Genève], Université de Genève (UNIGE)-Faculté de médecine [Genève], Universiteit Gent = Ghent University [Belgium] (UGENT), Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), European Bioinformatics Institute [Hinxton] (EMBL-EBI), EMBL Heidelberg, Eidgenössische Technische Hochschule - Swiss Federal Institute of Technology [Zürich] (ETH Zürich), Université de Genève = University of Geneva (UNIGE)-Faculté de médecine [Genève], Universiteit Gent = Ghent University (UGENT), and Bertacchi Griffi, Nathalie

Subjects
0301 basic medicine, Proteomics, Databases, Factual, Proteome, Computer science, [SDV]Life Sciences [q-bio], Guidelines as Topic, Biochemistry, Mass Spectrometry, Article, 03 medical and health sciences, Error analysis, Human proteome project, Humans, ddc:576, Databases, Protein, ComputingMilieux_MISCELLANEOUS, NeXtProt, Comparability, Data interpretation, General Chemistry, Data science, Checklist, [SDV] Life Sciences [q-bio], 030104 developmental biology, Protein identification, PeptideAtlas
Abstract

Every data-rich community research effort requires a clear plan for ensuring the quality of the data interpretation and comparability of analyses. To address this need within the Human Proteome Project (HPP) of the Human Proteome Organization (HUPO), we have developed through broad consultation a set of mass spectrometry data interpretation guidelines that should be applied to all HPP data contributions. For submission of manuscripts reporting HPP protein identification results, the guidelines are presented as a one-page checklist containing fifteen essential points followed by two pages of expanded description of each. Here, we present an overview of the guidelines and provide an in-depth description of each of the fifteen elements to facilitate understanding of the intentions and rationale behind the guidelines, both for authors and for reviewers. Broadly, these guidelines provide specific directions regarding how HPP data are to be submitted to mass spectrometry data repositories, how error analysis should be presented, and how detection of novel proteins should be supported with additional confirmatory evidence. These guidelines, developed by the HPP community, are presented to the broader scientific community for further discussion.

Published
2016

22. Looking for Missing Proteins in the Proteome of Human Spermatozoa: An Update

Author

Yves Vandenbrouck, Marlène Marcellin, Thomas Fréour, Anne-Marie Hesse, Karima Chaoui, Christine Carapito, Christophe Bruley, Anne Gonzalez de Peredo, Karine Rondel, Emmanuelle Mouton-Barbosa, Thibault Robin, Jérôme Garin, Charlotte Macron, Charles Pineau, Lydie Lane, Sarah Cianférani, Paula D. Duek, Loren Méar, Cecilia Lindskog, Myriam Ferro, Emmanuelle Com, Alain Gateau, Yohann Couté, Odile Burlet-Schiltz, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Département de science des protéines humaines [Genève], Université de Genève = University of Geneva (UNIGE)-Faculté de médecine [Genève], Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Swiss Institute of Bioinformatics [Genève] (SIB), Institut de recherche en santé, environnement et travail (Irset), Université d'Angers (UA)-Université de Rennes (UR)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Proteomics Core Facility (Protim), Université de Rennes (UR)-Plateforme Génomique Santé Biogenouest®, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Institut de pharmacologie et de biologie structurale (IPBS), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche en Transplantation et Immunologie (U1064 Inserm - CRTI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Uppsala University, ANR-10-INBS-08, Agence Nationale de la Recherche, Conseil Régional de Bretagne, Biogenouest, ANR-10-INBS-0008,ProFI,Infrastructure Française de Protéomique(2010), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université de Genève (UNIGE)-Faculté de médecine [Genève], Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Université d'Angers (UA)-Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-École des Hautes Études en Santé Publique [EHESP] (EHESP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Plateforme Génomique Santé Biogenouest®, Université de Strasbourg (UNISTRA)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Bertacchi Griffi, Nathalie, and Infrastructure Française de Protéomique - - ProFI2010 - ANR-10-INBS-0008 - INBS - VALID

Subjects
Male, Proteomics, 0301 basic medicine, human proteome project, Proteome, [SDV]Life Sciences [q-bio], Peptide, Computational biology, Biology, Bioinformatics, Biochemistry, 03 medical and health sciences, Expression pattern, Tandem Mass Spectrometry, Testis, Protein purification, Human proteome project, Humans, ddc:576, Databases, Protein, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, 030102 biochemistry & molecular biology, NeXtProt, cilia, General Chemistry, data mining, bioinformatics, Spermatozoa, mass spectrometry proteomics, [SDV] Life Sciences [q-bio], 030104 developmental biology, chemistry, spermatozoon, immunohistochemistry, Human testis, missing proteins, Chromatography, Liquid
Abstract

The Chromosome-Centric Human Proteome Project aims to identify proteins classed as « missing » in the neXtProt knowledgebase. In this article, we present an in-depth proteomics analysis of the human sperm proteome to identify testis-enriched missing proteins. Using a range of protein extraction procedures and LC-MS/MS analysis, we detected a total of 235 proteins (PE2-PE4) for which no previous evidence of protein expression was annotated. Through a combination of LC-MS/MS and LC-PRM analysis, data mining and immunohistochemistry, we were able to confirm the expression of 206 missing proteins (PE2-4) in line with current HPP guidelines (version 2.0). Parallel Reaction Monitoring (PRM) acquisition combined with synthetic heavy labeled peptides was used to target 36 « one-hit wonder » candidates selected on the basis of prior PSM assessment. Of this subset of candidates, 24 were validated with additional predicted and specifically targeted peptides. Evidence was found for a further 16 missing proteins using immunohistochemistry on human testis sections. The expression pattern for some of these proteins was specific to the testis, and they could potentially be valuable markers with applications in fertility assessment. Strong evidence was also found for the existence of 4 proteins labeled as "uncertain" (PE5); the status of these proteins should therefore be re-examined. Our results show how the use of a range of sample preparation techniques combined with MS-based analysis, expert knowledge and complementary antibody-based techniques can produce data of interest to the community. All MS/MS data are available via ProteomeXchange under identifier PXD003947. In addition to contributing to the Chromosome-Centric Human Proteome Project, we hope the availability of these data will stimulate the continued exploration of the sperm proteome.

Published
2016

23. Calibration plot for proteomics: A graphical tool to visually check the assumptions underlying FDR control in quantitative experiments

Author

Giai Gianetto, Quentin, Combes, Florence, Ramus, Claire, Bruley, Christophe, Coute, Yohann, Burger, Thomas, Etude de la dynamique des protéomes (EDyP ), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Bertacchi Griffi, Nathalie

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2016

24. Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics

Author

Joël Lachuer, Christian Delphin, Xavier Gidrol, Pascal Stuelsatz, Jacques Baudier, Nicole Assard, Benoit Gilquin, Bertacchi Griffi, Nathalie, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), INSERM U836, équipe 1, Physiopathologie du cytosquelette, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Métabolisme et pathologies du cerveau : imagerie et modélisation (GMPIB), Université Paris-Sud - Paris 11 (UP11), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
RefilinA, 0301 basic medicine, QH301-705.5, Science, [SDV]Life Sciences [q-bio], Arp2/3 complex, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, General Biochemistry, Genetics and Molecular Biology, Cell membrane, Protein filament, 03 medical and health sciences, Filamin binding, Actin remodeling of neurons, 0302 clinical medicine, FilaminA, medicine, Actin-binding protein, Biology (General), FAM101B, FAM101A, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Actin, ComputingMilieux_MISCELLANEOUS, RefilinB, biology, Cell biology, [SDV] Life Sciences [q-bio], 030104 developmental biology, medicine.anatomical_structure, biology.protein, Lamellipodium, General Agricultural and Biological Sciences, 030217 neurology & neurosurgery, Research Article, Polydendrocyte
Abstract

Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling., Summary: The newly identified Refilin protein family are unique short-lived proteins that complex with Filamin to regulate plasma membrane dynamics though the promotion of Actin filament bundling.

Published
2016

25. The Many Roles of Ubiquitin in NF-κB Signaling

Author

Gilles Courtois, Marie-Odile Fauvarque, Genetics and Chemogenomics (GenChem), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Bertacchi Griffi, Nathalie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], Cell, Medicine (miscellaneous), Inflammation, Review, medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Autoimmunity, 03 medical and health sciences, Ubiquitin, In vivo, ubiquitin, medicine, lcsh:QH301-705.5, ComputingMilieux_MISCELLANEOUS, biology, Cell growth, 3. Good health, Cell biology, [SDV] Life Sciences [q-bio], Transformation (genetics), ubiquitination/deubiquitination, 030104 developmental biology, medicine.anatomical_structure, lcsh:Biology (General), biology.protein, Signal transduction, medicine.symptom, nuclear factor κB, signal transduction
Abstract

The nuclear factor κB (NF-κB) signaling pathway ubiquitously controls cell growth and survival in basic conditions as well as rapid resetting of cellular functions following environment changes or pathogenic insults. Moreover, its deregulation is frequently observed during cell transformation, chronic inflammation or autoimmunity. Understanding how it is properly regulated therefore is a prerequisite to managing these adverse situations. Over the last years evidence has accumulated showing that ubiquitination is a key process in NF-κB activation and its resolution. Here, we examine the various functions of ubiquitin in NF-κB signaling and more specifically, how it controls signal transduction at the molecular level and impacts in vivo on NF-κB regulated cellular processes.

Published
2018

26. Force et spécificité du criblage pour des molécules bioactives au CMBA-Grenoble

Author

Barette, Caroline, Soleilhac, Emmanuelle, Charavay, Céline, Cochet, Claude, Fauvarque, Marie-Odile, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Genetics and Chemogenomics (GenChem), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Biologie du Cancer et de l'Infection (BCI ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM), Bertacchi Griffi, Nathalie, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio], ComputingMilieux_MISCELLANEOUS
Abstract

International audience

Published
2015

27. Les cellules acineuses différenciées se dupliquent pour régénérer les acinus salivaires

Author

Claudie Lemercier, Bertacchi Griffi, Nathalie, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
2. Zero hunger, Colorectal cancer, Aberrant methylation, business.industry, [SDV]Life Sciences [q-bio], Cancer, General Medicine, medicine.disease, Microbial dysbiosis, Molecular biology, General Biochemistry, Genetics and Molecular Biology, 3. Good health, Colon carcinogenesis, [SDV] Life Sciences [q-bio], Increased risk, medicine, Anaerobic bacteria, Faecal occult blood test, business, ComputingMilieux_MISCELLANEOUS
Abstract

m/s n° 8-9, vol. 31, aout-septembre 2015 DOI : 10.1051/medsci/20153108003 9. Marchesi JR, Dutilh BE, Hall N, et al. Towards the 661 human colorectal cancer microbiome. PLoS One 2011 ; 6 : e20447. 10. Warren RL, Freeman DJ, Pleasance S, et al. Co-occurrence of anaerobic bacteria in colorectal carcinomas. Microbiome 2013 ; 1 : 16. 11. Sobhani I, Amiot A, Le Baleur Y, et al. Microbial dysbiosis and colon carcinogenesis: could colon cancer be considered a bacteria-related disease? Therap Adv Gastroenterol 2013 ; 6 : 215-29. 12. Keku TO, Dulal S, Deveaux A, et al. The gastrointestinal microbiota and colorectal cancer. Am J Physiol Gastrointest Liver Physiol 2015 ; 308 : G351-63. 13. Zeller G, Tap J, Voigt AY, et al. Potential of fecal microbiota for early-stage detection of colorectal cancer. Mol Syst Biol 2014 ; 10 : 766. 14. Amiot A, Mansour H, Baumgartner I, et al. The detection of methylated Wif-1 gene is more accurate for identifying advanced adenomas and colorectal cancer than faecal occult blood test. PLoS One 2014 ; 9 : e99233. 15. Roperch JP, Incitti R, Forbin S, et al. Aberrant methylation of NPY, PENK, and WIF1 as a promising marker for blood-based diagnosis of colorectal cancer. BMC Cancer 2013 ; 13 : 566. 2. Samadder NJ, Curtin K, Tuohy TM, et al. Increased risk of colorectal neoplasia among family members of patients with colorectal cancer: a populationbased study in Utah. Gastroenterology 2014 ; 147 : 814-21.

Published
2015

28. High-throughput monitoring of major cell functions by means of lensfree video microscopy

Author

Fabien Momey, Xavier Gidrol, S. Vinjimore Kesavan, Nelly Dubrulle, Eric Sulpice, Jean-Marc Dinten, Brigitte David-Watine, Olivier Cioni, Bernard Chalmond, D. Freida, Spencer L. Shorte, Cédric Allier, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Imagerie Dynamique (Plate-Forme) (PFID), Institut Pasteur [Paris] (IP), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Centre de Mathématiques et de Leurs Applications (CMLA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Laboratoire d'Electronique et des Technologies de l'Information ( CEA-LETI ), Université Grenoble Alpes ( UGA ) -Direction de Recherche Technologique (CEA) ( DRT (CEA) ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Imagerie Dynamique (Plate-Forme) ( PFID ), Université de Cergy Pontoise ( UCP ), Université Paris-Seine, Centre de Mathématiques et de Leurs Applications ( CMLA ), École normale supérieure - Cachan ( ENS Cachan ) -Centre National de la Recherche Scientifique ( CNRS ), and Bertacchi Griffi, Nathalie

Subjects
Programmed cell death, Cell division, [SDV]Life Sciences [q-bio], Primary Cell Culture, Cell, Population, Video Recording, Cell Count, Video microscopy, Biology, Article, Cell Line, Tumor, Cell Adhesion, medicine, Humans, RNA, Small Interfering, education, [ SDV.IB.IMA ] Life Sciences [q-bio]/Bioengineering/Imaging, education.field_of_study, Microscopy, Video, Osteoblasts, Multidisciplinary, Cell Death, Mesenchymal stem cell, [ SDV.BC.BC ] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Mesenchymal Stem Cells, Transfection, Fibroblasts, Cell biology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Cell culture, Cell Division
Abstract

International audience; Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 - 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells.

Published
2014

29. Quantitative imaging of cell dynamics Parallelized contact imaging and automated analysis of cell migration dynamics

Author

Ghorbel, Itebeddine, Bertacchi, Nathalie, Gidrol, Xavier, Haguet, Vincent, Bertacchi Griffi, Nathalie, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio]
Abstract

International audience; We show the capacity to carry out screens for cell migrations using 96 wound healing assays achieved in 96-well microtiter plates based on a new optical technique developed by our group. Dynamics of the wound closures were obtained by parallelized time lapse contact imaging microscopy and dedicated automated image analysis. Our plate reader relies on an array of 96 image sensors, namely the planar arrangement of 12×8 image sensors, placed under the transparent flat-bottomed 96-well microtiter plate so that each well can be imaged by the image sensor placed underneath. Week-long monitoring of live cell populations showed long-term imaging position stability and no focus drift in any image series, which makes our time-lapse plate reader very competitive in comparison to conventional video microscopy equipment. The 96 wound closure dynamics were extracted from the images using a specifically developed automated segmentation method. Robust localization of the wound edges in low contrast images was achieved by global segmentation algorithms based on Markov random fields and active contours even with non-uniform illumination conditions. A parallel double snake was used to model the approximate parallelism between the two edges of a wound. The performance of global segmentation was validated on a set of images showing wounds in confluent epithelial cell cultures. Automated wound localization was compared with manual segmentation performed by seven cell biology experts by determining the root-mean-square error between the segmented interfaces and region-oriented analysis. Evaluations of intra and inter-biologist variabilities showed that automated segmentations are as accurate and robust as the cell biologist's ones. Wound closure dynamics was applied to measure and compare the motility of four affiliated prostate cell lines representing various grades of prostate cancer development.

Published
2014

30. Comprehensive analysis of human cells motion under an irrotational AC electric field in an electro-microfluidic chip

Author

Vaillier, Clarisse, Honegger, Thibault, Kermarrec, Frederique, Gidrol, Xavier, Peyrade, David, Docoslis, Aristides, Laboratoire des technologies de la microélectronique (LTM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Massachusetts Institute of Technology. Department of Electrical Engineering and Computer Science, Honegger, Thibault, and Bertacchi Griffi, Nathalie

Subjects
[SDV]Life Sciences [q-bio], lcsh:Medicine, 02 engineering and technology, 01 natural sciences, Electrokinetic phenomena, Electricity, Cell Movement, Molecular Cell Biology, lcsh:Science, ComputingMilieux_MISCELLANEOUS, Cellular Stress Responses, [PHYS]Physics [physics], Physics, Multidisciplinary, Classical Mechanics, Microfluidic Analytical Techniques, 021001 nanoscience & nanotechnology, [SDV] Life Sciences [q-bio], Cell Processes, Physical Sciences, Engineering and Technology, Electrohydrodynamics, 0210 nano-technology, Biological system, Research Article, Cell Physiology, Rotation, Biophysics, Dielectric, Fluid Mechanics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Continuum Mechanics, Cell Line, Electrical resistivity and conductivity, Electrostatics, Electric field, Humans, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Mechanical Engineering, 010401 analytical chemistry, HEK 293 cells, lcsh:R, Biology and Life Sciences, Cell Biology, Dielectrophoresis, 0104 chemical sciences, Membrane Trafficking, lcsh:Q, Cytometry, Voltage
Abstract

AC electrokinetics is a versatile tool for contact-less manipulation or characterization of cells and has been widely used for separation based on genotype translation to electrical phenotypes. Cells responses to an AC electric field result in a complex combination of electrokinetic phenomena, mainly dielectrophoresis and electrohydrodynamic forces. Human cells behaviors to AC electrokinetics remain unclear over a large frequency spectrum as illustrated by the self-rotation effect observed recently. We here report and analyze human cells behaviors in different conditions of medium conductivity, electric field frequency and magnitude. We also observe the self-rotation of human cells, in the absence of a rotational electric field. Based on an analytical competitive model of electrokinetic forces, we propose an explanation of the cell self-rotation. These experimental results, coupled with our model, lead to the exploitation of the cell behaviors to measure the intrinsic dielectric properties of JURKAT, HEK and PC3 human cell lines.

Published
2014

31. Therapeutic siRNAs targeting JAK/STAT signalling pathway in inflammatory bowel diseases

Author

Flora Clément, Adrien Nougarède, Stéphanie Combe, Frédérique Kermarrec, Arindam K Dey, Patricia Obeid, Arnaud Millet, Fabrice P Navarro, Patrice N Marche, Eric Sulpice, Xavier Gidrol, Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Biomicrotechnologie et génomique fonctionnelle (BIOMICS), BioSanté (UMR BioSanté), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), and Bertacchi Griffi, Nathalie

Subjects
Small interfering RNA, Filgotinib, [SDV]Life Sciences [q-bio], Pharmacology, inflammatory bowel diseases, 03 medical and health sciences, 0302 clinical medicine, RNA interference, Humans, Medicine, Gene silencing, RNA, Small Interfering, Janus Kinases, 030304 developmental biology, 0303 health sciences, Tofacitinib, JAK inhibitors, business.industry, Kinase, Gastroenterology, Therapeutic siRNA, JAK-STAT signaling pathway, General Medicine, 3. Good health, [SDV] Life Sciences [q-bio], 030211 gastroenterology & hepatology, business, Janus kinase, Signal Transduction
Abstract

Background and Aims Inflammatory bowel diseases are highly debilitating conditions that require constant monitoring and life-long medication. Current treatments are focused on systemic administration of immunomodulatory drugs, but they have a broad range of undesirable side-effects. RNA interference is a highly specific endogenous mechanism that regulates the expression of the gene at the transcript level, which can be repurposed using exogenous short interfering RNA [siRNA] to repress expression of the target gene. While siRNA therapeutics can offer an alternative to existing therapies, with a high specificity critical for chronically administrated drugs, evidence of their potency compared to chemical kinase inhibitors used in clinics is still lacking in alleviating an adverse inflammatory response. Methods We provide a framework to select highly specific siRNA, with a focus on two kinases strongly involved in pro-inflammatory diseases, namely JAK1 and JAK3. Using western-blot, real-time quantitative PCR and large-scale analysis, we assessed the specificity profile of these siRNA drugs and compared their efficacy to the most recent and promising kinase inhibitors for Janus kinases [Jakinibs], tofacitinib and filgotinib. Results siRNA drugs can reach higher efficiency and selectivity at lower doses [5 pM vs 1 µM] than Jakinibs. Moreover, JAK silencing lasted up to 11 days, even with 6 h pulse transfection. Conclusions The siRNA-based drugs developed hold the potential to develop more potent therapeutics for chronic inflammatory diseases.

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32. Organoids-based High Content Screening in Oncology

Author

Xavier Gidrol, Laperrousaz, B., Patricia Obeid, Porte, S., Gerbeau, S., Kermarrec, F., Vincent Haguet, Dolega, M., Christophe Le Priol, cedric allier, Nathalie Picollet-d'Hahan, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Bertacchi Griffi, Nathalie

Subjects
[SDV] Life Sciences [q-bio], [SDV]Life Sciences [q-bio]
Abstract

International audience; To analyze the phenotypic consequences of genetic perturbation in mammalian cells with drugs, RNAi or expression vectors, there are increasing needs for systematic cell-based high content screening (HCS) approaches, particularly in oncology. Although several groups are performing HCS in human cell lines, the real challenge in translational research remains screening on a reduced number of primary cells directly obtained from patients in a microenvironment that would resemble the original tissues. While standard monolayer two-dimensional culture conditions or even spheroids resulting form the forced aggregation of cells are poor mimics of the tumour natural history, microfabricated systems enable three-dimensional clonal organoids cultures and have the potential to provide biological insight not achievable before. We use microfluidics and MEMS (MicroElectroMechanical Systems) to analyze the phenotypic consequences of genetic perturbations (RNAi-based HCS) in 3D organoids. Specifically, we present a novel approach based on a flowfocusing microfluidic system that encapsulates either single prostatic or mammary cell in Matrigel beads and assay for development of organoids. Furthermore, we developed new imaging technology to monitor live organoids self-assembly and inter organoids cell trafficking.

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