Exploring the ubiquitin system during yeast sporulation
Supervision: Gwenaël Rabut
Rabut Lab - Ubiquitylation system
When facing poor growth conditions, many microorganisms form specialized quiescent cells called spores. This sporulation process is characterized by a sharp reduction of the cell and nuclear volume, a shut off of gene expression and the formation of a thick protecting cell wall. These changes enable spores to survive up to several years in very hostile environments.
In this PhD project, we propose to explore the function of the ubiquitin system during yeast sporulation. The ubiquitin system is known to regulate the abundance and activity of thousands of proteins in vegetative cells, but its roles during sporulation remain poorly characterized. This project will be conducted in the frame of the broader ANR EpiSpores project that aims to investigate the mechanisms that regulate gene expression and genome compaction during yeast sporulation. We will use molecular sensors to systematically monitor the partners of ubiquitin conjugating and ligating enzymes at different stages of sporulation. The results we will obtain will enable us to better understand the molecular mechanisms at play during sporulation and could help, in the long term, to develop original antifungal therapies.
Ubiquitin, Proteasome, Protein-protein interaction
Marie Le Boulch, Audrey Brossard, Gaëlle Le Dez, Sébastien Léon, Gwenaël Rabut. Sensitive detection of protein ubiquitylation using a protein-fragment complementation assay. Journal of Cell Science, Company of Biologists, 2020, 133 (12), pp.jcs240093. hal-02635146v1
Ewa Blaszczak, Natalia Lazarewicz, Aswani Sudevan, Robert Wysocki, Gwenaël Rabut. Protein-fragment complementation assays for large-scale analysis of protein-protein interactions. Biochemical Society Transactions, Portland Press, 2021, 49 (3), pp.1337-1348. hal-03282309v1
Marc Melkonian, Camille Juigné, Olivier Dameron, Gwenaël Rabut, Emmanuelle Becker. Towards a reproducible interactome: semantic-based detection of redundancies to unify protein-protein interaction databases. Bioinformatics, Oxford University Press (OUP), 2022, pp.1-7. hal-03522989v1
Structural characterization of the mechanism of action of trans-translation inhibitors
Supervision: Reynald Gillet, Emmanuel Giudice
Gillet Lab - Quality Control in Protein Synthesis
The development of antibiotic-resistant microorganisms is one of the major public health issues of our century. In this context, the present project is to characterize the mechanism of action of antimicrobial molecules aimed at a new target: trans-translation.
Trans-translation refers to a molecular process which allows for the release of ribosomes stalled on faulty mRNAs that lack stop codons as well as the elimination of these mRNAs and mistranslated peptides. The process is performed by the hybrid transfer-messenger RNA (tmRNA) and a small basic protein: SmpB. Essential for the survival and/or virulence of many pathogenic bacteria and absent in eukaryotes, trans-translation is a particularly attractive targets for new antibiotics. Using our recently developed HTS screening assays, we discovered a family of heterocyclic compounds that specifically targets trans-translation. The PhD student will be recruited to characterize biophysically (EMSA, RPS) and structurally (cryo-EM, X-ray) these inhibitor’s on their molecular targets, in order to understand their mode of action and improve their efficiency and specificity.
Ribosome, Trans-translation, Antibiotics, Cryo-EM, SAR
Charlotte Guyomar, Marion Thepaut, Sylvie Nonin-Lecomte, Agnès Méreau, Renan Goude et al. Reassembling green fluorescent protein for in vitro evaluation of trans-translation. Nucleic Acids Research, Oxford University Press, 2020, 48 (4), pp.e22-e22. hal-02440937v1
Charlotte Guyomar, Gaetano D’urso, Sophie Chat, Emmanuel Giudice, Reynald Gillet. Structures of tmRNA and SmpB as they transit through the ribosome. Nature Communications, Nature Publishing Group, 2021, 12 (1), pp.4909. hal-03343248v1
Rodrigo Campos-Silva, Gaetano d'Urso, Olivier Delalande, Emmanuel Giudice, Alexandre Jose Macedo et al. Trans-Translation Is an Appealing Target for the Development of New Antimicrobial Compounds. Microorganisms, 2022, 10 (1). hal-03575603v1
Automated intelligent microscopy for monitoring mitochondrial functions in living cells by multiplex imaging and high content of FRET biosensors
Supervision: Marc Tramier
Tramier Lab - Microscopy for Cell Biosensing
The "Microscopy for Cell Biosensing" team develops techniques and methods for the dynamic monitoring of biochemical activities in living cells. The team has developed a fastFLIM prototype, a FRET biosensor of AurkA kinase activities making it possible to discover new functions of this kinase at the mitochondria, FRET approaches by FLIM in multiplex, and a screening methodology for inhibitors of the AurkA kinase by HCS-FLIM. We are developing the Roboscope prototype able to analyze in real time acquired images using deep learning algorithms and to feed back to the microscope control module informations to adjust the acquisition sequence (patent pending, collaboration J Pécréaux and Inscoper). The proposed project is to integrate the development of the Roboscope in the pipeline of HCS-FLIM. It fits perfectly with the team's project which consists in implementing an automated platform which will allow the simultaneous monitoring of different mitochondrial functions (G Bertolin CRCN project) and more particularly in cancer cells (collaboration MD Galibert). It will be a first step in the development of quantitative automated microscopy for translational research, and pave the way for its use in diagnosis or personalized medicine.
Intelligent Microscopy, Artificial Intelligence, Quantitative Fluorescence Microscopy, FRET by FLIM, High Content Screening
Strong interest in fluorescence microscopy applied to biology and in machine learning for image analysis
Giulia Bertolin, Anne-Laure Bulteau, Marie-Clotilde Alves-Guerra, Agnes Burel, Marie-Thérèse Lavault et al. Aurora kinase A localises to mitochondria to control organelle dynamics and energy production. eLife, eLife Sciences Publication, 2018, 7, pp.e38111. hal-01862597v1
Claire Demeautis, Francois Sipieter, Julien Roul, Catherine Chapuis, Sergi Padilla-Parra et al. Multiplexing PKA and ERK1&2 kinases FRET biosensors in living cells using single excitation wavelength dual colour FLIM. Scientific Reports, Nature Publishing Group, 2017, 7 (1), pp.41026. hal-01478382v1
Florian Sizaire, Gilles Le Marchand, Jacques Pecreaux, Otmane Bouchareb, Marc Tramier. Automated screening of AURKA activity based on a genetically encoded FRET biosensor using fluorescence lifetime imaging microscopy. Methods and Applications in Fluorescence, IOP Science, 2020, 8 (2), pp.024006. hal-02500623v1
Which mechanisms account for robust partitioning of chromosomes during cell division? How do spindle mechanics and plasticity contribute to cell division?
Supervision: Jacques Pécréaux
Pécréaux Lab - Cell Division Reverse Engineering
While most of the players in the mitotic spindle are known, how they dynamically combine to ensure a robust cell division is yet to be found. Such robustness is ambivalent, allowing the development of multicellular organisms and permitting various developmental diseases like malformations of cortical development and cancer. In this latter case, aneuploid cells can divide, although not faithfully, and resulting chromosomal instability increases drug resistance. We foresee that spindle function is also robust because of mechanism design, as for spindle positioning. The nematode model, established and convenient for biophysics, exhibits viable and fertile tetraploid strains. Recently, we found unexpected fluxes of spindle microtubules and identified some of the related players. Previously, using C. elegans one-cell embryo, we could fingerprint the spindle mechanics and found that it slowly drifts rather than being at quasi-equilibrium, supporting its adaptability. We foresee a connection between fluxes and adaptability and set: (i) to investigate half-spindle mechanics by including chromosome motion; (ii) to identify the list of the players of spindle mechanics, likely overlapping partly with the one of the fluxes; (iii) to recapitulate the results into a continuous mechanical model (coll. L. Le Marrec, IRMAR, Rennes) and (iv) to validate the findings in human cells using our autonomous microscope. It will pave the way to interfere with this robustness to potentialise anti-mitotic drugs.
Cell Division Robustness, Nematode Model Organism, Continuous Mechanics Modelling
Training in cell biology and basic knowledge in interdisciplinary approaches, if possible, including image processing and biophysical modelling. Experience in optical microscopy, data science or statistics considered very favorably
Hélène Bouvrais, Laurent Chesneau, Yann Le Cunff, Danielle Fairbrass, Nina Soler et al. The coordination of spindle-positioning forces during the asymmetric division of the C. elegans zygote. EMBO Reports, EMBO Press, 2021, 22 (5), pp.e50770. hal-03000200v1
Benjamin Mercat. Analyse temps-fréquence en mécanique cellulaire et adaptabilité du fuseau mitotique. 2016. PhD thesis.
Maël Balluet, Florian Sizaire, Youssef El Habouz, Thomas Walter, Jérémy Pont et al. Neural network fast‐classifies biological images through features selecting to power automated microscopy. Journal of Microscopy, Wiley, 2022, 285 (1), pp.3-19. hal-03408803v1
Interplay between actomyosin and microtubules networks on the regulation of epithelial cell shape and fate
Supervision: Roland Le Borgne
Le Borgne Lab - Epithelia Dynamics and Mechanics
Animal cells exhibit a large diversity of shape and mechanics. Control of cell shape and fate are intimately linked, and each of the cell morphologies appears to be best suited in terms of geometry, surface, connectivity and plasticity to perform its function in tissues. Cell shape is determined by a balance of internal and external forces involving the cytoskeleton and its associated adhesion structures. The single layer epithelium of Drosophila pupal notum is composed of epidermal cell with regular pentagonal and hexagonal shapes and of stereotyped star-shaped sensory organ precursor (SOP) having more neighbors and curved interfaces. In the plane of adhesion structures, SOPs have a denser actomyosin meshwork, more microtubules and more spectraplakin, a linker of actin and microtubule cytoskeleton. ShapeFate aims to characterize their subcellular localization and dynamics using superresolution microscopy, and to perturb the system using genetic and biophysical means to decrypt their contributions. Shape Fate aims to determine their respective functions in the acquisition of mechanical properties and shape of SOPs and to decipher how this impacts fate.
Epithelium, Cytoskeleton, Asymmetric Cell Division, Live-imaging, Biophysics
Elise Houssin, Mathieu Pinot, Karen Bellec, Roland Le Borgne. Bazooka/Par3 cooperates with Sanpodo for the assembly of Notch clusters following asymmetric division of Drosophila sensory organ precursor cells. eLife, eLife Sciences Publication, 2021, 10:e66659. hal-03368144v1
Thomas Esmangart de Bournonville, Roland Le Borgne. Interplay between Anakonda, Gliotactin, and M6 for Tricellular Junction Assembly and Anchoring of Septate Junctions in Drosophila Epithelium. Current Biology - CB, Elsevier, 2020, 30 (21), pp.4245-4253. hal-02929725v1
Lene Malerød*, Roland Le Borgne*, Anette Lie-Jensen, Åsmund Husabø Eikenes, Andreas Brech et al. Centrosomal ALIX regulates mitotic spindle orientation by modulating astral microtubule dynamics
EMBO Journal, EMBO Press, 2018, 37 (13), pp.e97741. hal-01812517v1
Muscle stem cells dynamics; decoding the Notch signal
Supervision: Hadi Boukhatmi
Boukhatmi Lab - Muscle Development and Repair
Muscle stem cells (MuSCs) are essential for muscle growth, maintenance and repair. The mechanisms underlying MuSC differentiation have been thoroughly studied. However, one fascinating and incompletely understood question is how MuSCs sense and respond to instructive signals provided by their environment. One key signaling is the Notch pathway, which is required for both maintenance and proliferation of MuSCs. Notch is also involved in the fate-shifting choice between stemness and differentiation. This project aims to shed new light on how MuSCs sense and respond to Notch during the multistep process of the muscle repair. The recent discovery of Drosophila MuSCs, allowed us to design novel transgenic tools and an original live transcriptional imaging method (MS2-MCP) that will enable to track MuSC dynamics and identify, with unprecedented precision, the interplay with their microenvironment. This project will both provide new fundamental knowledge in muscle stem cell biology and contribute to adjustment of stem cell-based therapies needed to restore skeletal muscle function in humans when this process is failing.
Stem cells, Satellite cells, Regeneration, Muscle, Drosophila.
Hadi Boukhatmi. Drosophila, an Integrative Model to Study the Features of Muscle Stem Cells in Development and Regeneration. Cells, MDPI, 2021, 10 (8), pp.2112. hal-03355068v1
Hadi Boukhatmi, Torcato Martins, Zoe Pillidge, Tsveta Kamenova, Sarah Bray. Notch mediates inter-tissue communication to promote tumorigenesis. Current Biology, 2020, 30 (10), pp1809. pmid-32275875
Hadi Boukhatmi, Sarah Bray. A population of adult satellite-like cells in Drosophila is maintained through a switch in RNA-isoforms. eLife, 7, e35954. pmc-5919756
Regulation of AhR transcription factor in resistance to cancer therapies
Supervision: Sébastien Corre
Galibert Lab - Gene Expression and Oncogenesis
Understanding the mechanisms of resistance in cancer is therefore a fundamental question in order to consider new innovative therapeutic strategies. We propose in this project to deepen the mechanisms of AhR transcription factor regulation and to extend it to cytarabine resistance in acute myeloblastic leukemia. By using the acute myeloblastic leukemia (AML) tumor model, the questions asked will be:
- Understand the involvement of AhR in the control of the gene expression program and the resistance phenotype (Ara-C; BRAFi / MEKi).
- Decipher the mode of activation of AhR by its various ligands (tryptophan pathway) and by the signals from the tumor environment.
- Propose new therapeutic strategies to counteract resistance by targeting the activity (antagonists) or expression of AhR (antisense oligonucleotides).
All stages of the project should allow us to understand how AhR participates in response to signals from the tumor environment to adapt to resistance to targeted therapy. The therapeutic strategy is then to prevent or reverse this phenotypic switching by controlling AhR and thus prolong the effectiveness of existing therapies to prevent relapses in patients.
Cancer, Resistance, AhR
Experience in molecular and cellular biology, knowledge on cancer, fluency in English
Yan Jia, Juan Guo, Youshan Zhao, Zheng Zhang, Lei Shi, Ying Fang, Dong Wu, Lingyun Wu, Chunkang Chang. AHR signaling pathway reshapes the metabolism of AML/MDS cells and potentially leads to cytarabine resistance. Acta Biochim Biophys Sin (Shanghai), 2021, 53, pp492. pmid-33709099
Sébastien Corre, Nina Tardif, Nicolas Mouchet, Heloise M. Leclair, Lise Boussemart et al. Sustained activation of the Aryl hydrocarbon Receptor transcription factor promotes resistance to BRAF-inhibitors in melanoma. Nature Communications, Nature Publishing Group, 2018, 9 (1), pp.4775. hal-01972443v1
Anaïs Paris, Nina Tardif, Marie-Dominique Galibert, Sébastien Corre. AhR and Cancer: From Gene Profiling to Targeted Therapy. International Journal of Molecular Sciences, MDPI, 2021, 22 (2). hal-03134830v1
Characterization of canine oral melanomas as preclinical models for human mucosal melanomas
Supervision: Catherine André & Benoit Hédan
André Lab - Dog Genetics
Mucosal melanoma is a rare subtype of melanomas in humans and it is characterized by a poor prognosis due to its strong local aggressiveness, its high metastatic propensity but also due to the lack of effective therapy. On the contrary, in dogs, oral melanoma is the first tumor of the oral cavity, frequent and very severe in dogs, thus making a relevant natural model for studying human mucosal melanoma (Prouteau & André, review 2019). In fact, these two species show many similarities concerning the characteristics of oral melanoma, both epidemiologically, pathologically and genetically, with the identification of comparable recurrent amplifications and deletions in both species, some being linked to survival (Prouteau et al., 2020). Our previous work demonstrated, from 70 cases of well-characterized canine oral melanomas, the existence of two molecular subtypes, distinguished by their rate of chromosomal rearrangements (Prouteau et al., 2022). Expression analyzes on 30 cases made it possible to correlate these 2 subgroups with expression profiles of specific genes: the first (low levels of rearrangement) overexpressing genes involved in immunity and the second (high levels of rearrangements) overexpressing oncogenes and pigmentation genes. Our hypothesis is that the differences in “immunological” and “oncogenic” molecular signatures between the 2 tumor subgroups will allow canine patients to be stratified to offer more suitable therapies: immunotherapy for the first group and targeted therapies for the second group.
Therefore, the objective of the PhD project is to study the similarities between canine oral melanomas and human mucosal melanomas (oropharyngeal and ano-genital), on the immunohistochemical and genetic levels. The specific aims of the project are:
- To better investigate the microenvironment of the 2 types of oral melanomas, by immunohistochemistry and single cell RNAseq
- To better characterize the over-expressed genes (immunity vs pigmentation/oncogenes) through long read RNA sequencing.
These analyzes will be performed in dog oral melanomas, owing to the many samples available and breed specificities and then transferred to human mucosal melanomas, for the benefit of human and veterinary medicine, both for diagnosis and treatments.
Dog, Melanomas, Genetics, Chromosomal Rearrangements, Transcriptomics
Anaïs Prouteau, Catherine André. Canine Melanomas as Models for Human Melanomas Clinical, Histological, and Genetic Comparison. Genes, MDPI, 2019, 10 (7), pp.E501. hal-02179209v1
Anais Prouteau, Stephanie Mottier, Aline Primot, Edouard Cadieu, Laura Bachelot et al. Canine Oral Melanoma Genomic and Transcriptomic Study Defines Two Molecular Subgroups with Different Therapeutical Targets. Cancers, MDPI, 2022, 14 (2), pp.276. hal-03555450v1
Ronan Ulvé, Mélanie Rault, Mathieu Bahin, Laetitia Lagoutte, Jérôme Abadie et al. Discovery of Human-Similar Gene Fusions in Canine Cancers. Cancer Research, American Association for Cancer Research, 2017, 77 (21), pp.5721-5727. hal-01647128v1
Characterization and pre-clinical manipulation of a conserved host-microbiota interaction
Supervision: François Legoux
Legoux Lab - Host-Microbiota Interactions
The gut microbiota plays essential roles in regulating host biology, but few molecular interactions have been characterized to date. Mucosal Associated Invariant T (MAIT) cells are T lymphocytes recognizing metabolites from the microbial vitamin B2 pathway presented by the host protein MR1. MAIT cells are very abundant in humans and are programmed to respond quickly upon stimulation, making them attractive targets for clinical intervention.
Preliminary data from my group at Institut Curie indicate that MAIT cells become activated and produce tissue-repair molecules upon intestinal inflammation. Mice lacking MAIT cells (Mr1-/-) develop less severe intestinal inflammation, indicating that MAIT cells are protective in this setting. The role of microbiota-derived antigens in this protection is unknown. We are starting a new lab at IGDR to continue our research on host-microbiota interactions.
The goals of this PhD project are:
- To determine how MAIT-microbiota interactions provide host protection during intestinal inflammation.
- To manipulate MAIT cells to alleviate this pathology in mouse models.
Immunology, T cells, Microbiota, Colitis
Curiosity and enthusiasm for science. Previous experience with mouse work would be appreciated
François Legoux#, Déborah Bellet, Celine Daviaud, Yara El Morr, Aurélie Darbois et al. Microbial metabolites control the thymic development of mucosal-associated invariant T cells. Science, 2019, 366 (6464), pp.494-499. #Co-corresponding authors. hal-02349402v1
François Legoux*#, Jules Gilet*, Emanuele Procopio, Klara Echasserieau, Karine Bernardeau, Olivier Lantz#. Molecular mechanisms of lineage decisions in metabolite-specific T cells. Nature immunology, 2019, 20, pp.1244-1255. *Equal contribution. #Co-corresponding authors. pmid-31431722
Marion Salou*, François Legoux*, Jules Gilet*, Aurélie Darbois, Anastasia du Halgouet, Ruby Alonso, Wilfrid Richer, Anne-Gaëlle Goubet, Céline Daviaud, Laurie Menger, Emanuele Procopio, Virginie Premel, Olivier Lantz. A common transcriptomic program acquired in the thymus defines tissue residency of MAIT and NKT subsets. Journal of Experimental Medicine, 2019, 216, pp.133-151. *Equal contribution. pmc-6314520