IMCBio International PhD Program

Skeletal muscle cells present a fascinating challenge in cell biology due to their syncytial structure - thousands of nuclei sharing a single cytoplasm. To manage this complexity, these cells create distinct functional domains within themselves. Although numerous inherited and acquired muscle pathologies exist, the mechanisms underlying most of these disorders remain poorly understood. This is largely because we lack the tools to study molecular events in specific muscle domains, even though many diseases originate from these regions. To overcome this limitation, our team has developed a set of innovative tools enabling domain-specific genetic manipulation within muscle cells. The recruited student will apply these tools to investigate pathological mechanisms in severe congenital dystrophies and advance gene therapy by targeting the precise muscle regions involved. Additionally, our team continues to discover new muscle domains and myonuclear subtypes linked to various processes such as aging, exercise, and cancer, utilizing single-nucleus transcriptome technology. With our unique domain-specific tools, we can now explore these regions in unprecedented detail.

Keywords: Skeletal muscle, muscle domains, mouse genetics, muscle dystrophy and myopathy, gene therapy, disease mechanism

Relevant publications:

- Kim, M*., Franke, V*., Brandt, B., Lowenstein, E.D., Schowel, V., Spuler S., Akaline, A., and Birchmeier, C. (2020) Single-nucleus transcriptomics reveals functional compartmentalization in syncytial skeletal muscle cells. Nature Communications. 11, 6375. *, co-first authors.

- Bavat Bornstein, Lia Heinemann-Yerushalm, iSharon Krief, Ruth Adler, Bareket Dassa, Dena Leshkowitz, Minchul Kim, Guy Bewick, Robert W Banks, and Elazar Zelzer (2023). Molecular characterization of the intact mouse muscle spindle using a multi-omics approach. eLIFE. Feb 6, https://doi.org/10.7554/eLife.81843

- Cristofer Calvo*, Coalesco Smith*, Taejeong Song, Sakthivel Sadayappan, Douglas P. Millay# and Minchul Kim#. Loss of Ufsp1 is compatible with embryogenesis and causes subtle structural changes of the neuromuscular junction. (Under review)

Cell motion is involved in a variety of phenomena during development. Its origins are diverse and they often result from self-organisation of tissues. Specifically, groups of cells can undergo rotation and the mechanisms at play can be addressed and understood by studies at the Interfaces between Physics and Biology.

These dynamics of directed motion can be reproduced in vitro with controlled conditions in 2D and in 3D with organoids. In this context, the team has established that tug-of-war between cell polarities pilot the onset of rotation in 2D rings (Ref. 1) and in cell doublets in 3D (Ref. 2). The acto-myosin and microtubule machinery together with adhesion complexes play a key role in regulating motions and in setting cell shapes (Ref. 3). Cells compete with each other and this can result in a global rotation. However the minimal protein machinery involved in the process needs to be determined and characterised. The PhD will consist in triggering rotation in stem cells by the expression of relevant proteins and their dosages guided by theoretical principles. It will involve stem cell biology, microfabrication, quantitative biology, theoretical physics, single cell sequencing and spatial transcriptomics. The project will be associated with international collaborators.

Keywords: organoids, quantitative biology, cytoskeleton, physical biology, single cell sequencing

Relevant publications:

- S. Lo Vecchio, O. Pertz, M. Szopos, L. Navoret, **  D. Riveline**  (2024). Spontaneous rotations in epithelia as an interplay between cell polarity and boundaries. BioRxiv, Nature Physics 20:322.

- Linjie Lu, Tristan Guyomar, Quentin Vagne, Rémi Berthoz, Alejandro Torres-Sánchez, Michèle Lieb, Cecilie Martin-Lemaitre, Kobus van Unen, Alf Honigmann, Olivier Pertz, Daniel Riveline,** Guillaume Salbreux,** (2024) Polarity-driven three-dimensional spontaneous rotation of a cell doublet, BioRxiv, Nature Physics 20:1194.

- Markus Mukenhirn, Chen-Ho Wang, Tristan Guyomar, Matthew J. Bovyn, Michael F. Staddon, Riccardo Maraspini, Linjie Lu, Cecilie Martin-Lemaitre, Masaki Sano, Tetsuya Hiraiwa, Daniel Riveline,** Alf Honigmann,** (2024) Tight junctions regulate lumen morphology via hydrostatic pressure and junctional tension, BioRxiv, Developmental Cell 59:1–16.

The inflammasome is an intracellular multiprotein complex that senses sterile tissue damage and infectious agents to initiate innate immune responses. Distinct inflammasomes containing specific sensing molecules exist. The NLRP3 inflammasome is unique as it detects a broad range of cellular stress signals but a primary and converging sensing mechanism initiating inflammasome assembly remains ill-defined. We found that NLRP3 binds altered endomembranes as a result of disruption of inter-organelle contact sites in response to danger signals. However, little is known about this fundamentally new mechanism of pattern recognition linking organelle spatial organization and innate immunity. The organelle-generated signals sensed by NLRP3 and the mechanisms underlying membrane recruitment and activation of the inflammasome remain largely unexplored and thus will be subject of this proposal. The major limit hampering their identification is the difficulty to disentangle the complex cell response to the variety of stimuli leading to NLRP3 activation. We aim to push this limit through an unprecedented combination of approaches ranging from in vitro reconstitution studies with isolated organelles and artificial liposomes and proteo-lipidomics, to cryo-FIB and cryo-ET imaging, molecular modelling, and in vivo testing of the physiological relevance of in vitro findings. This project will lay the foundation for how altered endomembranes serve as danger-associated molecular patterns to trigger innate immune responses.

Keywords: Inflammasome, NLRP3, endosome, PI4P, innate immunity, macrophages, inflammation

Relevant publications:

- KCNN4 links Piezo-dependent mechanotransduction to NLRP3 inflammasome activation. Li R, et al. Science Immunolog. 2023

-  Distinct changes in endosomal composition promote NLRP3 inflammasome activation. Zhang Z, et al. Nat Immunol. 2023

- Protein kinase D at the Golgi controls NLRP3 inflammasome activation. Zhang Z, et al. J. Exp. Med. 2017

Prostate cancer is the second most commonly diagnosed neoplasia in men worldwide and one of the major causes of cancer-related deaths. Advances in the past decade have revealed that tumours comprise a very heterogenous coevolving cellular ecosystem. Prostate tumours are considered as “cold tumours“, as their microenvironment is highly immunosuppressive. Current immunotherapies include Sipuleucel-T and pembrolizumab, and are approved for only a limited subset of patients.

To characterise the cascade of molecular and cellular events involved in prostate tumour progression, we generated genetically-engineered Pten(i)pe-/- mice in which Pten is selectively inactivated in prostatic epithelial cells at adulthood. We have shown that such mice develop prostatic intraepithelial neoplasia within a few months and adenocarcinoma at later stage, as seen in human prostate cancer. Moreover, our single-cell RNA-sequencing analyses revealed a high prostatic tumoral cell heterogeneity, and a complex tumour microenvironment, composed of various cancer-associated fibroblasts, tumour-infiltrating immune cells and endothelial cells.

The goal of the project is to determine the dynamic of the various infiltrating immune cell populations during tumour progression and the communications between the various cell populations. Moreover, the impact of stimulation or inhibition of relevant immune cells will be determined. Finally, the infiltrating immune cells will be determined in human prostate tumours, and their profile will be correlated with disease outcome.

Thus, the project will open new avenues to boost the antitumor immunity and develop biomarkers to improve patient stratification and the selection of immunotherapies for prostate cancer.

Keywords: prostate cancer, mouse models, tumour-infiltrating immune cells, single-cell transcriptomics

Relevant publications:

- M. A. Abu el Maaty, E. Grelet, C. Keime, A.-I. Rerra, J. Gantzer, C. Emprou, J. Terzic, R. Lutzing, J.-M. Bornert, G. Laverny, D. Metzger (2021). Single-cell analyses unravel cell type–specific responses to a vitamin D analog in prostatic precancerous lesions. Sci. Adv. 7, eabg5982. PMID: 34330705.

- Mohamed A. Abu el Maaty, Julie Terzic, Céline Keime, Daniela Rovito, Régis Lutzing, Darya Yanushko, Maxime Parisotto, Elise Grelet, Izzie Jacques Namer, Véronique Lindner, Gilles Laverny and Daniel Metzger (2022). Hypoxia-mediated stabilization of HIF1A in prostatic intraepithelial neoplasia promotes cell plasticity and malignant progression. Science advances 8, eabo2295. PMID: 35867798.

- Julie Terzic, Mohamed A. Abu el Maaty, Régis Lutzing, Alexandre Vincent, Rana El Bizri, Matthieu Jung, Céline Keime and Daniel Metzger (2023). Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors. EMBO Molecular Medicine. e17209. https://doi.org/10.15252/emmm.202217209. PMID: 37070472.  

Dyneins are a large family of motor proteins that are involved in the minus end directed transport along microtubules. Dyneins fulfil important biological tasks and their versatile cargos include mRNA, whole organelles like mitochondria and nuclei as well as cilia components. Dyneins also carry out crucial functions during mitosis. Defects in dynein function have been linked to a variety of diseases including neurodevelopmental disorders, infertility and chronic lung infections.

Dyneins are multi-protein complexes with a molecular weight of around 1.4 MDa. In all dynein isoforms a    ̴3500 amino-acid motor domain within this complex is responsible for the movement along microtubules. ATP hydrolysis in its hexameric AAA+ ring drives the remodelling of the dynein linker domain, which creates the force for the movement along the microtubule. In this project, we will investigate the role of conserved structural elements in the AAA+ ring during linker remodeling. The student will clone dynein motor domain mutants, express them in insect cells, purify the constructs and characterize these constructs by high-resolution cryoEM. Preliminary data demonstrating the feasibility of the project is already available.

Keywords: Motor protein, dynein, cytoskeleton, AAA+ protein, cryoEM

Relevant publications:

- Remodelling of Rea1 linker domain drives the removal of assembly factors from pre-ribosomal particles, 2024, Nature Communications, accepted for publication

- Fagiewicz R., Crucifix C., Klos T., Deville C., Kieffer B., Nominé Y., Busselez J., Rossolillo P., Schimdt H. In vitro characterization of the full-length human dynein-1 cargo adaptor BicD2, 2022, Structure

- Schimdt H., Zalyte R., Urnavicius L., Carter A. P., Structure of human cytoplasmic dynein-2 primed for its power stroke, 2015, Nature

This project is affiliated to a team self-funded fellowship.

Our group investigates how peculiar genetic mutations, namely microsatellite expansions, which are DNA sequences typically composed of more than 50 repeats of 2 to 6 nucleotides-long DNA motifs (for example (CGG)100x, (CAG)29x, etc.) cause muscle and/or neurodegenerative diseases. Importantly these mutations are located in genomic regions annotated as “noncoding” (5’ and 3’UTRs, introns, lncRNAs, etc.). We notably focus on Neuronal Intranuclear Inclusion Disease (NIID), OcculoPharyngoDistal Myopathy (OPDM) and Amyotrophic Lateral Sclerosis (ALS), which affects specifically motor neurons and that is the 3rd most common neurodegenerative disease worldwide.

Our work shows that despite being localized in “non-coding” sequences, these microsatellites repeat expansions are nonetheless translated into novel and toxic proteins (Sellier et al., Neuron 2017, Boivin et al., EMBO Journal 2020, Boivin et al., Neuron 2021, etc.).

Thus, the PHD candidate will continue and deepen this work and investigate how microsatellites repeat expansions causing neuromuscular and neurodegenerative diseases are potentially translated into novel and toxic proteins using a wide range of molecular and cellular approaches, as well as develop novel animal models expressing these mutations.

Overall, this proposal will help to better understand the cause of muscle and neuronal dysfunctions to define therapeutic strategies for these devastating diseases.

This work will take place at Institute of Genetics and Biology Molecular and Cellular (IGBMC, http://www.igbmc.fr/) a large public research laboratory comprising 40 research groups and 12 technological platforms, including all instrument, expertise and common services essential to the present project.

Techniques and approaches employed during this PHD will comprise the following: Clonage, RT-qPCR, cell culture and cell transfection, western blotting, immunoprecipitation, immunofluorescence, FACS, fluorescence microscopy, AAV injection, mouse locomotor phenotyping, histology, IHC, etc.

Keywords: human genetic diseases, muscle and neuronal diseases, non-coding sequences

Relevant publications:

- Charlet N. An unexpected polyglycine route to spinocerebellar ataxia. Nature Genetics. 2024 Jun;56(6):1039-1041.

- Boivin et al., Translation of GGC repeat expansions into a toxic polyglycine protein in NIID defines a novel class of human genetic disorders. Neuron. 2021; 109(11):1825.

- Boivin et al. C9ORF72 haploinsufficiency synergizes DPR proteins toxicity, a double hit mechanism that can be prevented by drugs activating autophagy. EMBO J. 2020; 39(4).

The intricate cognitive functions of the cerebral cortex stem from a diverse network of neurons and glial cells, all generated from a uniform pool of progenitors during corticogenesis. This project seeks to elucidate how these progenitors sequentially produce distinct neuronal and glial progeny over time, shaping brain development.

While transcriptional programs have been recognized as key drivers of neuronal diversity, emerging evidence indicates that certain neuronal mRNAs are present in progenitor cells long before their corresponding proteins appear. This observation suggests that translation mechanisms may play a pivotal role in defining cell identity, alongside transcription.

We propose that dynamic regulation of tRNA pools significantly influences translation during cortical patterning, affecting progenitor competency. Our preliminary findings indicate that tRNA availability varies across cell types and developmental stages in the mouse cortex. Through cutting-edge cell labelling and sequencing techniques (tRNA sequencing, (quantitative measurement of translation efficiency), the PhD candidate will investigate whether fluctuations in tRNA abundance correlate with the progenitor's capacity to produce diverse daughter cells and determine whether tRNA abundance correlates with a change in translational programs.

By uncovering these mechanisms, this project not only advances our understanding of neural diversity but also has potential implications for neurodevelopmental disorders.

Keywords: brain development, neuronal diversity, translation, omic analysis, transfer RNA

Relevant publications:

- Asselin L, et al. Mutations in the KIF21B kinesin gene cause neurodevelopmental disorders through imbalanced canonical motor activity. .Nature Communication (2020) 11: 2441

- Ramos-Morales E et al. The structure of the mouse ADAT2/ADAT3 complex reveals the molecular basis for mammalian tRNA wobble adenosine-to-inosine deamination. Nucleic Acids Res (2021) 49: 6529-6548

- Rivera Alvarez J et al. The kinesin Kif21b regulates radial migration of cortical projection neurons through a non-canonical function on actin cytoskeleton. Cell reports (2023) 42, 112744

Mitochondrial diseases are a clinically heterogeneous group of disorders caused by mitochondrial dysfunctions. They may be caused by mutations of either the nuclear or the mitochondrial genomes and are mostly incurable. In the scope of the present project we are considering two frequent situations: (1) mutations within a single tRNA lead to several disorders and (2) mutations convert a sense codon into a non-sense one leading to premature termination (PTC). In both, we propose that the targeting of a single tRNA into mitochondria will allow counteracting the molecular defects at the origin of the diseases. In the first situation, the therapeutic agent is a replacement tRNA, whose role is to switch the ratio mutant tRNA/wild-type tRNA below the fateful threshold where the disease is triggered. In the second, it is a suppressor tRNA, able to decode a nonsense codon, to read-through the PTC and thus restore the synthesis of a full-length protein. Basing on recent discoveries, we propose to model gene therapy in cultured immortalized and human patients' cells. The design of therapeutic tRNAs will be rationalized according to our knowledge in tRNA identity and codon/anticodon recognition rules. Proof of concept series of experiments will assess for the efficacy of model cells' transfection with the therapeutic tRNA. Finally, rescue of phenotypes in cybrids and patient-derived cells upon tRNA transfection will demonstrate a therapeutic potential of tRNA PTC suppressor or replacement methodologies.

Keywords: Mitochondrial diseases / Mitochondrial DNA mutations / suppressor tRNA / gene therapy modeling

Relevant publications:

- D. Jeandard, A. Smirnova, A. M. Fasemore, L. Coudray, N. Entelis, K. U. Forstner, I. Tarassov, A. Smirnov, CoLoC-seq probes the global topology of organelle transcriptomes. Nucleic acids research 51, e16 (2023); published online EpubFeb 22 (10.1093/nar/gkac1183).

- K. Aure, G. Fayet, I. Chicherin, B. Rucheton, S. Filaut, A. M. Heckel, J. Eichler, F. Caillon, Y. Pereon, N. Entelis, I. Tarassov, A. Lombes, Homoplasmic mitochondrial tRNA(Pro) mutation causing exercise-induced muscle swelling and fatigue. Neurology. Genetics 6, e480 (2020); published online EpubAug (10.1212/NXG.0000000000000480).

- O. A. Kolesnikova, N. S. Entelis, H. Mireau, T. D. Fox, R. P. Martin, I. A. Tarassov, Suppression of mutations in mitochondrial DNA by tRNAs imported from the cytoplasm. Science 289, 1931-1933 (2000); published online EpubSep 15 (10.1126/science.289.5486.1931).

Financial support of this subject is guaranteed by the Cluster / LabEx MitoCross

Prostate cancer (PCa) is the second most commonly diagnosed cancer worldwide, the fifth cause of cancer-related mortality. As a slow-growing disease, PCa takes decades to elicit clinical symptoms. Moreover, chemical or surgical androgen deprivation are the main treatments offered to patients facing advanced PCa. Unfortunately, the disease progresses to castration-resistant PCa for which chemotherapies are poorly effective. It is therefore crucial to identify new molecular players in PCa to develop better and more precise treatments. PCa cells exhibit a unique metabolic reprogramming, drastically affecting various aspects of lipid metabolism from lipid biosynthesis and storage, fatty acids (FA) b-oxidation, to membrane remodeling, and supports cancer growth, survival, metastasis and therapy resistance. This project proposes to identify the physical and functional interplays between organelles involved in lipid metabolism (lipid droplets, mitochondria and peroxisomes), and how they contribute to PCa development, growth and metastasis. To do so, we will establish at the transcriptome and proteome levels components involved in FA metabolism and organelles contacts by multi-omics approaches (RNAseq, Proteomics). We will also characterize LD-mitochondria-peroxisomes dynamics and contacts by microscopy (TEM, confocal) using patient-derived tissues grown in 2D and 3D (organoids) as model. Best candidates will serve as proxies to design new therapeutic drugs targeting organelles contact sites and FA metabolism using genetically-modified mice and patient-derived organoids. 

Keywords: organoids, lipid droplet, mitochondria, peroxisomes, fatty acid, prostate cancer, organelles, contact sites, specialized medicine

Relevant publications:

- M. A. Abu el Maaty, E. Grelet, C. Keime, A.-I. Rerra, J. Gantzer, C. Emprou, J. Terzic, R. Lutzing, J.-M. Bornert, G. Laverny, D. Metzger (2021). Single-cell analyses unravel cell type–specific responses to a vitamin D analog in prostatic precancerous lesions. Sci. Adv. 7, eabg5982. PMID: 34330705.

- Mohamed A. Abu el Maaty, Julie Terzic, Céline Keime, Daniela Rovito, Régis Lutzing, Darya Yanushko, Maxime Parisotto, Elise Grelet, Izzie Jacques Namer, Véronique Lindner, Gilles Laverny and Daniel Metzger (2022). Hypoxia-mediated stabilization of HIF1A in prostatic intraepithelial neoplasia promotes cell plasticity and malignant progression. Science advances 8, eabo2295. PMID: 35867798.

- Julie Terzic, Mohamed A. Abu el Maaty, Régis Lutzing, Alexandre Vincent, Rana El Bizri, Matthieu Jung, Céline Keime and Daniel Metzger (2023). Hypoxia-inducible factor 1A inhibition overcomes castration resistance of prostate tumors. EMBO Molecular Medicine. e17209. https://doi.org/10.15252/emmm.202217209. PMID: 37070472.  

This project is for an inter-cluster fellowship.