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The branched-chain amino acids: leucine, isoleucine and valine occupy a special place among the essential amino acids because of their importance not only in the structure of proteins but also in general and cerebral metabolism. Among the first amino acids absorbed after food intake, they play a major role in the regulation of protein synthesis and insulin secretion. They are involved in the modulation of brain uptake of monoamine precursors with which they may compete for occupancy of a common transporter. In the brain, branched-chain amino acids are involved not only in protein synthesis but also in the metabolic cycles of GABA and Glutamate, and in energy metabolism. In particular, they can affect GABAergic neurons and the excitation/inhibition balance. Branched-chain amino acids are known for the 24-hour rhythmicity of their plasma concentrations, which is remarkably conserved in rodent models. This rhythmicity is partly circadian, independent of sleep and food. Moreover, their concentration increases when sleep is disturbed and in obesity and diabetes. The mechanisms regulating these rhythms and their physiological impact remain poorly understood. In this context, the Drosophila model has not yet been widely used, but it is highly relevant and the first results indicate that it can generate new concepts. The elucidation of the metabolism and fluxes of branched-chain amino acids is beginning to shed light on the mysterious connections between clock, sleep, and metabolism, opening the possibility of new therapies., Les acides aminés branchés : leucine, isoleucine et valine occupent une place particulière parmi les acides aminés essentiels de par leur importance non seulement dans la structure des protéines mais aussi dans le métabolisme général et cérébral. Ils sont parmi les premiers acides aminés absorbés après la prise alimentaire et jouent un rôle majeur dans la régulation de la synthèse protéique et de la sécrétion d’insuline. Ils participent à la modulation de l’import cérébral des précurseurs de monoamines avec lesquels ils peuvent entrer en compétition pour l’occupation d’un transporteur commun. Dans le cerveau, les acides aminés branchés interviennent également dans les cycles métaboliques du GABA et du Glutamate, et dans le métabolisme énergétique. Ils peuvent notamment affecter les neurones GABAergiques et la balance excitation/inhibition. Les acides aminés branchés sont connus pour la rythmicité sur 24h de leurs concentrations plasmatiques qui est remarquablement conservée chez les modèles rongeurs. Cette rythmicité est en partie circadienne, indépendante du sommeil et de l’alimentation. Par ailleurs leur concentration augmente lorsque le sommeil est perturbé ainsi que dans l’obésité et le diabète. Les mécanismes régulant ces rythmes et leur impact physiologique restent mal compris. Dans ce contexte, le modèle drosophile a encore été peu utilisé, mais il a toute sa pertinence et les premiers résultats indiquent qu’il peut générer de nouveaux concepts. L’élucidation du métabolisme et des flux des acides aminés branchés commence à éclairer les connections mystérieuses qui existent entre horloge, sommeil, et métabolisme, ouvrant la possibilité de nouvelles thérapies.

Les nanopores biologiques sont d’étonnantes machines moléculaires. Ils remplissent une grande variété de fonctions, allant du tri des biomolécules à la transmission des signaux dans nos neurones et au repliement des protéines nouvellement produites. Le membre le plus surprenant de ce club est le pore nucléaire. Il régule le flux de molécules entre le noyau et l’intérieur de la cellule. Ses performances, mesurées par son efficacité énergétique, sa directionnalité ou sa sélectivité, n’ont pas d’équivalent dans les systèmes artificiels. Nous verrons que la compréhension de son fonctionnement permet d’appréhender des phénomènes physiques nouveaux et d’imaginer des systèmes de filtration sélectifs, ainsi que des pompes moléculaires.

Although several mechanisms of autophagy have been dissected in the last decade, following this pathway in real time remains challenging. Among the early events leading to its activation, the ATG4B protease primes the key autophagy player LC3B. Given the lack of reporters to follow this event in living cells, we developed a Förster’s Resonance Energy Transfer (FRET) biosensor responding to the priming of LC3B by ATG4B. The biosensor was generated by flanking LC3B within a pH-resistant donor-acceptor FRET pair, Aquamarine/tdLanYFP. We here showed that the biosensor has a dual readout. First, FRET indicates the priming of LC3B by ATG4B and the resolution of the FRET image allows to characterize the spatial heterogeneity of the priming activity. Second, quantifying the number of Aquamarine-LC3B puncta determines the degree of autophagy activation. We then showed that there are pools of unprimed LC3B uponATG4Bdownregulation, and that the priming of the biosensor is abolished inATG4Bknockout cells. The lack of priming can be rescued with the wild-type ATG4B or with the partially active W142A mutant, but not with the catalytically dead C74S mutant. Moreover, we screened for commercially-available ATG4B inhibitors, and we illustrated their differential mode of action by implementing a spatially-resolved, broad-to-sensitive analysis pipeline combining FRET and the quantification of autophagic puncta. Finally, we uncovered the CDK1-dependent regulation of the ATG4B-LC3B axis at mitosis. Therefore, the LC3B FRET biosensor paves the way for a highly-quantitative monitoring of the ATG4B activity in living cells and in real time, with unprecedented spatiotemporal resolution.

Irradiated cells can propagate signals to neighboring cells. Manifestations of these so-called bystander effects (BEs) are thought to be relatively more important after exposure to low- vs high-dose radiation and can be mediated via the release of secreted molecules, including inflammatory cytokines, from irradiated cells. Thus, BEs can potentially modify the inflammatory environment of irradiated cells. To determine whether these modifications could affect the functionality of bystander immune cells and their inflammatory response, we analyzed and compared the in vitro response of primary human fibroblasts and keratinocytes to low and high doses of radiation and assessed their ability to modulate the inflammatory activation of peripheral blood mononuclear cells (PBMCs). Only high-dose exposure resulted in either up- or down-regulation of selected inflammatory genes. In conditioned culture media transfer experiments, radiation-induced bystander signals elicited from irradiated fibroblasts and keratinocytes were found to modulate the transcription of inflammatory mediator genes in resting PBMCs, and after activation of PBMCs stimulated with lipopolysaccharide (LPS), a strong inflammatory agent. Radiation-induced BEs induced from skin cells can therefore act as a modifier of the inflammatory response of bystander immune cells and affect their functionality.

International audience; The presence of CD8+ T cells with a memory phenotype in nonimmunized mice has been noted for decades, but it was not until about 2 decades ago that they began to be studied in greater depth. Currently called virtual memory CD8+ T cells, they consist of a heterogeneous group of cells with memory characteristics, without any previous contact with their specific antigens. These cells were identified in mice, but a few years ago, a cell type with characteristics equivalent to the murine ones was described in healthy humans. In this review, we address the different aspects of its biology mainly developed in murine models and what is currently known about its cellular equivalent in humans.

Epithelia act as a barrier against environmental stress and abrasion and in vivo they are continuously exposed to environments of various mechanical properties. The impact of this environment on epithelial integrity remains elusive. By culturing epithelial cells on 2D hydrogels, we observe a loss of epithelial monolayer integrity through spontaneous hole formation when grown on soft substrates. Substrate stiffness triggers an unanticipated mechanical switch of epithelial monolayers from tensile on soft to compressive on stiff substrates. Through active nematic modelling, we find unique patterns of cell shape texture called nematic topological defects that underpin large isotropic stress fluctuations at certain locations thereby triggering mechanical failure of the monolayer and hole opening. Our results show that substrate stiffness provides feedback on monolayer mechanical state and that topological defects can trigger stochastic mechanical failure, with potential application towards a mechanistic understanding of compromised epithelial integrity in bacterial infection, tumor progression and morphogenesis.

International audience; Signaling through the inflammasome is important for the inflammatory response. Low concentrations of intracellular K+ are associated with the specific oligomerization and activation of the NLRP3 inflammasome, a type of inflammasome involved in sterile inflammation. After NLRP3 oligomerization, ASC protein binds and forms oligomeric filaments that culminate in large protein complexes named ASC specks. ASC specks are also initiated from different inflammasome scaffolds, such as AIM2, NLRC4, or Pyrin. ASC oligomers recruit caspase-1 and then induce its activation through interactions between their respective caspase activation and recruitment domains (CARD). So far, ASC oligomerization and caspase-1 activation are K+-independent processes. Here, we found that when there is low intracellular K+, ASC oligomers change their structure independently of NLRP3 and make the ASCCARD domain more accessible for the recruitment of the pro-caspase-1CARD domain. Therefore, conditions that decrease intracellular K+ not only drive NLRP3 responses but also enhance the recruitment of the pro-caspase-1 CARD domain into the ASC specks.

International audience; Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder, characterised by cognitive and behavioural impairment, in addition to the typical muscle pathology. Imaging studies revealed widespread white matter lesions in DM1 patients, which may be partially explained by local demyelination. DM1 is caused by the abnormal expansion of non-coding CTG trinucleotide repeat, which is transcribed into toxic CUG RNA that accumulates in nuclear RNA foci and disrupts critical RNA-binding proteins. To investigate the impact of RNA toxicity on myelin biology, we used a transgenic mouse model of DM1, known as the DMSXL mice, which express expanded CUG transcripts in multiple tissues and cell types.We found delayed axon myelination of the corpus callosum at two weeks of age, which later recovered at four months. Early myelin defects were linked to an overall reduction in the number of oligodendroglia cells, specifically myelinating oligodendrocytes (OL), with a concurrent increase in oligodendrocyte progenitor cells (OPC). These phenotypes were accompanied by abundant RNA foci and splicing dysregulation in oligodendroglia, which were more pronounced at two weeks. Taking advantage of primary cell models, we investigated the mechanisms triggered by toxic CUG RNA in oligodendroglia. OPC isolated from DMSXL newborns exhibited impaired differentiation into fully ramified OLs in culture, a defect we confirmed in human oligodendroglia derived from DM1 patients. RNA sequencing revealed expression and splicing changes in DMSXL OL that affect transcripts related to the cytoskeleton and cell differentiation. Importantly, the transcriptomic defects of DMSXL OL recreated expression and splicing profiles typical of immature OPC.In conclusion, toxic CUG RNA disrupts the molecular program of oligodendroglia differentiation, impairing the transcriptome changes occurring during the OPC-OL transition and leading to transient hypomyelination in mice.

Microtubules are cytoskeleton components with unique mechanical and dynamic properties. They are rigid polymers that alternate phases of growth and shrinkage. Nonetheless, the cells can display a subset of stable microtubules, but it is unclear whether microtubule dynamics and mechanical properties are related. Recent in vitro studies suggest that microtubules have mechano-responsive properties, being able to stabilize their lattice by self-repair on physical damage. Here we study how microtubules respond to cycles of compressive forces in living cells and find that microtubules become distorted, less dynamic and more stable. This mechano-stabilization depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. This process seems to be instrumental for cell migration in confined spaces. Overall, these results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected, being a central mediator of cellular mechano-responses.

The coordinating role of nuclear factor erythroid-2-related factor 2 (Nrf2) in cellular function is undeniable. Evidence indicates that this transcription factor exerts massive regulatory functions in multiple signaling pathways concerning redox homeostasis and xenobiotics, macromolecules, and iron metabolism. Being the master regulator of antioxidant system, Nrf2 controls cellular fate, influencing cell proliferation, differentiation, apoptosis, resistance to therapy, and senescence processes, as well as infection disease success. Because Nrf2 is the key coordinator of cell defence mechanisms, dysregulation of its signaling has been associated with carcinogenic phenomena and infectious and age-related diseases. Deregulation of this cytoprotective system may also interfere with immune response. Oxidative burst, one of the main microbicidal mechanisms, could be impaired during the initial phagocytosis of pathogens, which could lead to the successful establishment of infection and promote susceptibility to infectious diseases. There is still a knowledge gap to fill regarding the molecular mechanisms by which Nrf2 orchestrates such complex networks involving multiple pathways. This review describes the role of Nrf2 in non-pathogenic and pathogenic cells.

Sexuality is the most ancestral and common reproductive system but several forms of reproduction without sex exist. This requires modifications of female meiosis, for the production of unreduced gametes. The genomic consequences of asexuality have been well studied but much less is known about the cellular and molecular changes at the origin of new types of meiosis in asexuals. In my thesis, I first explored how asexual females are produced in the nematode species Mesorhabditis belari. I discovered that during meiosis, homologous chromosomes undergo crossing-overs. However, meiosis I is abortive due to a default in anaphase B. Meiosis II next proceeds normally giving rise to diploid oocytes with an assortment of non-sister chromatids. The theory predicts that species undergoing such modified meiosis with recombination should have a widely homozygous genome. However, our collaborators demonstrated that the genome of M. belari is widely heterozygous, which raised a paradox. Using a combination of cytology and genomics we uncovered this species undergoes a new type of meiosis, which we named Directed Chromatid Assortment (DCA), whereby the two recombinant chromatids of a given pair of chromosomes co-segregate during the meiotic division. We demonstrated that DCA allows the maintenance of genome-wide heterozygosity in an asexual, despite recombination. In parallel, I searched for genes involved in oocyte determination, using molecular and cytological tools. In doing so, I developed the CRISPR Cas9 technique in M. belari. Finally, I wrote a review which summarizes my readings and thoughts on the importance of cytological approaches for the study of asexuality.; La sexualité est le mode reproduction le plus ancien et le plus courant, pourtant de nombreuses formes de reproduction sans sexualité existent. Celles-ci requièrent des modifications de la méiose chez les femelles, afin de produire des gamètes non réduits. Si les conséquences génomiques de l’asexualité ont été extensivement étudiées, nous ne savons que peu de choses sur les changements cellulaires et moléculaires à l’origine des nouveaux types de méiose chez les asexués. Lors de ma thèse, j’ai en premier lieu exploré comment les femelles asexuées sont produites chez l’espèce de nématode Mesorhabditis belari. J’ai découvert que durant la méiose, les chromosomes homologues effectuent des crossing-overs et donc recombinent. De plus, la méiose I est abortive, ceci étant dû à un défaut au court de l’anaphase B. Par la suite, la méiose II se déroule classiquement et génère des ovocytes diploïdes contenant un assortiment de chromatides non sœurs. La théorie prédit que dans une espèce soumise à une telle méiose avec des évènements de recombinaison, le génome de cette espèce devrait être largement homozygote. Or, nos collaborateurs ont démontré qu’en réalité M. belari possède un génome hétérozygote, ce qui, de ce fait, constitue un paradoxe. En combinant des approches de cytologie et de génomique, nous avons révélé que cette espèce subit une méiose d’un nouveau type que nous avons nommé « Directed Chromatid Assortment » (DCA). Selon le DCA, les deux chromatides recombinantes d’une paire donnée de chromosomes co-ségrègent ensemble durant la division méiotique. En somme, nous avons démontré que le DCA permet le maintien de l’hétérozygotie du génome de l’espèce en dépit de la présence de recombinaison. En parallèle de ce travail, j’ai recherché les gènes impliqués dans la détermination du type d’ovocytes chez M. belari en usant d’outils moléculaires et cytologiques. Pour cela, j’ai développé la technique de transgénèse CRISPR Cas9 chez M. belari. Enfin, j’ai rédigé une revue qui référence mes lectures et mes réflexions sur l’importance de l’approche cytologique dans l’étude de l’asexualité.

International audience; Glioblastomas (GBM) are heterogeneous tumors with high metabolic plasticity. Their poor prognosis is linked to the presence of glioblastoma stem cells (GSC), which support resistance to therapy, notably to temozolomide (TMZ). Mesenchymal stem cells (MSC) recruitment to GBM contributes to GSC chemoresistance, by mechanisms still poorly understood. Here, we provide evidence that MSCs transfer mitochondria to GSCs through tunneling nanotubes, which enhances GSCs resistance to TMZ. More precisely, our metabolomics analyses reveal that MSC mitochondria induce GSCs metabolic reprograming, with a nutrient shift from glucose to glutamine, a rewiring of the tricarboxylic acid cycle from glutaminolysis to reductive carboxylation and increase in orotate turnover as well as in pyrimidine and purine synthesis. Metabolomics analysis of GBM patient tissues at relapse after TMZ treatment documents increased concentrations of AMP, CMP, GMP, and UMP nucleotides and thus corroborate our in vitro analyses. Finally, we provide a mechanism whereby mitochondrial transfer from MSCs to GSCs contributes to GBM resistance to TMZ therapy, by demonstrating that inhibition of orotate production by Brequinar (BRQ) restores TMZ sensitivity in GSCs with acquired mitochondria. Altogether, these results identify a mechanism for GBM resistance to TMZ and reveal a metabolic dependency of chemoresistant GBM following the acquisition of exogenous mitochondria, which opens therapeutic perspectives based on synthetic lethality between TMZ and BRQ.Significance: Mitochondria acquired from MSCs enhance the chemoresistance of GBMs. The discovery that they also generate metabolic vulnerability in GSCs paves the way for novel therapeutic approaches.

International audience; Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the dysfunction of pulmonary endothelial cells (ECs) and obstructive vascular remodeling. The non-receptor tyrosine kinase c-Abelson (cAbl) plays central roles in regulating cell-cycle arrest, apoptosis, and senescence after cellular stress. We hypothesized that cAbl is down-activated in experimental and human PAH, thus leading to reduced DNA integrity and angiogenic capacity of pulmonary ECs from PAH patients (PAH-ECs). We found cAbl and phosphorylated cAbl levels to be lower in the endothelium of remodeled pulmonary vessels in the lungs of PAH patients than controls. Similar observations were obtained for the lungs of sugen+hypoxia (SuHx) and monocrotaline (MCT) rats with established pulmonary hypertension. These in situ abnormalities were also replicated in vitro, with cultured PAH-ECs displaying lower cAbl expression and activity and an altered DNA damage response and capacity of tube formation. Downregulation of cAbl by RNA-interference in Control-ECs or its inhibition with dasatinib resulted in genomic instability and the failure to form tubes, whereas upregulation of cAbl with DPH reduced DNA damage and apoptosis in PAH-ECs. Finally, we establish the existence of crosstalk between cAbl and bone morphogenetic protein receptor type II (BMPRII). This work identifies the loss of cAbl signaling as a novel contributor to pulmonary EC dysfunction associated with PAH.

International audience; Treating large bone defects or fragile patients may require enhancing the bone regeneration rate to overcome a weak contribution from the body. This work investigates the osteogenic potential of nutrient fisetin, a flavonoid found in fruits and vegetables, as a doping agent inside the structure of a SiO 2 − CaO bioactive glass−poly(caprolactone) (BG−PCL) hybrid scaffold. Embedded in the full mass of the BG− PCL hybrid during one-pot synthesis, we demonstrate fisetin to be delivered sustainably; the release follows a first-order kinetics with active fisetin concentration being delivered for more than 1 month (36 days). The biological effect of BG−PCL−fisetin-doped scaffolds (BG−PCL−Fis) has been highlighted by in vitro and in vivo studies. A positive impact is demonstrated on the adhesion and the differentiation of rat primary osteoblasts, without an adverse cytotoxic effect. Implantation in critical-size mouse calvaria defects shows bone remodeling characteristics and remarkable enhancement of bone regeneration for fisetin-doped scaffolds, with the regenerated bone volume being twofold that of nondoped scaffolds and fourfold that of a commercial trabecular bovine bone substitute. Such highly bioactive materials could stand as competitive alternative strategies involving biomaterials loaded with growth factors, the use of the latter being the subject of growing concerns.

Posté le 17 septembre 2021.; International audience; ObjectivesA nationwide cross-sectional epidemiological survey was conducted to capture the true extent of coronavirus disease 2019 (COVID-19) exposure in Senegal.MethodsMulti-stage random cluster sampling of households was performed between October and November 2020, at the end of the first wave of COVID-19 transmission. Anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies were screened using three distinct ELISA assays. Adjusted prevalence rates for the survey design were calculated for each test separately, and thereafter combined. Crude and adjusted prevalence rates based on test performance were estimated to assess the seroprevalence. As some samples were collected in high malaria endemic areas, the relationship between SARS-CoV-2 seroreactivity and antimalarial humoral immunity was also investigated.ResultsOf the 1463 participants included in this study, 58.8% were female and 41.2% were male; their mean age was 29.2 years (range 0.20–84.8.0 years). The national seroprevalence was estimated at 28.4% (95% confidence interval 26.1–30.8%). There was substantial regional variability. All age groups were impacted, and the prevalence of SARS-CoV-2 was comparable in the symptomatic and asymptomatic groups. An estimated 4 744 392 (95% confidence interval 4 360 164–5 145 327) were potentially infected with SARS-CoV-2 in Senegal, while 16 089 COVID-19 RT-PCR laboratory-confirmed cases were reported by the national surveillance. No correlation was found between SARS-CoV-2 and Plasmodium seroreactivity.ConclusionsThese results provide a better estimate of SARS-CoV-2 dissemination in the Senegalese population. Preventive and control measures need to be reinforced in the country and especially in the south border regions.

Different strains of a microorganism growing in the same environment display a wide variety of growth rates and growth yields. We developed a coarse-grained model to test the hypothesis that different resource allocation strategies, corresponding to different compositions of the proteome, can account for the observed rate-yield variability. The model predictions were verified by means of a database of hundreds of published rate-yield and uptake-secretion phenotypes ofEscherichia colistrains grown in standard laboratory conditions. We found a very good quantitative agreement between the range of predicted and observed growth rates, growth yields, and glucose uptake and acetate secretion rates. These results support the hypothesis that resource allocation is a major explanatory factor of the observed variability of growth rates and growth yields across different bacterial strains. An interesting prediction of our model, supported by the experimental data, is that high growth rates are not necessarily accompanied by low growth yields. The resource allocation strategies enabling high-rate, high-yield growth ofE. colilead to a higher saturation of enzymes and ribosomes, and thus to a more efficient utilization of proteomic resources. Our model thus contributes to a fundamental understanding of the quantitative relationship between rate and yield inE. coliand other microorganisms. It may also be useful for the rapid screening of strains in metabolic engineering and synthetic biology.

International audience; A signature feature of the animal kingdom is the presence of epithelia: sheets of polarized cells that both insulate the organism from its environment and mediate interactions with it. Epithelial cells display a marked apico-basal polarity, which is highly conserved across the animal kingdom, both in terms of morphology and of molecular regulators. How did this architecture first evolve? Although the last eukaryotic common ancestor almost certainly possessed a simple form of apico-basal polarity (marked by the presence of one or several flagella at a single cellular pole), comparative genomics and evolutionary cell biology reveal that the polarity regulators of animal epithelial cells have a surprisingly complex and stepwise evolutionary history. Here, we retrace their evolutionary assembly. We suggest that the "polarity network" that polarized animal epithelial cells evolved by integration of initially independent cellular modules that evolved at distinct steps of our evolutionary ancestry. The first module dates back to the last common ancestor of animals and amoebozoans and involved Par1, extracellular matrix proteins, and the integrin-mediated adhesion complex. Other regulators, such as Cdc42, Dlg, Par6 and cadherins evolved in ancient unicellular opisthokonts, and might have first been involved in F-actin remodeling and filopodial dynamics. Finally, the bulk of "polarity proteins" as well as specialized adhesion complexes evolved in the metazoan stem-line, in concert with the newly evolved intercellular junctional belts. Thus, the polarized architecture of epithelia can be understood as a palimpsest of components of distinct histories and ancestral functions, which have become tightly integrated in animal tissues.

Lamin A/C is a well-established key contributor to nuclear stiffness and its role in nucleus mechanical properties has been extensively studied. However, its impact on whole cell mechanics has been poorly addressed, even less so in terms of measurable physical parameters. In the present study, microfluidic experiments combined with theoretical analyses were performed to provide a quantitative estimation of the whole cell mechanical properties. This allowed the characterization of mechanical cell changes induced by lamin A/C alterations resulting from Atazanavir treatment or lipodystrophy-associated LMNA R482W pathogenic variant. Results unveil an increase in the long-time viscosity as a signature of cells affected by lamin A/C alterations. In addition, they show that the whole cell response to mechanical stress is driven not only by the nucleus but also by the nucleo-cytoskeleton links and the microtubule network. This enhanced cell viscosity assessed by our microfluidic device could represent a useful diagnosis marker for lamin-related diseases.

SummaryAlthough oncogene-induced senescence (OIS) is a potent tumor-suppressor mechanism, recent studies revealed that cells can escape from OIS with features of transformed cells. However, the mechanisms that promote OIS escape remain unclear, and evidence of post-senescent cells in human cancers is missing. Here, we unravel the regulatory mechanisms underlying OIS escape using dynamic multidimensional profiling. We demonstrate a critical role for AP1 and POU2F2 transcription factors for escape from OIS and identify ‘senescence-associated chromatin scars (SACS)’ as an epigenetic memory of OIS, detectable during colorectal cancer progression. POU2F2 levels are elevated already in precancerous lesions and as cells escape from OIS, and its expression and binding activity to cis-regulatory elements are associated with decreased patient survival. Our results support a model in which POU2F2 exploits a precoded enhancer landscape to promote senescence escape and reveal POU2F2 gene signatures and SACS as valuable biomarkers with diagnostic and prognostic potential.

International audience; The difficulty of retrieving high-resolution, in vivo evidence of the proliferative and migratory processes occurring in neural germinal zones has limited our understanding of neurodevelopmental mechanisms. Here, we used a connectomic approach using a high-resolution, serial-sectioning scanning electron microscopy volume to investigate the laminar cytoarchitecture of the transient external granular layer (EGL) of the developing cerebellum, where granule cells coordinate a series of mitotic and migratory events. By integrating image segmentation, three-dimensional reconstruction, and deep-learning approaches, we found and characterized anatomically complex intercellular connections bridging pairs of cerebellar granule cells throughout the EGL. Connected cells were either mitotic, migratory, or transitioning between these two cell stages, displaying a chronological continuum of proliferative and migratory events never previously observed in vivo at this resolution. This unprecedented ultrastructural characterization poses intriguing hypotheses about intercellular connectivity between developing progenitors and its possible role in the development of the central nervous system.

Toxoplasma gondii is a member of Apicomplexa phylum, which is charaterzied by the presence of three parasite’s specific secretory organelles named micronemes, rhoptries and dense granules. While some Apicomplexa parasites can infect either only animals or humans cells, Toxoplasma gondii infects both animals and humans to induce toxoplasmosis. Like other Apicomplexa parasites such as Plasmodium SP, Cryptosporidium SP..., the virulence and intracellular survival of T. gondii depend on the MICs, ROPs/RONs and GRAs proteins that are the virulence factors contained in micronemes, rhoptries and dense granules respectively. The trafficking of this factors for the biogenesis of the parasite organelles as well as their secretion during invasion depend on other essential factors such as TgVPS35 and TgSORT which are partners of each other and which allowed to identify TgREMIND (T. gondii REgulator of Membrane Interacting Domain) which is the project of my thesis. The goal of this thesis is to elucidate the biological functions of the TgREMIND protein in intracellular trafficking and biogenesis of specific secretory organelles in T. gondii. We first showed by bioinformatics that TgREMIND has a N-terminus F-BAR domain known to bind membranes by inducing vesicle formation and a novel C-terminus domain that we named REMIND for Regulator of Membrane Interacting Domain. We subsequently showed by Lipid Overlay assay that TgREMIND binds several phosphatidylinositol phosphate (PIP2 species). These data confirm the predicted interaction of TgREMIND with the membrane. We also identified by GST-pull down TgREMIND partners, such as some trafficking factors like TgVPS35, TgSORT, SECs etc..., some lipid factors as START domaining containing protein and phosphatidylinositol kinases, and dense granules proteins and rhoptries proteins, which are in the majority of all partners. Moreover, by testing the anti-TgREMIND antibody on wild type parasites, we first identified by confocal microscopy the signal of TgREMIND which is localized as punctated siganls in the cytoplasm of the parasites and includes the golgi and post-golgi compartments, endosomal and at the apical end where rhoptries are located. Then we determined by western blot, TgREMIND native size that is about 140 kDa. Moreover, we created an inducible knock-out mutant of TgREMIND and showed that in the absence of TgREMIND protein, there are morphological changes of the rhoptries, disappearance of dense granules and inhibition of the secretion of these organelles. This severely affected the motility of the parasites resulting in their inability to invade mammalian cells to cause the infection. These defects were restored by complementation of the TgREMIND mutants. We thus conclude that TgREMIND protein is probably involved in the formation of membrane vesicules that are required for the biogenesis of the specific secretory organelles of T. gondii. The second part of my thesis focused on the TgSTART protein, lipid partner of TgREMIND. We have characterized TgSTART from transgenic parasites and the Knockdown mutant of the TgSTART gene tagged with the HA epitope.; Toxoplasma gondii est membre des parasites Apicomplexa qui sont caractérisés non seulement par un développement intracellulaire obligatoire, mais aussi par trois organites sécrétoires spécifiques à savoir les micronèmes, rhoptries et les granules denses. Pendant que certains parasites Apicomplexa sont capables d’infecter soit uniquement les animaux ou les humains, Toxoplasma gondii infecte à la fois les animaux et les humains en causant la toxoplasmose. Tout comme les autres parasites Apicomplexa (Plasmodium SP, Cryptosporidium SP…) la virulence et la survie intracellulaire de T. gondii dépendent des facteurs de virulence MICs, ROPs/RONs et GRAs que renferment respectivement les micronèmes, les rhoptries et les granules denses. Le trafic des facteurs pour la biogénèse des organites du parasite ainsi que leur sécrétion lors de l’invasion dépendent d’autres facteurs indispensables dont TgVPS35 et TgSORT qui sont partenaires l’une de l’autre et qui ont permis d’identifier TgREMIND (T. gondii REgulator of Membrane Interacting Domain) qui a fait l’objet de ma thèse. Le but principal de cette thèse était d’élucider les fonctions biologiques de la protéine TgREMIND dans le trafic intracellulaire et la biogénèse des organites sécrétoires spécifiques chez T. gondii. Nous avons dans un premier temps montré par bio-informatique que TgREMIND possède un premier domaine F-BAR connu pour fixer les membranes en induisant la formation de vésicules et un second domaine original de fonctions inconnues que nous avons appelé REMIND (Regulator of Membrane Interacting Domain). Ce domaine a donc donné le nom de la protéine entière TgREMIND. Nous avons montré par la suite grâce aux tests de Lipid Overlay que TgREMIND fixe plusieurs phosphoinositols (PIP2) confirmant ainsi l’interaction prédite de TgREMIND avec la membrane. Nous avons aussi identifié par GST-pull down les partenaires de TgREMIND, comme certains facteurs du trafic (TgVPS35, TgSORT, SECs etc…), certains facteurs lipidiques (START domaining containing protein et des phosphatidylinositols kinases) et des protéinesdes granules denses et celles des rhoptries qui sont majoritaires de toutes les partenaires. Par ailleurs, en testant l’anticorps anti TgREMIND sur les parasites sauvages, nous avons d’abord identifié en microscopie confocale le signal de TgREMIND qui est un signal ponctiforme cytoplasmique dans différents compartiments subcellulaires y compris les compartiments golgiens et post-golgiens, endosomal et à l’extrémité apical du parasite donc à proximité des rhoptries. Nous avons déterminé par western blots que la taille native est d’environ 140 kDa de TgREMIND. Ensuite, nous avons créé un mutant knock-out inductible de TgREMIND à partir duquel nous avons montré qu’en absence de TgREMIND chez le mutant, qu’il y a des changements morphologiques des rhoptries, disparition des granules denses et absence de la sécrétion de leur contenu. Cela a affecté sévèrement la motilité des parasites, ce qui a pour conséquence une incapacité sévère des parasites à envahir les cellules de mammifères pour provoquer une infection toxoplasmique. Les différents défauts ont été corrigés par complémentation du mutant TgREMIND. Nous avons ainsi conclu que la protéine TgREMIND est probablement impliquée dans la formation de la membrane vésiculaire requise pour la biogenèse des organites sécrétoires spécifiques de T. gondii. La deuxième partie de ma thèse était portée sur la protéine TgSTART, partenaire lipidique de TgREMIND. Nous avons caractérisé TgSTART à partir des parasites transgéniques et du mutant Knockdown du gène TgSTART taggués avec l’épitope HA.

Prokineticin-1 (PROK1) protein, also known as EG-VEGF (Endocrine gland derived-vascular endothelial growth factor) belongs to the prokineticin (PROK) family, whose members are directly involved in the control of key processes such as inflammation and angiogenesis. PROK1 acts via two GPCR receptors, PROKR1 and PROKR2 and regulates several physiological and pathological processes, including cancer and cystic fibrosis (CF). My research team recently demonstrated that the expression of PROK1 and PROKR2 were increased in the female reproductive cancer, gestational choriocarcinoma (GC) and in CF. The antagonization of PROKR2 in an animal model of GC decreased tumor growth and its progression, suggesting that inhibition of the PROK1/PROKR2 system may constitute a promising therapeutic target for all PROK1-dependent cancers and pro-inflammatory diseases. However, anti-PROKR2 antagonists are not specific for this receptor, exhibit long-term toxicity and may be associated with the development of side effects as they block a shared receptor with other members of the PROKs family.The objectives of my thesis project aimed at i) developing a new strategy that targets the inhibition of the ligand, PROK1, and ii) characterizing the molecular and cellular features that differentiate PROKR1 from PROKR2 in order to propose new therapeutic strategies that will specifically target one or the other receptor.The development of the PROK1 blocking strategy required the cloning of the human Prok1 sequence in different plasmids, the implementation of several eukaryotic or prokaryotic expression and production systems, and the development of numerous purification techniques in order to resolve its three-dimensional structure (STD). In silico analyses were also performed to accelerate the STD resolution of PROK1, to characterize the sites of its interaction with PROKR2, and to perform molecular docking in order to identify chemical molecules able to inhibit the PROK1-PROKR2 interaction. A functional assay was also developed to test in vitro the most promising molecules. The functional test required the development of two stable cell lines expressing PROKR1 or PROKR2.The cellular and molecular characterization of PROKR1 and PROKR2 revealed post-translational differences regarding the receptor’s half-life, its stability, trafficking and folding levels. Based on these observations, we initiate the investigation of the mechanisms behind these differences, firstly by targeting the roles of N- and C-terminus in these processes, particularly in the dimerizations of PROKR1and PROKR2.Altogether my thesis project allowed the production of a recombinant PROK1 protein, the resolution of its structure and the identification of the amino acids that are involved in its interaction with PROKR2. The development of cell lines expressing PROKR1 or PROKR2 allowed, i) the set-up of a functional test of the activity of prokineticins, ii) the identification of three inhibiting molecules of the PROK1 ligand and iii) the demonstration that, in spite the existence of a huge identity between PROKR1 and PROKR2, PROKR1 would be more stable at the plasma membrane; undergo less degradation at the lysosome level and its C-terminal part may confer the dimeristaion between PROKRs receptors.In conclusion, my thesis project established new molecular tools for the study of the prokineticins system; identified new inhibiting molecules of the PROK1-PROKR2 interaction, and compared for the first time PROKRs cellular and biochemical features. All these data will allow better targeting of PROKRs and their ligands in order to propose more specific pharmacotherapies for PROK-dependent cancers and inflammatory diseases, such as preeclampsia and cystic fibrosis.; Le facteur, PROK1 aussi dénommé EG-VEGF appartient à la famille des prokinéticines (PROK), dont les membres sont directement impliqués dans le contrôle de l’inflammation et l’angiogenèse. Ces facteurs agissent via deux récepteurs de la famille des RCPG, PROKR1 et PROKR2 et régulent plusieurs processus physiologiques et pathologiques, y compris le cancer. Mon équipe de recherche a récemment démontré que l’expression de PROK1 et PROKR2 était augmentée dans le cancer du système reproducteur féminin, le choriocarcinome gestationnel (CG), et dans la mucoviscidose. L’antagonisation de PROKR2 dans un modèle animal de CG diminue le développement et la progression de ce cancer et réduit la réponse inflammatoire, suggérant que l'inhibition du système PROK1/PROKR2 pourrait constituer une cible thérapeutique prometteuse pour les cancers et les pathologies pro-inflammatoires PROK1-dépendantes. Cependant, les antagonistes anti-PROKR2 ne sont pas spécifiques pour ce récepteur, présentent une toxicité à long terme et peuvent être associés au développement d’effets secondaires. Ainsi, les objectifs de ma thèse étaient, i) de développer une nouvelle stratégie d’inhibition en ciblant le ligand, PROK1, et ii) de mettre en évidence les différences moléculaires et cellulaires des récepteurs PROKR1 et PROKR2 afin de proposer, à long terme, des stratégies thérapeutiques ciblant spécifiquement l’un ou l’autre récepteur. Le développement de la stratégie du blocage du ligand PROK1 a nécessité, le clonage de la séquence humaine de prok1 dans différents plasmides, la mise en place de plusieurs systèmes « eucaryotes ou procaryotes » d’expression et de production, et le développement de nombreuses techniques de purification afin de résoudre sa structure tridimensionnelle (STD). Des analyses in silico ont également été réalisées ; pour accélérer la résolution de sa STD, caractériser les interfaces de son interaction avec PROKR2, et réaliser le docking moléculaire dans le but d’identifier des molécules chimiques capables d’inhiber l’interaction PROK1-PROKR2. Un test fonctionnel a aussi été développé pour évaluer in vitro les molécules les plus prometteuses. Pour la réalisation de ce test fonctionnel, nous avons généré deux lignées stables exprimant PROKR1 ou PROKR2. La caractérisation cellulaire et moléculaire de PROKR1 et PROKR2 a mis en évidence l’existence de différences post-traductionnelles concernant les demi-vies des récepteurs, leur stabilité, leur mode d’adressage à la membrane et leurs niveaux de repliement. Partant de ce constat, nous avons débuté l’étude des mécanismes sous-jacents à ces différences en ciblant prioritairement les extrémités N- et C-ter et leurs implications dans le processus de dimérisation potentielle de PROKR1 et PROKR2.L’ensemble des résultats de ma thèse ont permis de produire la protéine PROK1, de résoudre sa structure et d’identifier les acides aminés impliqués dans son interaction avec le PROKR2. Le développement des lignées exprimant PROKR1 ou PROKR2 a permis, i) de mettre en place un test fonctionnel de l’activité des prokinéticines, ii) d’identifier trois molécules inhibitrices du ligand PROK1 et iii) de démontrer qu’en dépit de la faible différence d’identité entre PROKR1 et PROKR2, que PROKR1serait plus stable au niveau de la membrane, subirait moins de dégradation au niveau du lysosome, et que sa partie C-ter confèrerait la dimérisation entre récepteurs PROKRs.En conclusion, ma thèse a apporté des outils moléculaires pour l’étude des prokinéticines, a permis d’identifier de nouvelles molécules inhibitrices du ligand PROK1 et de son interaction avec PROKR2 et à comparer pour la première fois les deux récepteurs de cette famille. L’ensemble de ces données nous permettra de mieux cibler les récepteurs PROKRs et leurs ligands afin de proposer une pharmacothérapie plus spécifique des cancers PROK-dépendants et des maladies inflammatoires dans lesquelles ils sont dérégulés, comme la prééclampsie et la mucoviscidose.

International audience; The analysis of multidimensional time-varying datasets faces challenges, notably regarding the representation of the data and the visualization of temporal variations. We propose an extension of the well-known Space-Time Cube (STC) visualization technique in order to visualize time-varying 3D spatial data, taking advantage of the interaction capabilities of Virtual Reality (VR). First, we propose the Space-Time Hypercube (STH) as an abstraction for 3D temporal data, extended from the STC concept. Second, through the example of embryo development imaging dataset, we detail the construction and visualization of a STC based on a user-driven projection of the spatial and temporal information. This projection yields a 3D STC visualization, which can also encode additional numerical and categorical data. Additionally, we propose a set of tools allowing the user to filter and manipulate the 3D STC which benefits the visualization, exploration and interaction possibilities offered by VR. Finally, we evaluated the proposed visualization method in the context of 3D temporal cell imaging data analysis, through a user study (n = 5) reporting the feedback from five biologists. These domain experts also accompanied the application design as consultants, providing insights on how the STC visualization could be used for the exploration of complex 3D temporal morphogenesis data.

Noxo1, the organizing element of the Nox1-dependent NADPH oxidase complex responsible for producing reactive oxygen species, has been described to be degraded by the proteasome. We mutated a D-box in Noxo1 to express a protein with limited degradation and capable of maintaining Nox1 activation. Wild-type (wt) and mutated Noxo1 (mut1) proteins were expressed in different cell lines to characterize their phenotype, functionality, and regulation. Mut1 increases ROS production through Nox1 activity affects mitochondrial organization and increases cytotoxicity in colorectal cancer cell lines. Unexpectedly the increased activity of Noxo1 is not related to a blockade of its proteasomal degradation since we were unable in our conditions to see any proteasomal degradation either for wt or mut1 Noxo1. Instead, D-box mutation mut1 leads to an increased translocation from the membrane soluble fraction to a cytoskeletal insoluble fraction compared to wt Noxo1. This mut1 localization is associated in cells with a filamentous phenotype of Noxo1, which is not observed with wt Noxo1. We found that mut1 Noxo1 associates with intermediate filaments such as keratin 18 and vimentin. In addition, Noxo1 D-Box mutation increases Nox1-dependent NADPH oxidase activity. Altogether, Nox1 D-box does not seem to be involved in Noxo1 degradation but rather related to the maintenance of the Noxo1 membrane/cytoskeleton balance.

Cell migration depends on the dynamic organization of the actin cytoskeleton and assembly and disassembly of focal adhesions (FA). However the precise mechanisms coordinating these processes remain poorly understood. We previously identified the estrogen-related receptor α (ERRα) as a major regulator of cell migration. Here, we show that loss of ERRα leads to abnormal accumulation of actin filaments that is associated with an increase in the level of inactive form of the actin-depolymerizing factor cofilin. We further show that ERRα depletion decreases cell adhesion and promotes defective FA formation and turnover. Interestingly, specific inhibition of the RhoA-ROCK-LIMK-cofilin pathway rescues the actin polymerization defects resulting from ERRα silencing, but not cell adhesion. Instead we found that MAP4K4 is a direct target of ERRα and down-regulation of its activity rescues cell adhesion and FA formation in the ERRα-depleted cells. Altogether, our results highlight a crucial role of ERRα in coordinating the dynamic of actin network and focal adhesion through the independent regulation of the RhoA and MAP4K4 pathways.

International audience; Tracking the pH variation of intracellular vesicles throughout the endocytosis pathway is of prior importance to better assess the cell trafficking and metabolism of cells. Small molecular fluorescent pH probes are valuable tools in bioimaging but are generally not targeted to intracellular vesicles or are directly targeted to acidic lysosomes, thus not allowing the dynamic observation of the vesicular acidification. Herein, we designed Mem-pH, a fluorogenic ratiometric pH probe based on chromenoquinoline with appealing photophysical properties, which targets the plasma membrane (PM) of cells and further accumulates in the intracellular vesicles by endocytosis. The exposition of Mem-pH toward the vesicle's lumen allowed to monitor the acidification of the vesicles throughout the endocytic pathway and enabled the measurement of their pH via ratiometric imaging.

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International audience; Tumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Background: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt–Oram syndrome (also known as hand–heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. Methods: We analyzed ECGs of an extended family pedigree of Holt–Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome ( Tbx5 G125R ) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). Results: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt–Oram syndrome patients. Tbx5 G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5 G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. Conclusions: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

Ubiquitylation is one of the most common post-translational modifications (PTMs) of proteins that frequently targets substrates for proteasomal degradation. However it can also result in non-proteolytic events which play important functions in cellular processes such as intracellular signaling, membrane trafficking, DNA repair and cell cycle. Emerging evidence demonstrates that dysfunction of non-proteolytic ubiquitylation is associated with the development of multiple human diseases. In this review, we summarize the current knowledge and the latest concepts on how non-proteolytic ubiquitylation pathways are involved in cellular signaling and in disease-mediating processes. Our review, may advance our understanding of the non-degradative ubiquitylation process.

International audience; Mitochondria are essential to cell homeostasis, and alterations in mitochondrial distribution, segregation, and turnover have been linked to complex pathologies such as neurodegenerative diseases and cancer. Understanding how these functions are coordinated in specific cell types is a major challenge to discover how mitochondria globally shape cell functionality. In this review, we first describe how mitochondrial transport and dynamics are regulated throughout the cell cycle in yeast and in mammals. Second, we explore the functional consequences of mitochondrial transport and partitioning on cell proliferation, fate acquisition, and stemness and on the way cells adapt their metabolism. Finally, we focus on how mitochondrial clearance programs represent a further layer of complexity for cell differentiation or in the maintenance of stemness. Defining how mitochondrial transport, dynamics, and clearance are mutually orchestrated in specific cell types may help our understanding of how cells can transition from a physiological to a pathological state.

International audience; Colchicine, the main active alkaloid from Colchicum autumnale L., is a potent tubulin binder and represents an interesting lead structure for the development of potential anticancer chemotherapeutics. We report on the synthesis and investigation of potentially reactive colchicinoids and their surprising biological activities. In particular, the previously undescribed colchicinoid PT-100, a Bring contracted 6-exomethylene colchicinoid, exhibits extraordinarily high antiproliferative and apoptosis-inducing effects on various types of cancer cell lines like acute lymphoblastic leukemia (Nalm6), acute myeloid leukemia (HL-60), Burkitt-like lymphoma (BJAB), human melanoma (MelHO), and human breast adenocarcinoma (MCF7) cells at low nanomolar concentrations. Apoptosis induction proved to be especially high in multidrug-resistant Nalm6derived cancer cell lines, while healthy human leukocytes and hepatocytes were not affected by the concentration range studied. Furthermore, caspase-independent initiation of apoptosis via an intrinsic pathway was observed. PT-100 also shows strong synergistic effects in combination with vincristine on BJAB and Nalm6 cells. Cocrystallization of PT-100 with tubulin dimers revealed its (noncovalent) binding to the colchicine-binding site of β-tubulin at the interface to the α-subunit. A pronounced effect of PT-100 on the cytoskeleton morphology was shown by fluorescence microscopy. While the reactivity of PT-100 as a weak Michael acceptor toward thiols was chemically proven, it remains unclear whether this contributes to the remarkable biological properties of this unusual colchicinoid.

International audience; Single cell multimodal analysis is at the frontier of single cell research: it defines the roles and functions of distinct cell types through simultaneous analysis to provide unprecedented insight into cellular processes. Current single cell approaches are rapidly moving toward multimodal characterizations. It replaces one-dimensional single cell analysis, for example by allowing for simultaneous measurement of transcription and post-transcriptional regulation, epigenetic modifications and/or surface protein expression. By providing deeper insights into single cell processes, multimodal single cell analyses paves the way to new understandings in various cellular processes such as cell fate decisions, physiological heterogeneity or genotype–phenotype linkages. At the forefront of this, microfluidics is key for high-throughput single cell analysis. Here, we present an overview of the recent multimodal microfluidic platforms having a potential in biomedical research, with a specific focus on their potential clinical applications.

International audience; At first glance, the structure of a microtubule is simple. Globular α- and β-tubulin subunits form constitutive heterodimers that align head-to-tail in protofilaments. In the most common configuration, 13 protofilaments associate laterally with a slight longitudinal stagger that results in a left-handed 3-start helix featuring lateral associations between tubulin subunits. This seemingly straightforward description is actually based on almost half a century of research aimed at understanding how tubulin dimers interact within the microtubule lattice. But while we start to have a good overview of their architecture in vitro, our knowledge of microtubule-lattice organization in vivo is nowhere near to being complete.

Viruses are key players in marine ecosystems where they infect abundant marine microbes. RNA viruses are emerging as key members of the marine virosphere. They have recently been identified as a potential source of mortality in diatoms, a group of microalgae that accounts for roughly 40% of the primary production in the ocean. Despite their likely importance, their impacts on host populations and ecosystems remain difficult to assess. In this study, we introduce an innovative approach that combines automated 3D confocal microscopy with quantitative image analysis and physiological measurements to expand our understanding of viral infection. We followed different stages of infection of the bloom-forming diatom Guinardia delicatula by the RNA virus GdelRNAV-04 until the complete lysis of the host. From 20h after infection, we observed quantifiable changes in subcellular host morphology and biomass. Our microscopy monitoring also showed that viral infection of G. delicatula induced the formation of auxospores as a probable defense strategy against viruses. Our method enables the detection of discriminative morphological features on the subcellular scale and at high throughput for comparing populations, making it a promising approach for the quantification of viral infections in the field in the future.

How does the interplay between selection, mutation and horizontal gene transfer modify the phenotypic distribution of a bacterial or cell population? While horizontal gene transfer, which corresponds to the exchange of genetic material between individuals, has a major role in the adaptation of many organisms, its impact on the phenotypic density of populations is not yet fully understood. We study an elliptic integro-differential equation describing the evolutionary equilibrium of the phenotypic density of an asexual population. In a regime of small mutational variance, we characterize the solution which results from the balance between competition for a resource, mutation and horizontal gene transfer. We show that in a certain range of parameters polymorphic equilibria exist, which means that the phenotypic density may concentrate around several dominant traits. Such polymorphic equilibria result from an antagonist interplay between horizontal gene transfer and selection, while similar models which neglect the transfer lead only to monomorphic equilibria.; Comment l'interaction entre sélection, mutation et transfert horizontal de gènes modifie-t-elle la distribution phénotypique d'une population bactérienne ou cellulaire ? Si le transfert horizontal de gènes, qui correspond à l'échange de matériel génétique entre individus, joue un rôle majeur dans l'adaptation de nombreux organismes, son impact sur la densité phénotypique des populations n'est pas encore totalement connu. Nous étudions une équation elliptique intégro-différentielle décrivant l'équilibre évolutif de la densité phénotypique d'une population asexuée. Dans un régime de faible variance mutationnelle, nous caractérisons la solution qui résulte de l'équilibre entre compétition pour une ressource, mutation et transfert horizontal de gènes. Nous montrons que dans une certaine gamme de paramètres existent des équilibres polymorphes, ce qui signifie que la densité phénotypique peut se concentrer autour de plusieurs traits dominants. De tels équilibres polymorphes résultent d'un jeu antagoniste entre transfert horizontal de gènes et sélection, alors que des modèles similaires qui négligent le transfert ne conduisent qu'à des équilibres monomorphes.

International audience; This work is in the framework of SMMiL-E activities (a joint project of CNRS, Institute of Industrial Science,Centre Oscar Lambret, and the University of Lille). The authors acknowledge the French State-Region plan (CPER IRICL, Lille Interdisciplinary research Institute against cancer) for financial support and IRCL for hosting SMMiL-E. M.C. Tarhan and Q. Rezard acknowledge I-SITE ULNE.

International audience; Integrin endocytosis is essential for many fundamental cellular processes. Whether and how the internalization impacts cellular mechanics remains elusive. Whereas previous studies reported the contribution of the integrin activator, talin, in force development, the involvement of inhibitors is less documented. We identified ICAP-1 as an integrin inhibitor involved in mechanotransduction by co-working with NME2 to control clathrin-mediated endocytosis of integrins at the edge of focal adhesions (FA). Loss of ICAP-1 enables β3-integrin-mediated force generation independently of β1 integrin. β3-integrin-mediated forces were associated with a decrease in β3 integrin dynamics stemming from their reduced diffusion within adhesion sites and slow turnover of FA. The decrease in β3 integrin dynamics correlated with a defect in integrin endocytosis. ICAP-1 acts as an adaptor for clathrin-dependent endocytosis of integrins. ICAP-1 controls integrin endocytosis by interacting with NME2, a key regulator of dynamin-dependent clathrin-coated pits fission. Control of clathrin-mediated integrin endocytosis by an inhibitor is an unprecedented mechanism to tune forces at FA.

One of the most abundant DNA lesions induced by Reactive oxygen species (ROS) is 8-oxoG, a highly mutagenic lesion that compromises genetic instability when not efficiently repaired. 8-oxoG is specifically recognized by the DNA-glycosylase OGG1 that excises the base and initiates the Base Excision Repair pathway (BER). Furthermore, OGG1 has not only a major role in DNA repair but it is also involved in transcriptional regulation. Cancer cells are particularly exposed to ROS, thus challenging their capacity to process oxidative DNA damage has been proposed as a promising therapeutic strategy for cancer treatment. Two competitive inhibitors of OGG1 (OGG1i) have been identified, TH5487 and SU0268, which bind to the OGG1 catalytic pocket preventing its fixation to the DNA. Early studies with these inhibitors show an enhanced cellular sensitivity to cytotoxic drugs and a reduction in the inflammatory response. Our study uncovers two unreported off-targets effects of these OGG1i that are independent of OGG1. In vitro and in cellulo approaches have unveiled that OGG1i TH5487 and SU0268, despite an unrelated molecular structure, are able to inhibit some members of the ABC family transporters, in particular ABC B1 (MDR1) and ABC G2 (BCRP). The inhibition of these efflux pumps by OGG1 inhibitors results in a higher intra-cellular accumulation of various fluorescent probes and drugs, and largely contributes to the enhanced cytotoxicity observed when the inhibitors are combined with cytotoxic agents. Furthermore, we found that SU0268 has an OGG1-independent anti-mitotic activity—by interfering with metaphase completion—resulting in a high cellular toxicity. These two off-target activities are observed at concentrations of OGG1i that are normally used for in vivo studies. It is thus critical to consider these previously unreported non-specific effects when interpreting studies using TH5487 and SU0268 in the context of OGG1 inhibition. Additionally, our work highlights the persistent need for new specific inhibitors of the enzymatic activity of OGG1.

International audience; Metastatic melanoma develops once transformed melanocytic cells begin to de-differentiate into migratory and invasive melanoma cells with neural crest cell (NCC)-like and epithelial-tomesenchymal transition (EMT)-like features. However, it is still unclear how transformed melanocytes assume a metastatic melanoma cell state. Here, we define DNA methylation changes that accompany metastatic progression in melanoma patients and discover Nuclear Receptor Subfamily 2 Group F, Member 2 – isoform 2 (NR2F2-Iso2) as an epigenetically regulated metastasis driver. NR2F2-Iso2 is transcribed from an alternative transcriptional start site (TSS) and it is truncated at the N-terminal end which encodes the NR2F2 DNA-bindingdomain. We find that NR2F2-Iso2 expression is turned off by DNA methylation when NCCs differentiate into melanocytes. Conversely, this process is reversed during metastatic melanoma progression, when NR2F2-Iso2 becomes increasingly hypomethylated and re-expressed. Our functional and molecular studies suggest that NR2F2-Iso2 drives metastatic melanomaprogression by modulating the activity of full-length NR2F2 (Isoform 1) over EMT- and NCC-associated target genes. Our findings indicate that DNA methylation changes play a crucial role during metastatic melanoma progression, and their control of NR2F2 activity allows transformed melanocytes to acquire NCC-like and EMT-like features. This epigenetically regulated transcriptional plasticity facilitates cell state transitions and metastatic spread.

A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression and/or release of stored or newly synthesized bioactive molecules, and (2) membrane blebbing followed by release of microvesicles. An ultimate response, namely the formation of extracellular traps by neutrophils (NETs), is outside the scope of this work. The main objective of this article is to provide an overview on the mechanism of plasminogen reception and activation at the surface of cell-derived microvesicles, new actors in fibrinolysis and proteolysis. The role of microvesicle-bound plasmin in pathological settings involving inflammation, atherosclerosis, angiogenesis, and tumour growth, remains to be investigated. Further studies are necessary to determine if profibrinolytic microvesicles are involved in a finely regulated equilibrium with pro-coagulant microvesicles, which ensures a balanced haemostasis, leading to the maintenance of vascular patency., Un certain nombre de facteurs de stress et de médiateurs inflammatoires (cytokines, protéases, médiateurs du stress oxydatif) libérés pendant l'inflammation ou l'ischémie stimulent et activent les cellules du sang, de la paroi vasculaire ou des tissus. Les réponses fonctionnelles et phénotypiques les plus connues des cellules activées sont (1) l'expression et/ou la libération immédiate de molécules bioactives stockées ou nouvellement synthétisées, et (2) le décollement de la membrane suivi de la libération de microvésicules. Une réponse ultime, à savoir la formation de pièges extracellulaires par les neutrophiles (NETs), sort du cadre de ce travail. L'objectif principal de cet article est de donner un aperçu du mécanisme de réception et d'activation du plasminogène à la surface des microvésicules dérivées des cellules, nouveaux acteurs de la fibrinolyse et de la protéolyse. Le rôle de la plasmine liée aux microvésicules dans des contextes pathologiques impliquant l'inflammation, l'athérosclérose, l'angiogenèse et la croissance tumorale, reste à étudier. D'autres études sont nécessaires pour déterminer si les microvésicules profibrinolytiques sont impliquées dans un équilibre finement régulé avec les microvésicules pro-coagulantes, qui assure une hémostase équilibrée, conduisant au maintien de la perméabilité vasculaire.

Matrix remodeling is a salient feature of idiopathic pulmonary fibrosis (IPF). Targeting cells driving matrix remodeling could be a promising avenue for IPF treatment. Analysis of transcriptomic database identified the mesenchymal transcription factor PRRX1 as upregulated in IPF. PRRX1, strongly expressed by lung fibroblasts, was regulated by a TGF-β/PGE2 balance in vitro in control and IPF human lung fibroblasts, while IPF fibroblast-derived matrix increased PRRX1 expression in a PDGFR-dependent manner in control ones. PRRX1 inhibition decreased human lung fibroblast proliferation by downregulating the expression of S phase cyclins. PRRX1 inhibition also impacted TGF-β driven myofibroblastic differentiation by inhibiting SMAD2/3 phosphorylation through phosphatase PPM1A upregulation and TGFBR2 downregulation, leading to TGF-β response global decrease. Finally, targeted inhibition of Prrx1 attenuated fibrotic remodeling in vivo with intra-tracheal antisense oligonucleotides in bleomycin mouse model of lung fibrosis and ex vivo using human and mouse precision-cut lung slices. Our results identified PRRX1 as a key mesenchymal transcription factor during lung fibrogenesis.

International audience; Microtubule self-repair has been studied both in vitro and in vivo as an underlying mechanism of microtubule stability. The turnover of tubulin dimers along the microtubule has challenged the pre-existing dogma that only growing ends are dynamic. However, although there is clear evidence of tubulin incorporation into the shaft of polymerized microtubules in vitro, the possibility of such events occurring in living cells remains uncertain. In this study, we investigated this possibility by microinjecting purified tubulin dimers labeled with a red fluorophore into the cytoplasm of cells expressing GFP-tubulin. We observed the appearance of red dots along the pre-existing green microtubule within minutes. We found that the fluorescence intensities of these red dots were inversely correlated with the green signal, suggesting that the red dimers were incorporated into the microtubules and replaced the pre-existing green dimers. Lateral distance from the microtubule center was similar to that in incorporation sites and in growing ends. The saturation of the size and spatial frequency of incorporations as a function of injected tubulin concentration and post-injection delay suggested that the injected dimers incorporated into a finite number of damaged sites. By our low estimate, within a few minutes of the injections, free dimers incorporated into major repair sites every 70 μm of microtubules. Finally, we mapped the location of these sites in micropatterned cells and found that they were more concentrated in regions where the actin filament network was less dense and where microtubules exhibited greater lateral fluctuations.

The Human Immunodeficiency Virus type 1 (HIV-1) is an intracellular pathogen whose replication cycle strictly depends on the host cell molecular machinery. HIV-1 crosses twice the plasma membrane, to get in and to get out of the cell. Therefore, the first and the last line of intracellular component encountered by the virus is the cortical actin network. Here, we review the role of actin and actin-related proteins in HIV-1 entry, assembly, budding and release. We first highlight the mechanisms controlling actin polymerization at the entry site that promote the clustering of HIV-1 receptors, a crucial step for the virus to fuse with the plasma membrane. Then, we describe how actin is transiently depolymerized locally to allow the capsid to cross the actin cortex, before migrating toward the nucleus. Finally, we review the role of several actin-binding proteins in actin remodeling events required for membrane deformation and curvature at the viral assembly site as well as for virus release. Strikingly, it appears that common actin-regulating pathways are involved in viral entry and exit. However, while the role of actin remodeling during entry is well understood, this is not yet the case during exit. We discuss remaining challenges regarding the actin-dependent mechanisms involved in HIV-1 entry and exit, and how they could be overcome. This article is protected by copyright. All rights reserved.

International audience; Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

International audience; Methods to follow in real time complex processes occurring along living cell membranes such as cell permeabilization are rare. Here, the terahertz spectroscopy reveals early events in plasma membrane alteration generated during photodynamic therapy (PDT) protocol, events which are not observable in any other conventional biological techniques performed in parallel as comparison. Photodynamic process is examined in Madin-Darby canine kidney cells using Pheophorbide (Pheo) photosensitizer alone or alternatively encapsulated in poly(ethylene oxide)-block-poly(ε-caprolactone) micelles for drug delivery purpose. Terahertz spectroscopy (THz) reveals that plasma membrane permeabilization starts simultaneously with illumination and is stronger when photosensitizer is encapsulated. In parallel, the exchange of biological species is assessed. Over several hours, this conventional approach demonstrates significant differences between free and encapsulated Pheo, the latter leading to high penetration of propidium iodide, Na+ and Ca2+ ions, and a high level of leakage of K+, ATP, and lactate dehydrogenase. THz spectroscopy provides, in a single measurement, the relative number of defects per membrane surface created after PDT, which is not achieved by any other method, providing early, sensitive real-time information. THz spectroscopy is therefore a promising technique and can be applied to any biological topic requiring the examination of short-term plasma membrane permeabilization.

International audience; The main estrogen, 17β-estradiol (E2), exerts several beneficial vascular actions through estrogen receptor α (ERα) in endothelial cells. However, the impact of other natural estrogens such as estriol (E3) and estetrol (E4) on arteries remains poorly described. In the present study, we report the effects of E3 and E4 on endothelial healing after carotid artery injuries in vivo. After endovascular injury, which preserves smooth muscle cells (SMCs), E2, E3, and E4 equally stimulated reendothelialization. By contrast, only E2 and E3 accelerated endothelial healing after perivascular injury that destroys both endothelial cells and SMCs, suggesting an important role of this latter cell type in E4’s action, which was confirmed using Cre/lox mice inactivating ERα in SMCs. In addition, E4 mediated its effects independently of ERα membrane-initiated signaling, in contrast with E2. Consistently, RNA sequencing analysis revealed that transcriptomic and cellular signatures in response to E4 profoundly differed from those of E2. Thus, whereas acceleration of endothelial healing by estrogens had been viewed as entirely dependent on endothelial ERα, these results highlight the very specific pharmacological profile of the natural estrogen E4, revealing the importance of dialogue between SMCs and endothelial cells in its arterial protection.

International audience; Obesity is characterized by systemic low-grade inflammation associated with disturbances of intestinal homeostasis and microbiota dysbiosis. Mitochondrial metabolism sustains epithelial homeostasis by providing energy to colonic epithelial cells (CEC) but can be altered by dietary modulations of the luminal environment. Our study aimed at evaluating whether the consumption of an obesogenic diet alters the mitochondrial function of CEC in mice. Mice were fed for 22 weeks with a 58% kcal fat diet (diet-induced obesity [DIO] group) or a 10% kcal fat diet (control diet, CTRL). Colonic crypts were isolated to assess mitochondrial function while colonic content was collected to characterize microbiota and metabolites. DIO mice developed obesity, intestinal hyperpermeability, and increased endotoxemia. Analysis of isolated colonic crypt bioenergetics revealed a mitochondrial dysfunction marked by decreased basal and maximal respirations and lower respiration linked to ATP production in DIO mice. Yet, CEC gene expression of mitochondrial respiration chain complexes and mitochondrial dynamics were not altered in DIO mice. In parallel, DIO mice displayed increased colonic bile acid concentrations, associated with higher abundance of Desulfovibrionaceae. Sulfide concentration was markedly increased in the colon content of DIO mice. Hence, chronic treatment of CTRL mouse colon organoids with sodium sulfide provoked mitochondrial dysfunction similar to that observed in vivo in DIO mice while acute exposure of isolated mitochondria from CEC of CTRL mice to sodium sulfide diminished complex IV activity. Our study provides new insights into colon mitochondrial dysfunction in obesity by revealing that increased sulfide production by DIO-induced dysbiosis impairs complex IV activity in mouse CEC.

The SorLA protein, encoded by the SORL1 gene, is a major player in Alzheimer’s disease (AD) pathophysiology. Functional and genetic studies demonstrated that SorLA deficiency results in increased production of Aβ peptides, and thus a higher risk of AD. A large number of SORL1 missense variants have been identified in AD patients, but their functional consequences remain largely undefined. Here, we identified a new pathophysiological mechanism, by which rare SORL1 missense variants identified in AD patients result in altered maturation and trafficking of the SorLA protein. An initial screening, based on the overexpression of 70 SorLA variants in HEK293 cells, revealed that 15 of them (S114R, R332W, G543E, S564G, S577P, R654W, R729W, D806N, Y934C, D1535N, D1545E, P1654L, Y1816C, W1862C, P1914S) induced a maturation and trafficking-deficient phenotype. Three of these variants (R332W, S577P, and R654W) and two maturation-competent variants (S124R and N371T) were further studied in details in CRISPR/Cas9-modified hiPSCs. When expressed at endogenous levels, the R332W, S577P, and R654W SorLA variants also showed a maturation defective profile. We further demonstrated that these variants were largely retained in the endoplasmic reticulum, resulting in a reduction in the delivery of SorLA mature protein to the plasma membrane and to the endosomal system. Importantly, expression of the R332W and R654W variants in hiPSCs was associated with a clear increase of Aβ secretion, demonstrating a loss-of-function effect of these SorLA variants regarding this ultimate readout, and a direct link with AD pathophysiology. Furthermore, structural analysis of the impact of missense variants on SorLA protein suggested that impaired cellular trafficking of SorLA protein could be due to subtle variations of the protein 3D structure resulting from changes in the interatomic interactions.

International audience; Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.