Barcoding T Cell Calcium Response Diversity with Methods for Automated and Accurate Analysis of Cell Signals (MAAACS)

International audienceWe introduce a series of experimental procedures enabling sensitive calcium monitoring in T cell populations by confocal video-microscopy. Tracking and post-acquisition analysis was performed using Methods for Automated and Accurate Analysis of Cell Signals (MAAACS), a fully customized program that associates a high throughput tracking algorithm, an intuitive reconnection routine and a statistical platform to provide, at a glance, the calcium barcode of a population of individual T-cells. Combined with a sensitive calcium probe, this method allowed us to unravel the heterogeneity in shape and intensity of the calcium response in T cell populations and especially in naive T cells, which display intracellular calcium oscillations upon stimulation by antigen presenting cells

Influence du métabolisme mitochondrial dans l'hématopoïèse : Analyse de la réponse adaptative des cellules de la moelle osseuse et des thymocytes au dysfonctionnement de l’OXPHOS

By integrating different biochemical pathways and generating energy in form of ATP, through the electron transfer associated to oxidative phosphorylation (OXPHOS), mitochondria play a key role in cellular metabolism. In the hematopoietic cells, the mitochondrial metabolism appears implicated in proliferation, differentiation, activation and self-renewal regulation. In this context, the aim of my PhD work was to unravel the response of bone marrow (BM) cells, B-cells and thymocytes to OXPHOS dysfunction. To do that, we have developed two original hematopoietic cell-specific murine models deficient in the mitochondrial proteins AIF or NDUFS4. Severe (AIF KO) or moderate (NDUFS4 KO) OXPHOS dysfunction leads to pleiotropic consequences on hematopoietic development, including pancytopenia, BM aplasia, alterations in the development of the B-cell and erythroid lineages and T-cell developmental blockade at the immature stage. Strikingly, in response to OXPHOS dysfunction, BM cells stimulate anaerobic glycolysis and mitochondrial biogenesis, whereas thymocytes favor the assimilation and degradation of fatty acids. Overall my work, which included in vivo, ex vivo and in vitro approaches, underlines the relevance of OXPHOS and mitochondrial metabolism in the development of the hematopoietic cells.Les mitochondries sont des organelles qui jouent un rôle clé dans le métabolisme cellulaire en centralisant la production d'ATP à partir de nombreux substrats via la phosphorylation oxydative (OXPHOS). Les réactions enzymatiques impliquées dans ce processus régulent la prolifération, la différenciation, l'activation et l'auto renouvellement cellulaire. Le but de mon travail a été d'identifier le rôle de l'OXPHOS dans l'hématopoïèse et les mécanismes d'adaptation métabolique des cellules sanguines de la moelle, des lymphocytes B et des thymocytes à la dysfonction mitochondriale. L'atout majeur de cette étude est la génération de deux modèles murins déficients pour les protéines mitochondriales AIF ou NDUFS4 dans le système hématopoïétique. Nous avons observé que l'absence de ces protéines entraine des dysfonctions de l'OXPHOS sévère (AIF KO) ou modérée (NDUFS4 KO), entrainant des anomalies dans le développement hématopoïétique. Dans les deux modèles, en réponse au stress métabolique induit par la dysfonction de l'OXPHOS, les cellules de moelle activent la glycolyse anaérobie et la biogenèse mitochondriale tandis que les thymocytes favorisent l'assimilation et la dégradation des acides gras. Cette étude multiparamétrique, incluant des approches in vivo, ex vivo et in vitro, souligne l'importance de l'OXPHOS et du métabolisme mitochondrial dans le développement hématopoïétique

Influence du métabolisme mitochondrial dans l'hématopoïèse : Analyse de la réponse adaptative des cellules de la moelle osseuse et des thymocytes au dysfonctionnement de l’OXPHOS

By integrating different biochemical pathways and generating energy in form of ATP, through the electron transfer associated to oxidative phosphorylation (OXPHOS), mitochondria play a key role in cellular metabolism. In the hematopoietic cells, the mitochondrial metabolism appears implicated in proliferation, differentiation, activation and self-renewal regulation. In this context, the aim of my PhD work was to unravel the response of bone marrow (BM) cells, B-cells and thymocytes to OXPHOS dysfunction. To do that, we have developed two original hematopoietic cell-specific murine models deficient in the mitochondrial proteins AIF or NDUFS4. Severe (AIF KO) or moderate (NDUFS4 KO) OXPHOS dysfunction leads to pleiotropic consequences on hematopoietic development, including pancytopenia, BM aplasia, alterations in the development of the B-cell and erythroid lineages and T-cell developmental blockade at the immature stage. Strikingly, in response to OXPHOS dysfunction, BM cells stimulate anaerobic glycolysis and mitochondrial biogenesis, whereas thymocytes favor the assimilation and degradation of fatty acids. Overall my work, which included in vivo, ex vivo and in vitro approaches, underlines the relevance of OXPHOS and mitochondrial metabolism in the development of the hematopoietic cells.Les mitochondries sont des organelles qui jouent un rôle clé dans le métabolisme cellulaire en centralisant la production d'ATP à partir de nombreux substrats via la phosphorylation oxydative (OXPHOS). Les réactions enzymatiques impliquées dans ce processus régulent la prolifération, la différenciation, l'activation et l'auto renouvellement cellulaire. Le but de mon travail a été d'identifier le rôle de l'OXPHOS dans l'hématopoïèse et les mécanismes d'adaptation métabolique des cellules sanguines de la moelle, des lymphocytes B et des thymocytes à la dysfonction mitochondriale. L'atout majeur de cette étude est la génération de deux modèles murins déficients pour les protéines mitochondriales AIF ou NDUFS4 dans le système hématopoïétique. Nous avons observé que l'absence de ces protéines entraine des dysfonctions de l'OXPHOS sévère (AIF KO) ou modérée (NDUFS4 KO), entrainant des anomalies dans le développement hématopoïétique. Dans les deux modèles, en réponse au stress métabolique induit par la dysfonction de l'OXPHOS, les cellules de moelle activent la glycolyse anaérobie et la biogenèse mitochondriale tandis que les thymocytes favorisent l'assimilation et la dégradation des acides gras. Cette étude multiparamétrique, incluant des approches in vivo, ex vivo et in vitro, souligne l'importance de l'OXPHOS et du métabolisme mitochondrial dans le développement hématopoïétique

Influence of mitochondrial metabolism in hematopoieisis : Analysis of the adaptative response of bone marrow cells and thymocytes to OXPHOS dysfunction

Les mitochondries sont des organelles qui jouent un rôle clé dans le métabolisme cellulaire en centralisant la production d'ATP à partir de nombreux substrats via la phosphorylation oxydative (OXPHOS). Les réactions enzymatiques impliquées dans ce processus régulent la prolifération, la différenciation, l'activation et l'auto renouvellement cellulaire. Le but de mon travail a été d'identifier le rôle de l'OXPHOS dans l'hématopoïèse et les mécanismes d'adaptation métabolique des cellules sanguines de la moelle, des lymphocytes B et des thymocytes à la dysfonction mitochondriale. L'atout majeur de cette étude est la génération de deux modèles murins déficients pour les protéines mitochondriales AIF ou NDUFS4 dans le système hématopoïétique. Nous avons observé que l'absence de ces protéines entraine des dysfonctions de l'OXPHOS sévère (AIF KO) ou modérée (NDUFS4 KO), entrainant des anomalies dans le développement hématopoïétique. Dans les deux modèles, en réponse au stress métabolique induit par la dysfonction de l'OXPHOS, les cellules de moelle activent la glycolyse anaérobie et la biogenèse mitochondriale tandis que les thymocytes favorisent l'assimilation et la dégradation des acides gras. Cette étude multiparamétrique, incluant des approches in vivo, ex vivo et in vitro, souligne l'importance de l'OXPHOS et du métabolisme mitochondrial dans le développement hématopoïétique.By integrating different biochemical pathways and generating energy in form of ATP, through the electron transfer associated to oxidative phosphorylation (OXPHOS), mitochondria play a key role in cellular metabolism. In the hematopoietic cells, the mitochondrial metabolism appears implicated in proliferation, differentiation, activation and self-renewal regulation. In this context, the aim of my PhD work was to unravel the response of bone marrow (BM) cells, B-cells and thymocytes to OXPHOS dysfunction. To do that, we have developed two original hematopoietic cell-specific murine models deficient in the mitochondrial proteins AIF or NDUFS4. Severe (AIF KO) or moderate (NDUFS4 KO) OXPHOS dysfunction leads to pleiotropic consequences on hematopoietic development, including pancytopenia, BM aplasia, alterations in the development of the B-cell and erythroid lineages and T-cell developmental blockade at the immature stage. Strikingly, in response to OXPHOS dysfunction, BM cells stimulate anaerobic glycolysis and mitochondrial biogenesis, whereas thymocytes favor the assimilation and degradation of fatty acids. Overall my work, which included in vivo, ex vivo and in vitro approaches, underlines the relevance of OXPHOS and mitochondrial metabolism in the development of the hematopoietic cells

Giant Mimiviridae CsCl Purification Protocol

International audienceWhile different giant viruses' purification protocols are available, they are not fully described and they use sucrose gradient that does not reach an equilibrium. Here, we report a protocol for the purification of members of the Mimiviridae family virions resulting from Acanthamoeaba castellanii infections. Viruses are harvested after cell lysis and purified through a high density CsCl gradient to optimize the isolation of the virus from the cell debris or other potential contaminants. Due to the large size of the virion capsids, reaching half a micrometer diameter, the quality of the process can be monitored by light microscopy. The resulting purified particles can then be used to perform new infections, DNA extraction, structural studies, sugar composition analyses, sub-compartment characterization or proteomic experiments

Mitochondrial OXPHOS influences immune cell fate: lessons from hematopoietic AIF-deficient and NDUFS4-deficient mouse models

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Diversity and evolution of the emerging PandoraviridaePandoraviridae family

International audienceWith DNA genomes reaching 2.5 Mb packed in particles of bacterium-like shape and dimension, the first two Acanthamoeba-infecting pandoraviruses remained up to now the most complex viruses since their discovery in 2013. Our isolation of three new strains from distant locations and environments is now used to perform the first comparative genomics analysis of the emerging worldwide-distributed Pandoraviridae family. Thorough annotation of the genomes combining transcriptomic, proteomic, and bioinformatic analyses reveals many non-coding transcripts and significantly reduces the former set of predicted protein-coding genes. Here we show that the pandoraviruses exhibit an open pan-genome, the enormous size of which is not adequately explained by gene duplications or horizontal transfers. As most of the strain-specific genes have no extant homolog and exhibit statistical features comparable to intergenic regions, we suggest that de novo gene creation could contribute to the evolution of the giant pandoravirus genomes

H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis

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In-depth study of Mollivirus sibericum , a new 30,000-y-old giant virus infecting Acanthamoeba

International audienceAcanthamoeba species are infected by the largest known DNA viruses. These include icosahedral Mimiviruses, amphora-shaped Pandoraviruses, and Pithovirus sibericum, the latter one isolated from 30,000-y-old permafrost. Mollivirus sibericum, a fourth type of giant virus, was isolated from the same permafrost sample. Its approximately spherical virion (0.6-µm diameter) encloses a 651-kb GC-rich genome encoding 523 proteins of which 64% are ORFans; 16% have their closest homolog in Pandoraviruses and 10% in Acanthamoeba castellanii probably through horizontal gene transfer. The Mollivirus nucleocytoplasmic replication cycle was analyzed using a combination of “omic” approaches that revealed how the virus highjacks its host machinery to actively replicate. Surprisingly, the host’s ribosomal proteins are packaged in the virion. Metagenomic analysis of the permafrost sample uncovered the presence of both viruses, yet in very low amount. The fact that two different viruses retain their infectivity in prehistorical permafrost layers should be of concern in a context of global warming. Giant viruses’ diversity remains to be fully explored