Formation of blood platelets : mechanisms involved in megakaryocytes intravasation across medullar sinusoids

Les plaquettes sanguines sont vitales pour la prévention des hémorragies. Une étape clé et encore mal comprise dans leur formation est le passage de leurs précurseurs, les mégacaryocytes (MK) au travers de la barrière endothéliale des sinusoïdes médullaires, permettant de libérer des fragments cytoplasmiques dans le sang, lesquels se remodèleront ensuite en plaquettes dans la circulation. L’objectif de cette thèse a été de comprendre les mécanismes cellulaires et moléculaires impliqués dans l’intravasation des MKs.Dans la première partie, j’ai montré que les protrusions émises in situ par les MKs au contact des sinusoïdes sont des podosomes. Les podosomes s’invaginent profondément dans l’endothélium jusqu’à le traverser, permettant aux MKs d’emprunter une voie transcellulaire vers la circulation. Ils sont reliés par un réseau secondaire d’actomyosine, adoptant ainsi une organisation collective, interconnectée, permettant aux MKs de perforer l’endothélium en de multiples points. Le rôle essentiel du cytosquelette d’actomyosine dans la structuration des podosomes in vivo, a pu être confirmé en analysant l’impact des déficits en complexe Arp2/3 ou en myosine IIA dans la lignée mégacaryocytaire. Dans la deuxième partie de la thèse, nous nous sommes intéréssés au rôle des intégrines dans l’intravasation des MKs par l’utilisation de souris déficientes en intégrines β1 et β3. L’observation de la moelle de ces souris a mis en lumière le rôle essentiel des intégrines dans la prévention du passage de MKs entiers au travers des sinusoïdes. Les intégrines contrôlent l’intravasation des MKs en permettant leur ancrage dans la matrice extracellulaire et en modulant les propriétés physiques et structurales de leurs noyaux. Enfin, dans la troisième partie de la thèse, nous avons évalué la contribution des métalloprotéases dans ce processus. L’étude de la moelle osseuse de souris déficientes en MT1-MMP, nous a permis de démontrer que cette enzyme n’était pas nécessaire dans la formation des podosomes et au passage de la lame basale. Plus globalement, nos données révèlent que les MKs matures issus de la moelle n’ont pas d’activité de dégradation de la matrice et que l’activité des MMPs n’est pas nécessaire dans la formation des plaquettes in vivo.L'ensemble de nos résultats permet une meilleure compréhension des mécanismes cellulaires et moléculaires impliqués dans l'intravasation des MKs en identifiant l'un de ses principaux acteurs : le podosome. De nombreux types cellulaires forment ces structures d’adhésion particulières. Ce travail ouvre ainsi des perspectives plus larges en Biologie Cellulaire. Il amène des pistes pour explorer les mécanismes d’intravasation des autres cellules sanguines en générale ou dans un contexte plus pathologique de cellules tumorales.Blood platelets are vital for the prevention of bleeding. A key and still poorly understood step in their formation is the passage of their precursors, megakaryocytes (MKs), through the endothelial barrier of the medullary sinusoids, to release cytoplasmic fragments into the blood, which will remodel into platelets. The aim of the present thesis is to understand the cellular and molecular mechanisms involved in the intravasation of MKs. In the first part, I characterized the invasive protrusions emitted in situ by MKs at the sinusoidal contact. These protrusions can invaginate deep into the endothelium until they cross it, leading MKs to take a transcellular route to the circulation. These structures, connected by a lateral actomyosin network, adopt a collective, interconnected organization, allowing MKs to perforate the endothelium at multiple points. The essential role of the actomyosin cytoskeleton in the in vivo organization of podosomes has been confirmed by analyzing the impact of Arp2/3 complex or myosin IIA deficiency in the megakaryocytic lineage. In the second part of the thesis, we focused on the role of integrins in MKs intravasation by using 1 and 3 integrin deficient mice, demonstrating that they prevent the passage of whole MKs through the sinusoids. Integrins control MKs intravasation by mediating their anchoring in the extracellular matrix and by modulating the physical and structural properties of their nuclei. Finally, in the third part of the thesis, we evaluated the contribution of metalloproteinases in the process of MKs intravasation. By investigating the bone marrow of MT1-MMP deficient mice, we demonstrated that this enzyme was not required for podosome formation and basement membrane passage. More generally, our data reveal that mature MKs from marrow do not have matrix degradation activity and that MMP activity is not required for platelet formation in vivo.Our work has improved our understanding of a key step in thrombopoiesis, the transmigration of MKs through the sinusoids of the bone marrow. Many cell types elaborate these structures, including invasive cancer cells, macrophages and osteoclasts. This work therefore offers wider perspectives in cell biology. It provides insights into the mechanisms of intravasation of other blood cells as well as in a more pathological context of tumor cells

Formation of blood platelets : mechanisms involved in megakaryocytes intravasation across medullar sinusoids

Les plaquettes sanguines sont vitales pour la prévention des hémorragies. Une étape clé et encore mal comprise dans leur formation est le passage de leurs précurseurs, les mégacaryocytes (MK) au travers de la barrière endothéliale des sinusoïdes médullaires, permettant de libérer des fragments cytoplasmiques dans le sang, lesquels se remodèleront ensuite en plaquettes dans la circulation. L’objectif de cette thèse a été de comprendre les mécanismes cellulaires et moléculaires impliqués dans l’intravasation des MKs.Dans la première partie, j’ai montré que les protrusions émises in situ par les MKs au contact des sinusoïdes sont des podosomes. Les podosomes s’invaginent profondément dans l’endothélium jusqu’à le traverser, permettant aux MKs d’emprunter une voie transcellulaire vers la circulation. Ils sont reliés par un réseau secondaire d’actomyosine, adoptant ainsi une organisation collective, interconnectée, permettant aux MKs de perforer l’endothélium en de multiples points. Le rôle essentiel du cytosquelette d’actomyosine dans la structuration des podosomes in vivo, a pu être confirmé en analysant l’impact des déficits en complexe Arp2/3 ou en myosine IIA dans la lignée mégacaryocytaire. Dans la deuxième partie de la thèse, nous nous sommes intéréssés au rôle des intégrines dans l’intravasation des MKs par l’utilisation de souris déficientes en intégrines β1 et β3. L’observation de la moelle de ces souris a mis en lumière le rôle essentiel des intégrines dans la prévention du passage de MKs entiers au travers des sinusoïdes. Les intégrines contrôlent l’intravasation des MKs en permettant leur ancrage dans la matrice extracellulaire et en modulant les propriétés physiques et structurales de leurs noyaux. Enfin, dans la troisième partie de la thèse, nous avons évalué la contribution des métalloprotéases dans ce processus. L’étude de la moelle osseuse de souris déficientes en MT1-MMP, nous a permis de démontrer que cette enzyme n’était pas nécessaire dans la formation des podosomes et au passage de la lame basale. Plus globalement, nos données révèlent que les MKs matures issus de la moelle n’ont pas d’activité de dégradation de la matrice et que l’activité des MMPs n’est pas nécessaire dans la formation des plaquettes in vivo.L'ensemble de nos résultats permet une meilleure compréhension des mécanismes cellulaires et moléculaires impliqués dans l'intravasation des MKs en identifiant l'un de ses principaux acteurs : le podosome. De nombreux types cellulaires forment ces structures d’adhésion particulières. Ce travail ouvre ainsi des perspectives plus larges en Biologie Cellulaire. Il amène des pistes pour explorer les mécanismes d’intravasation des autres cellules sanguines en générale ou dans un contexte plus pathologique de cellules tumorales.Blood platelets are vital for the prevention of bleeding. A key and still poorly understood step in their formation is the passage of their precursors, megakaryocytes (MKs), through the endothelial barrier of the medullary sinusoids, to release cytoplasmic fragments into the blood, which will remodel into platelets. The aim of the present thesis is to understand the cellular and molecular mechanisms involved in the intravasation of MKs. In the first part, I characterized the invasive protrusions emitted in situ by MKs at the sinusoidal contact. These protrusions can invaginate deep into the endothelium until they cross it, leading MKs to take a transcellular route to the circulation. These structures, connected by a lateral actomyosin network, adopt a collective, interconnected organization, allowing MKs to perforate the endothelium at multiple points. The essential role of the actomyosin cytoskeleton in the in vivo organization of podosomes has been confirmed by analyzing the impact of Arp2/3 complex or myosin IIA deficiency in the megakaryocytic lineage. In the second part of the thesis, we focused on the role of integrins in MKs intravasation by using 1 and 3 integrin deficient mice, demonstrating that they prevent the passage of whole MKs through the sinusoids. Integrins control MKs intravasation by mediating their anchoring in the extracellular matrix and by modulating the physical and structural properties of their nuclei. Finally, in the third part of the thesis, we evaluated the contribution of metalloproteinases in the process of MKs intravasation. By investigating the bone marrow of MT1-MMP deficient mice, we demonstrated that this enzyme was not required for podosome formation and basement membrane passage. More generally, our data reveal that mature MKs from marrow do not have matrix degradation activity and that MMP activity is not required for platelet formation in vivo.Our work has improved our understanding of a key step in thrombopoiesis, the transmigration of MKs through the sinusoids of the bone marrow. Many cell types elaborate these structures, including invasive cancer cells, macrophages and osteoclasts. This work therefore offers wider perspectives in cell biology. It provides insights into the mechanisms of intravasation of other blood cells as well as in a more pathological context of tumor cells

Genome sequence-based identification of Enterobacter strains and description of Enterobacter pasteurii sp. nov.

International audienceABSTRACT Members of the Enterobacter cloacae complex (ECC) are almost ubiquitous in nature and can act as pathogens. In this study, we used a polyphasic taxonomy approach to establish the accurate taxonomic position of six strains within the ECC. Notably, the 16S rRNA gene-based phylogeny failed to group all Enterobacter species into a monophyletic cluster. As an alternative to this, we explored genome sequence-based phylogenetic approaches. The bac120 gene-based phylogeny successfully grouped all Enterobacter species into a monophyletic cluster, although some species-level clustering conflicted with average nucleotide identity (ANI) values. Furthermore, the Enterobacter -specific core gene phylogeny resolved all species, aligning with ANI results. Three strains were identified as Enterobacter asburiae , while strain P99 was classified as “ Enterobacter xiangfangensis ” and strain C45 as Enterobacter quasihormaechei . Conversely, strain A-8 T formed a distinct cluster in all phylogenies, with ANI and digital DNA-DNA hybridization (dDDH) values below the species threshold (<92% and <44%, respectively) with all known Enterobacter species. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry biotyper results confirmed that all strains belonged to the ECC. However, the comparison of mean spectrum profile of strain A-8 T with those of other strains revealed the presence of 18 unique peaks, highlighting its distinct protein profile. Based on comprehensive genotypic and phenotypic characterizations, we propose that strain A-8 T is a new species of the genus Enterobacter , which is named Enterobacter pasteurii sp. nov. The type strain is A-8 T (CIP 103550 T ; ATCC 23355 T ; DSM 26481 T ; and WDCM 00082 T ). This study advances our understanding of the ECC, emphasizing the need for multidimensional taxonomic techniques and contributing to the better management of microbial resource centers. IMPORTANCE Accurate taxonomy is essential for microbial biological resource centers, since the microbial resources are often used to support new discoveries and subsequent research. Here, we used genome sequence data, alongside matrix-assisted laser desorption/ionization time-of-flight mass spectrometer biotyper-based protein profiling, to accurately identify six Enterobacter cloacae complex strains. This approach effectively identified distinct species within the E. cloacae complex, including Enterobacter asburiae , “ Enterobacter xiangfangensis ,” and Enterobacter quasihormaechei . Moreover, the study revealed the existence of a novel species within the Enterobacter genus, for which we proposed the name Enterobacter pasteurii sp. nov. In summary, this study demonstrates the significance of adopting a genome sequence-driven taxonomy approach for the precise identification of bacterial strains in a biological resource center and expands our understanding of the E. cloacae complex

Use of electron microscopy to study megakaryocytes

Electron microscopy (EM) has a long history in megakaryocyte (MK) cellular biology. This chapter shows how the electron microscope, since its first appearance almost 90 years ago, has occupied center stage in the studies of MK morphology and function. It describes some of the more productive EM techniques that have shaped our understanding of the physiology of thrombopoiesis. These include the standard transmission and scanning EM techniques as well as the new imaging methods, correlative microscopy and volume EM which provide information on the 3D organization of MKs on different scales: single organelles, whole cells and tissues. For each technique, we list the advantages and limitations, the resolution that can be achieved, the technical difficulties and the applications in MK biology

Single-cell transcriptomic profiling of the mouse cochlea: An atlas for targeted therapies

International audienceFunctional molecular characterization of the cochlea has mainly been driven by the deciphering of the genetic architecture of sensorineural deafness. As a result, the search for curative treatments, which are sorely lacking in the hearing field, has become a potentially achievable objective, particularly via cochlear gene and cell therapies. To this end, a complete inventory of cochlear cell types, with an in-depth characterization of their gene expression profiles right up to their final differentiation, is indispensable. We therefore generated a single-cell transcriptomic atlas of the mouse cochlea based on an analysis of more than 120,000 cells on postnatal day 8 (P8), during the prehearing period, P12, corresponding to hearing onset, and P20, when cochlear maturation is almost complete. By combining whole-cell and nuclear transcript analyses with extensive in situ RNA hybridization assays, we characterized the transcriptomic signatures covering nearly all cochlear cell types and developed cell type–specific markers. Three cell types were discovered; two of them contribute to the modiolus which houses the primary auditory neurons and blood vessels, and the third one consists in cells lining the scala vestibuli. The results also shed light on the molecular basis of the tonotopic gradient of the biophysical characteristics of the basilar membrane that critically underlies cochlear passive sound frequency analysis. Finally, overlooked expression of deafness genes in several cochlear cell types was also unveiled. This atlas paves the way for the deciphering of the gene regulatory networks controlling cochlear cell differentiation and maturation, essential for the development of effective targeted treatments