In vivo topology converts competition for cell-matrix adhesion into directional migration

International audienceWhen migrating in vivo, cells are exposed to numerous conflicting signals: chemokines, repellents, extracellular matrix, growth factors. The roles of several of these molecules have been studied individually in vitro or in vivo, but we have yet to understand how cells integrate them. To start addressing this question, we used the cephalic neural crest as a model system and looked at the roles of its best examples of positive and negative signals: stromal-cell derived factor 1 (Sdf1/Cxcl12) and class3-Semaphorins. Here we show that Sdf1 and Sema3A antagonistically control cell-matrix adhesion via opposite effects on Rac1 activity at the single cell level. Directional migration at the population level emerges as a result of global Semaphorin-dependent confinement and broad activation of adhesion by Sdf1 in the context of a biased Fibronectin distribution. These results indicate that uneven in vivo topology renders the need for precise distribution of secreted signals mostly dispensable

A New Family of Receptor Tyrosine Kinases with a Venus Flytrap Binding Domain in Insects and Other Invertebrates Activated by Aminoacids

Background: Tyrosine kinase receptors (RTKs) comprise a large family of membrane receptors that regulate various cellular processes in cell biology of diverse organisms. We previously described an atypical RTK in the platyhelminth parasite Schistosoma mansoni, composed of an extracellular Venus flytrap module (VFT) linked through a single transmembrane domain to an intracellular tyrosine kinase domain similar to that of the insulin receptor. Methods and Findings: Here we show that this receptor is a member of a new family of RTKs found in invertebrates, and particularly in insects. Sixteen new members of this family, named Venus Kinase Receptor (VKR), were identified in many insects. Structural and phylogenetic studies performed on VFT and TK domains showed that VKR sequences formed monophyletic groups, the VFT group being close to that of GABA receptors and the TK one being close to that of insulin receptors. We show that a recombinant VKR is able to autophosphorylate on tyrosine residues, and report that it can be activated by L-arginine. This is in agreement with the high degree of conservation of the alpha amino acid binding residues found in many amino acid binding VFTs. The presence of high levels of vkr transcripts in larval forms and in female gonads indicates a putative function of VKR in reproduction and/or development. Conclusion: The identification of RTKs specific for parasites and insect vectors raises new perspectives for the control of human parasitic and infectious diseases

Molecular characterization of Venus Kinase Receptors : functional studies in the parasite Schistosoma mansoni

La famille des Venus Kinase Receptors (VKR) est une nouvelle famille de Récepteurs Tyrosine Kinase découverte au laboratoire chez le ver parasite Schistosoma mansoni (SmVKR1), puis chez 14 autres organismes invertébrés, principalement des insectes. Les études réalisées au laboratoire ont montré la présence des transcrits vkr dans les organes reproducteurs des organismes adultes mais aussi dans les stades larvaires suggérant un rôle dans le développement et/ou la reproduction de ces organismes. Ma thèse concerne la caractérisation structurale, biochimique et fonctionnelle des VKRs de deux organismes d’intérêt sanitaire majeur : le schistosome, parasite responsable de la bilharziose qui représente la seconde endémie parasitaire mondiale et l’anophèle, vecteur principal du paludisme en Afrique. Dans une première partie nous avons mis en évidence la présence d’un second VKR chez le schistosome, nommé SmVKR2. Nous avons montré que ses transcrits sont exprimés à tous les stades du cycle parasitaire et principalement dans les organes génitaux de la femelle au niveau des ovocytes immatures et de l’ootype. Nous avons exprimé SmVKR1 et SmVKR2 et montré qu’ils sont tous deux des récepteurs à activité tyrosine kinase, activables respectivement par la L-Arginine et par le calcium. Des expériences complémentaires semblent indiquer qu’un ligand naturel de SmVKR1 pourrait se trouver dans le canal gynécophore des vers appariés. Parallèlement, nous avons entrepris l’identification des partenaires cytosoliques de SmVKR1 et SmVKR2 grâce au criblage d’une banque d’ADNc de vers adultes par la technique de double hybride en levure en utilisant les domaines intracellulaires (DIC) des récepteurs comme appâts. L’analyse partielle des résultats obtenus montre que les DIC de SmVKR1 et SmVKR2 interagissent en autres avec des protéines du cytosquelette et avec des protéines cytoplasmiques pourvues de domaines d’interaction protéine-protéine SH2, acteurs de voies de signalisation classique des RTKs. Nous avons montré par ARNi que la diminution des transcrits SmVKR1 et SmVKR2 a un impact majeur sur la morphologie des organes génitaux de la femelle. Les sporocystes interférés pour les deux récepteurs présentent une diminution significative de leur taille comparée aux témoins.La deuxième partie de mes travaux de thèse a été centrée sur l’étude d’AgVKR, le récepteur d’Anopheles gambiae. En utilisant deux systèmes d’expression hétérologue, nous avons pu établir que ce récepteur était lui aussi catalytiquement actif et activable par la L-arg comme SmVKR1. L’étude de la fonction d’AgVKR a été abordée grâce à la découverte récente de son expression constitutive dans une lignée de cellules d’A. gambiae nommées SuA5B et de type hémocytaire. Des expériences d’ARNi ont été mises au point dans le but de visualiser l’impact d’une diminution de transcrits sur la physiologie des cellules. Dans leur ensemble, les résultats de ces travaux participent à la compréhension des mécanismes de régulation et de la fonction des VKRs, des récepteurs qui semblent d’une grande importance pour le développement et la reproduction des organismes.Venus Kinase Receptors (VKRs) form a new family of Receptor Tyrosine Kinases discovered for the first time in the parasite Schistosoma mansoni (SmVKR1), then in fourteen other invertebrate organisms and mainly in insects. In our laboratory, previous studies have shown that vkr transcripts are present in reproductive organs of adult organisms but also in larval stages, suggesting a role of VKRs in reproductive and development mechanisms. My thesis was concerned with the structural, biochemical, and functional characterization of the VKRs of two invertebrate organisms causing serious public health concerns: the worm S. mansoni responsible for the second human parasitic disease, and the principal malaria vector in Africa, Anopheles gambiae. At first, we have shown the existence of SmVKR2, a second VKR in the worm. Its transcripts are expressed in all the parasitic stages and localized in the immature oocytes and the ootype of the female worm. Recombinant SmVKR1 and SmVKR2 proteins showed a tyrosine kinase activity in vitro. Their catalytic activity could be induced by small molecules such as L-Arginine for SmVKR1 and calcium ions for SmVKR2. Preliminary experiments showed the presence of a potential natural ligand inside of the gynaecophoral duct of paired worms, able to activate SmVKR1 but not SmVKR2. To identify cytoplasmic partners of SmVKR1 and SmVKR2, we used intracellular domains (ICD) of each receptor as baits to screen an adult worm cDNA yeast library. We could show that SmVKR1 and SmVKR2 ICDs interact with various proteins, including cytoskeleton components and proteins containing SH2 protein-protein interaction domains, known to participate in classical signalling pathways of RTKs. We have also shown by RNAi that the diminution of smvkr1 and smvkr2 transcripts results in major changes in the morphology of genital organs of female worms. In the sporocyst larvae, RNAi of both SmVKR1 and SmVKR2 led to a significant decrease of the size of the parasites, as compared to the controls.The second part of my thesis work concerned the study of AgVKR from A. gambiae. Using two different expression systems, we have established that AgVKR was also catalytically active and activable by L-Arg as was SmVKR1. Functional studies of AgVKR could be facilitated by the recent discovery that the hemocyte-like SuA5B cell line of A. gambiae are constitutively expressing AgVKR. RNAi procedures have been designed to analyse the impact of a diminution of agvkr transcripts on the physiology of SuA5B cells.Taken together, these results already participate in a better knowledge of the mechanisms of VKR regulation and of their function, confirming their potential importance in growth and reproduction of invertebrate organisms

Caractérisation moléculaire des Récepteurs Venus Kinase : étude fonctionnelle chez le parasite Schistosoma mansoni

Venus Kinase Receptors (VKRs) form a new family of Receptor Tyrosine Kinases discovered for the first time in the parasite Schistosoma mansoni (SmVKR1), then in fourteen other invertebrate organisms and mainly in insects. In our laboratory, previous studies have shown that vkr transcripts are present in reproductive organs of adult organisms but also in larval stages, suggesting a role of VKRs in reproductive and development mechanisms. My thesis was concerned with the structural, biochemical, and functional characterization of the VKRs of two invertebrate organisms causing serious public health concerns: the worm S. mansoni responsible for the second human parasitic disease, and the principal malaria vector in Africa, Anopheles gambiae. At first, we have shown the existence of SmVKR2, a second VKR in the worm. Its transcripts are expressed in all the parasitic stages and localized in the immature oocytes and the ootype of the female worm. Recombinant SmVKR1 and SmVKR2 proteins showed a tyrosine kinase activity in vitro. Their catalytic activity could be induced by small molecules such as L-Arginine for SmVKR1 and calcium ions for SmVKR2. Preliminary experiments showed the presence of a potential natural ligand inside of the gynaecophoral duct of paired worms, able to activate SmVKR1 but not SmVKR2. To identify cytoplasmic partners of SmVKR1 and SmVKR2, we used intracellular domains (ICD) of each receptor as baits to screen an adult worm cDNA yeast library. We could show that SmVKR1 and SmVKR2 ICDs interact with various proteins, including cytoskeleton components and proteins containing SH2 protein-protein interaction domains, known to participate in classical signalling pathways of RTKs. We have also shown by RNAi that the diminution of smvkr1 and smvkr2 transcripts results in major changes in the morphology of genital organs of female worms. In the sporocyst larvae, RNAi of both SmVKR1 and SmVKR2 led to a significant decrease of the size of the parasites, as compared to the controls.The second part of my thesis work concerned the study of AgVKR from A. gambiae. Using two different expression systems, we have established that AgVKR was also catalytically active and activable by L-Arg as was SmVKR1. Functional studies of AgVKR could be facilitated by the recent discovery that the hemocyte-like SuA5B cell line of A. gambiae are constitutively expressing AgVKR. RNAi procedures have been designed to analyse the impact of a diminution of agvkr transcripts on the physiology of SuA5B cells.Taken together, these results already participate in a better knowledge of the mechanisms of VKR regulation and of their function, confirming their potential importance in growth and reproduction of invertebrate organisms.La famille des Venus Kinase Receptors (VKR) est une nouvelle famille de Récepteurs Tyrosine Kinase découverte au laboratoire chez le ver parasite Schistosoma mansoni (SmVKR1), puis chez 14 autres organismes invertébrés, principalement des insectes. Les études réalisées au laboratoire ont montré la présence des transcrits vkr dans les organes reproducteurs des organismes adultes mais aussi dans les stades larvaires suggérant un rôle dans le développement et/ou la reproduction de ces organismes. Ma thèse concerne la caractérisation structurale, biochimique et fonctionnelle des VKRs de deux organismes d’intérêt sanitaire majeur : le schistosome, parasite responsable de la bilharziose qui représente la seconde endémie parasitaire mondiale et l’anophèle, vecteur principal du paludisme en Afrique. Dans une première partie nous avons mis en évidence la présence d’un second VKR chez le schistosome, nommé SmVKR2. Nous avons montré que ses transcrits sont exprimés à tous les stades du cycle parasitaire et principalement dans les organes génitaux de la femelle au niveau des ovocytes immatures et de l’ootype. Nous avons exprimé SmVKR1 et SmVKR2 et montré qu’ils sont tous deux des récepteurs à activité tyrosine kinase, activables respectivement par la L-Arginine et par le calcium. Des expériences complémentaires semblent indiquer qu’un ligand naturel de SmVKR1 pourrait se trouver dans le canal gynécophore des vers appariés. Parallèlement, nous avons entrepris l’identification des partenaires cytosoliques de SmVKR1 et SmVKR2 grâce au criblage d’une banque d’ADNc de vers adultes par la technique de double hybride en levure en utilisant les domaines intracellulaires (DIC) des récepteurs comme appâts. L’analyse partielle des résultats obtenus montre que les DIC de SmVKR1 et SmVKR2 interagissent en autres avec des protéines du cytosquelette et avec des protéines cytoplasmiques pourvues de domaines d’interaction protéine-protéine SH2, acteurs de voies de signalisation classique des RTKs. Nous avons montré par ARNi que la diminution des transcrits SmVKR1 et SmVKR2 a un impact majeur sur la morphologie des organes génitaux de la femelle. Les sporocystes interférés pour les deux récepteurs présentent une diminution significative de leur taille comparée aux témoins.La deuxième partie de mes travaux de thèse a été centrée sur l’étude d’AgVKR, le récepteur d’Anopheles gambiae. En utilisant deux systèmes d’expression hétérologue, nous avons pu établir que ce récepteur était lui aussi catalytiquement actif et activable par la L-arg comme SmVKR1. L’étude de la fonction d’AgVKR a été abordée grâce à la découverte récente de son expression constitutive dans une lignée de cellules d’A. gambiae nommées SuA5B et de type hémocytaire. Des expériences d’ARNi ont été mises au point dans le but de visualiser l’impact d’une diminution de transcrits sur la physiologie des cellules. Dans leur ensemble, les résultats de ces travaux participent à la compréhension des mécanismes de régulation et de la fonction des VKRs, des récepteurs qui semblent d’une grande importance pour le développement et la reproduction des organismes

Aberrant neural crest development causes craniofacial and other malformations in an animal model of Musculocontractural Ehlers-Danlos syndrome.

Musculocontractural Ehlers-Danlos syndrome (MC-EDS) is a rare recessive disorder that is characterized by connective tissue fragility, distinct craniofacial features and congenital malformations. MC-EDS patients have defects in the enzymes dermatan sulfate epimerase-1 and dermatan 4-O-sulfotransferase-1, which are involved in the biosynthesis of iduronic acid in the chondroitin sulfate/dermatan sulfate (CS/DS) chains of proteoglycans (PGs). While the connective tissue defect is a result of disturbed collagen fibril assembly based on a decreased iduronic acid content of interacting CS/DS-PGs, the cause of the developmental malformations in MC-EDS is not well understood. This review focuses on a new role of CS/DS-PGs in the development of multipotent and highly migratory neural crest (NC) cells in the Xenopus embryo model of MC-EDS. Single iduronic acid residues in CS/DS-PGs are involved in the formation of NC-derived craniofacial structures by facilitating the migration and adhesion of NC cells to fibronectin. Our results suggest a defect in NC development as cause of the craniofacial and other congenital anomalies in MC-EDS patients, which might contribute to an improved diagnosis and etiology-based therapy

Neural crest delamination and migration: Looking forward to the next 150 years

International audienc

Active signals, gradient formation and regional specificity in neural induction

AbstractThe question of how the vertebrate embryo gives rise to a nervous system is of paramount interest in developmental biology. Neural induction constitutes the earliest step in this process and is tightly connected with development of the embryonic body axes. In the Xenopus embryo, perpendicular gradients of BMP and Wnt signals pattern the dorsoventral and anteroposterior body axes. Both pathways need to be inhibited to allow anterior neural induction to occur. FGF8 and IGF are active neural inducers that together with BMP and Wnt signals are integrated at the level of Smad 1/5/8 phosphorylation. Hedgehog (Hh) also contributes to anterior neural induction. Suppressor-of-fused plays an important role in intertwining the Hh and Wnt pathways. Distinct mechanisms are discussed that establish morphogen gradients and integrate retinoic acid and FGF signals during posterior development. These findings not only improve our understanding of regional specification in neural induction, but have profound implications for mammalian stem cell research and regenerative medicine

The venus kinase receptor (VKR) family: structure and evolution.

International audienceBACKGROUND: Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be highly expressed in the larval stages and gonads of several invertebrates, suggesting that it could have functions in development and/or reproduction. RESULTS: Analysis of recent genomic data has allowed us to extend the presence of VKR to five bilaterian phyla (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata) as well as to the Cnidaria phylum. The presence of NveVKR in the early-branching metazoan Nematostella vectensis suggested that VKR arose before the bilaterian radiation. Phylogenetic and gene structure analyses showed that the 40 receptors identified in 36 animal species grouped monophyletically, and likely evolved from a common ancestor. Multiple alignments of tyrosine kinase (TK) and VFT domains indicated their important level of conservation in all VKRs identified up to date. We showed that VKRs had inducible activity upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site. CONCLUSIONS: This study highlights the presence of VKR in a large number of invertebrates, including primitive metazoans like cnidarians, but also its absence from nematodes and chordates. This little-known RTK family deserves to be further explored in order to determine its evolutionary origin, its possible interest for the emergence and specialization of Metazoa, and to understand its function in invertebrate development and/or reproductive biology

Musculocontractural Ehlers-Danlos syndrome and neurocristopathies : Dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin

Of all live births with congenital anomalies, approximately one-third exhibit deformities ofthe head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers-Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused bydysfunction ofdermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 inconnective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1isimportant for the generationofisolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial-mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo. Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and metastasis in neuroblastoma and malignant melanoma suggest an association between DS and NC-derived cancers

Dual Targeting of Insulin and Venus Kinase Receptors of Schistosoma mansoni for Novel Anti-schistosome Therapy

Background: Chemotherapy of schistosomiasis relies on a single drug, Praziquantel (PZQ) and mass-use of this compoundhas led to emergence of resistant strains ofSchistosoma mansoni, therefore pointing out the necessity to find alternativedrugs. Through their essential functions in development and metabolism, receptor tyrosine kinases (RTK) could representvaluable drug targets for novel anti-schistosome chemotherapies. Taking advantage of the similarity between the catalyticdomains ofS. mansoniinsulin receptors (SmIR1 and SmIR2) and Venus Kinase Receptors (SmVKR1 and SmVKR2), we studiedthe possibility to fight schistosomes by targeting simultaneously the four receptors with a single drug.Methodology/Principal Findings: Several commercial RTK inhibitors were tested for their potential to inhibit the kinaseactivities of SmIR1, SmIR2, SmVKR1 and SmVKR2 intracellular domains (ICD) expressed inXenopusoocytes. We measured theinhibitory effect of chemicals on meiosis resumption induced by the active ICD of the schistosome kinases in oocytes. The IRinhibitor, tyrphostin AG1024, was the most potent inhibitory compound towards SmIR and SmVKR kinases.In vitrostudiesthen allowed us to show that AG1024 affected the viability of both schistosomula and adult worms ofS. mansoni.Atmicromolar doses, AG1024 induced apoptosis and caused schistosomula death in a dose-dependent manner. In adultworms, AG1024 provoked alterations of reproductive organs, as observed by confocal laser scanner microscopy. With 5mMAG1024, parasites were no more feeding and laying eggs, and they died within 48 h with 10mM.Conclusion/Significance: IRs and VKRs are essential inS. mansonifor key biological processes including glucose uptake,metabolism and reproduction. Our results demonstrate that inhibiting the kinase potential and function of these receptorsby a single chemical compound AG1024 at low concentrations, leads to death of schistosomula and adult worms. Thus,AG1024 represents a valuable hit compound for further design of anti-kinase drugs applicable to anti-schistosomechemotherapy