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Outre un cytosquelette cortical bien structuré et fortement ancré à la membrane, le globule rouge (GR) jouit d’une concentration en calcium intracellulaire ([Ca2+]i) finement contrôlée , impliquée dans sa déformation et sa destruction et dérégulée dans plusieurs formes d’anémies héréditaires. Grâce à l’imagerie confocale vitale de GRs, le laboratoire d’accueil a découvert que plusieurs classes de lipides du feuillet externe de la membrane plasmique (phosphatidylcholine [PC], sphingomyéline [SM], ganglioside GM1 et cholestérol) s’organisent en domaines submicrométriques partiellement reliés spatialement. Cette démonstration a été permise grâce (i) à l’insertion d’analogues lipidiques fluorescents (BODIPY-PC, -SM et - GM1) à la membrane plasmique, ou (ii) au marquage direct de lipides endogènes (SM et cholestérol) avec des fragments non toxiques de toxines spécifiques (lysénine et theta, respectivement). Mon projet s’intéresse à l’importance des domaines lipidiques pour la déformation des GRs, via le recrutement et/ou l’activation de protéines impliquées dans les échanges de Ca2+. Le premier objectif de mon mémoire a consisté à évaluer l’importance du Ca2+ pour l’organisation de la membrane des GRs en domaines lipidiques. J’ai tout d’abord déterminé en microscopie vitale à fluorescence l’abondance des domaines enrichis en SM (mis en évidence par le BODIPY-SM ou la lysénine) après stimulation de l’influx (par un ionophore, le A23187) ou de l’efflux de Ca2+ (par incubation dans un milieu dépourvu de Ca2+ par l’EGTA augmente d’un facteur trois l’abondance des domaines enrichis en SM. Cet effet est spécifique de certains domaines lipidiques puisque seuls les domaines enrichis en SM ou en ganglioside GM1 sont augmentés, mais pas ceux enrichis en cholestérol ou en PC. La suite de mon mémoire s’est donc focalisée sur les domaines enrichis en SM. J’ai ensuite étudié l’importance des domaines enrichis en SM pour les échanges de Ca2+. Dans ce but, j’ai tiré profit de l’observation que ces domaines (i) disparaissent suite au traitement à la sphingomyélinase ou à la métyl-B-cyclodextrine, des agents qui déplètent respectivement le contenu membranaire en SM et en cholestérol ; et à l’inverse (ii) augmentent en nombre suite au découplage de la membrane de son cytosquelette sous-jacent par activation de la PKC. J’ai ainsi pu montrer que la disparition des domines suite au traitement de la sphingolmyélinase ou à la cyclodextrine induit une augmentation de la [Ca2+]i. J’ai également observé que l’augmentation du nombre de domaines suite au découplage de la membrane de son cytosquelette sous-jacent s’accompagne d’une diminution de la [Ca2+]i. L’ensemble des résultats de ces deux premières parties montre qu’il existe une relation inverse entre l’abondance des domaines enrichis en SM et la [Ca2+]i. Pour ensuite tenter d’établir s’il existe une relation entre les domaines lipidiques, la [Ca2+]i et la déformabilité des GRs, j’ai travaillé sur des GRs de patients atteints d’elliptocytose et de sphérocytose héréditaires, deux maladies de fragilité membranaire des GRs. Alors que la régulation des échanges de Ca2+ est affectée chez les GRs elliptocytotiques, le nombre de domaines n’est pas significativement modifié. Chez les GRs sphérocytotiques, le nombre de domaines et les échanges de Ca2+ sont affectés de manière inversement proportionnelle, suggérant que l’organisation de la membrane en domaines enrichis en SM et la [Ca2+]i pourraient jouer un rôle dans la déformabilité des GRs. J’ai enfin commencé à rechercher par quel mécanisme Ca2+ pourrait moduler les domaines enrichis en SM. Avec l’aide des doctorants du groupe, j’ai notamment évalué si une modification de la [Ca2+]i pouvait avoir des conséquences sur (i) l’asymétrie membranaire (translocation de la phosphatidylsérine au feuillet externe mis en évidence par marquage à l’Annexine V fluorescente), ou (ii) la fluidité membranaire (mesurée par insertion du laurdan à la membrane plasmique du GR). En conclusion, les données obtenues au cours de ce mémoire ont permis de montrer la relation entre [Ca2+]i et l’organisation de la membrane en certains domaines lipidiques, ceux enrichis en SM et en ganglioside GM1. D’autre part, mes résultats suggèrent une relation entre les domaines, les échanges de Ca2+ et la déformabilité du GR. Cette étude ouvre de nombreuses perspectives en biophysique. Besides a well-structured cytoskeleton strongly anchored to the membrane, the red blood cell (RBC) exhibits an intracellular calcium concentration [Ca 2+]i finely controlled, involved in RBC deformation and destruction and deregulated in several forms of hereditary anemias. Using vital confocal imaging of RBCs, the host laboratory discovered that several types of lipids (phosphatidylcholine (PC), sphingomyelin (SM), ganglioside GM1 and cholesterol) are organized into spatially connected submicrometric domains at the outer plasma membrane leaflet. This discovery was made possible thanks to (i) incorporation at the plasma membrane of fluorescent lipid analogs (BODIPY-PC, -SM and –GM1), or (ii) direct labeling of endogenous lipids (SM and cholesterol) with non-toxic fragments of specific toxins (lysenin and theta, respectively). My project focuses on the importance of lipid domains for RBC plasticity, through the recruitment and/or activation of proteins involved in the exchange of Ca2+.The first aim of my master thesis was to assess the importance of Ca2+ in the organization of RBC membrane lipids into domains. I first determined by vital fluorescence microcopy the abundance of domains enriched in SM (highlighted by plasma membrane insertion of BODIPY-SM or decoration of endogenous SM by lysenin) after stimulation of the Ca2+ influx (by the ionopohore A23187) or efflux (by incubation in medium lacking Ca2+, in combination or not with a chelation by EGTA). Thanks to those agents, I showed that a stimulation of Ca2+ efflux by EGTA strongly increases the number of SM-enriched domains. This effect is specific to some lipid domains since only the ones enriched in SM or GM1 are increased, whereas those enriched in cholesterol or PC are not affected. The rest of my master thesis thereby focused on the domains enriched in SM. I subsequently studied the importance of SM-enriched domains for the exchanges of Ca2 To this end, I took advantage of the observation that these domains disappear upon treatment with sphingomyelinase or methyl-13-cyclodextrin, two agents that respectively deplete the membrane content in SM and cholesterol. I was able to show that the disappearance of SM­ enriched domains due to these treatments induces an increase in [Ca2+]i. 1 also showed that the increased domain abundance by plasma membrane: cytoskeleton uncoupling at 4.lR complexes is linked to a decrease in [Ca2+li· All these results show thatthere is an inverse relationship between the abundance of SM-enriched domains and [Ca2+]i. To next establish whether there is a relationship between lipid domains, [Ca2+]i and RBC deformability, 1 worked on RBCs of patients suffering from hereditary spherocytosis and elliptocytosis, two RBC membrane fragility diseases. ln elliptocytosis, whereas Ca2+ exchanges are modified, the number of domains is not significantly altered. ln contrast, in spherocytotic RBCs,ca2+ exchanges are favored and the abundance of domains is increased as compared to control RBCs, suggesting that membrane organization in SM-enriched domains and Ca 2+ exchanges could play a role in RBC deformability. I finally started to examine by which mechanism Ca2+ could modulate SM-enriched domains. With the help of PhD students in the group, 1 evaluated whether a modification of [Ca2+L could affect (i) membrane asymmetry (by probing the translocation of phosphatidylserine to the outer leaflet by labeling with fluorescent Annexin V); or (ii) membrane fluidity (by laurdan insertion at the RBC plasma membrane).ln conclusion, the data obtained in this master thesis have shown the relation between [Ca2+]i and the organization of the plasma membrane in domains enriched in SM (and ganglioside GM1). Moreover, my results suggest a relationship between domains, Ca2+ efflux and RBC plasticity. This study opens up many prospects in biophysics.

Lipid-Linked Proteins --- Calcium-Binding Proteins --- Calcium Signaling

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This eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contact

neuronal Ca2+ sensors --- calcium signaling --- molecular basis of disease --- calcium bindind proteins --- neurological disorders

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Written by expert contributors, this much-needed book emphasizes a quantitative and comprehensive perspective to explore the many dimensions of cellular Ca2+ signalling. Leading researchers in the field of calcium signaling present key aspects of this process in cells, starting with Ca2+ entry into the cell, its uptake from the cytosol by organelles, such as the endoplasmic reticulum and mitochondria, and the actions of Ca2+ in turn on a variety of important cellular regulatory processes. This research and reference text should prove useful to a wide readership, from students to researchers at many different levels, particularly in physiology, pharmacology, or biomedical engineering. Part of Biophysical Society-IOP series.

SCIENCE / Life Sciences / Biophysics. --- Calcium --- Cellular signal transduction. --- Calcium Signaling. --- Signal Transduction. --- Biophysics. --- Physiological effect.

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This eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contact

Science: general issues --- Neurosciences --- neuronal Ca2+ sensors --- calcium signaling --- molecular basis of disease --- calcium bindind proteins --- neurological disorders --- neuronal Ca2+ sensors --- calcium signaling --- molecular basis of disease --- calcium bindind proteins --- neurological disorders

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This eBook is a collection of articles from a Frontiers Research Topic. Frontiers Research Topics are very popular trademarks of the Frontiers Journals Series: they are collections of at least ten articles, all centered on a particular subject. With their unique mix of varied contributions from Original Research to Review Articles, Frontiers Research Topics unify the most influential researchers, the latest key findings and historical advances in a hot research area! Find out more on how to host your own Frontiers Research Topic or contribute to one as an author by contacting the Frontiers Editorial Office: frontiersin.org/about/contact

Science: general issues --- Biology, life sciences --- epigenetics --- calcium signaling and signal transduction --- cell biology and cell fate --- fertilisation --- Preimplantation development --- reprogramming