Thesis (M.D. with Distinction in Research) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Not embargoed. Vita. Bibliography: 52-62.

Made available in DSpace on 2015-07-22T20:49:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-25 / As membranas biológicas são formadas por uma mistura de várias classes de lipídeos, cujo empacotamento preferencial entre esfingolipídeos e esteróis formam junto com proteínas específicas, esta complexa organização conhecida como microdomínios de membrana. Com o intuito de verificar a presença de microdomínios de membrana em leveduras de fungos patogênicos como Paracoccidioides brasiliensis e Histoplasma capsulatum, as leveduras desses dois fungos foram lisadas com pérolas de vidro e incubadas com Brij 98 a 4°C. As frações de microdomínios de membrana foram separadas por ultracentrifugação em gradiente de sacarose e, seus componentes analisados por HPTLC, SDS-PAGE e “Western blotting”. As análises dos lipídeos de membrana mostram que aproximadamente 40% do ergosterol das duas espécies de leveduras analisadas estão presentes nos microdomínios de membrana. Enquanto as porcentagens de glicoesfingolipídeos presentes nessas frações correspondem a 42% e 25%, em leveduras de P. brasiliensis e H. capsulatum, respectivamente. Em conjunto com as análises lipídicas, verificou-se nas duas espécies, um enriquecimento protéico nas frações de microdomínios de membrana, entre estas proteínas podemos citar a Pma1p, uma proteína marcadora de microdomínios de fungos, e uma proteína de 30 kDa, capaz de se ligar a laminina. Para determinar a importância do ergosterol na manutenção da integridade dos microdomínios de membrana utilizou-se metil-beta-ciclodextrina (mβCD), que é capaz de complexar e retirar o ergosterol. Após o tratamento das leveduras com mβCD verificou-se a extração dos esteróis numa proporção de 80% e 70% para P. brasiliensis e H. capsulatum, respectivamente. No entanto, o perfil de distribuição dos glicoesfingolipídeos e fosfolipídeos, analisados por HPTLC, não apresentou mudanças significativas após o tratamento com a mβCD. As análises protéicas demonstraram o deslocamento de algumas proteínas para frações solúveis do gradiente de sacarose como Pma1p e uma proteína de 30 kDa. Por outro lado, a marcação com o anticorpo anti-α5-integrina mostra a presença de uma proteína de 50 kDa nos microdomínios de membrana, mesmo após o tratamento com a mβCD, sugerindo a existência de uma população de microdomínios de membrana que não depende do ergosterol para manutenção de sua integridade. A importância desses microdomínios de membrana foi testada na infectividade de macrófagos alveolares, onde uma redução de 53% na infectividade de macrófagos foi verificada após o tratamento das leveduras de H. capsulatum com mβCD. Os resultados apresentados nessa tese demonstram a existência de microdomínios de membrana em leveduras de P. brasiliensis e H. capsulatum, bem como sua importância para a interação levedura-macrófago. / Biological membranes are constituted by a mixture of several classes of lipids. In this enviroment, sphingolipids and sterols pack tightly to form together with specific proteins a complex membrane organization known as membrane microdomains. In order to detect the presence of membrane microdomains in yeast forms of pathogenic fungi, such as Paracoccidioides brasiliensis and Histoplasma capsulatum, yeast forms of both fungi were lysed by vortexing with glass beads and then incubated with Brij 98 at 4ºC. Fractions containing membrane microdomains were isolated by ultracentrifugation on sucrose gradient, and their components were analyzed by HPTLC, SDSPAGE and Western blotting. Analysis of membrane lipids showed that about 40% of ergosterol from both P. brasiliensis and H. capsulatum was present in the membrane microdomains, whereas the percentage of glycosphingolipids present in P. brasiliensis and H. capsulatum was 42% and 25%, respectively. Analysis by SDS-PAGE and Western blotting clearly showed a protein enrichment in the membrane microdomains fraction of both fungi. It is noteworthy the presence of Pma1p, a fungal microdomain marker, and also the presence of a 30 kDa (glyco)protein which binds to laminin. To investigate the requirement of ergosterol to mantain the integrity of membrane microdomains, it was used methylbeta- cyclodextrin (mβCD) an agent able to efficiently extract membrane sterols. After treatment of yeasts using mβCD, it was observed the removal of 80% and 70% of ergosterol in P. brasiliensis and H. capsulatum, respectively. After treatment with mβCD it was observed a shift of 25% of the glycosphingolipids from the insoluble to the soluble fraction, conversely the distribution profile of phospholipids remained unmodified after treatment with mβCD. The protein analysis showed the displacement of a few proteins to soluble fractions of the sucrose gradient, such as Pma1p and the (glyco)protein of 30 kDa. On the other hand, using an anti-α5-integrin antibody it was detected, even after the mβCD treatment, the presence of a 50 kDa protein in membrane microdomains, suggesting the existence of microdomains that do not depend on ergosterol for their integrity. These data strongly suggest the existence of two population of microdomains: i) dependent of ergosterol for integrity maintenance of microdomains and ii) microdomains non-dependent of ergosterol for the maintenance of their functional role. Furthermore, the biological importance of membrane microdomains was clearly demonstrated by a 53% reduction of infectivity of alveolar macrophage infectivity when yeast forms of H. capsulatum were treated with mβCD. / TEDE / BV UNIFESP: Teses e dissertações

Les jonctions adhérentes sont des jonctions intercellulaires essentielles à la morphogenèse et à la maintenance des tissus. Elles reposent sur l'assemblage de grands complexes multiprotéiques aux contacts intercellulaires, centrés sur des protéines transmembranaires appelées cadhérines. Nous avons découvert deux nouveaux partenaires des cadhérines N, E, M, P, R et 11, les flotillines. Nous avons caractérisé leur interaction avec la N-cadhérine et découvert qu'elle était constitutive à la membrane plasmique et vraisemblablement indirecte. Nous avons démontré que les flotillines sont essentielles à la stabilisation des jonctions adhérentes dans des cellules musculaires et épithéliales, ainsi qu'à des processus cellulaires dépendants des jonctions. Nous montrons qu'en effet, les flotillines sont nécessaires à l'interaction des cadhérines avec la p120-caténine, qui inhibe leur internalisation et leur dégradation. Nos expériences suggèrent que les flotillines seraient impliquées dans la formation d'un microdomaine membranaire particulier au niveau de la jonction en cours de maturation, permettant le recrutement de la p120-caténine. / Cadherins are essential in many fundamental processes such as tissue patterning during development and in the maintenance of adult tissue architecture. At regions of cell-cell contact, cadherins assemble into large macromolecular complexes named adherens junctions. Here we identify flotillin 1 and 2 as new partners of several classical cadherins. The interaction between flotillines and N-cadherin is constitutive at the plasma membrane and seems to require an intermediate partner. Knockdown of flotillins had a dramatic effect on N- and E-cadherin recruitment at the adherens junctions in both mesenchymal and epithelial cell types. At the molecular level, we show that flotillins stabilize cadherins at the PM hence allowing the coupling of 120 catenin, one of their main stabilizing partners. Our results suggest that flotillins might scaffold a membrane microdomaine at maturing junctions, allowing the recruitment of p120-catenin.

Le fer est essentiel à la croissance et au développement des plantes. Chez Arabidopsis thaliana, le transporteur IRT1 permet l’absorption du fer par les cellules épidermiques de la racine et est, par conséquent, un des acteurs majeurs de la nutrition en fer. IRT1 est cependant un transporteur peu spécifique qui transporte également des métaux non ferreux que sont le zinc (Zn), le manganèse (Mn) et le cobalt, qui constituent les substrats secondaires d’IRT1 et qui ont été récemment démontrés dans notre laboratoire comme régulant l’endocytose d’IRT1. Afin d’identifier des protéines potentiellement impliquées dans le trafic ou dans la régulation de l’activité d’IRT1, nous avons isolé des interactants de ce transporteur via des immunopurifications d’IRT1 combinées à des analyses de spectrométrie de masse. Cette approche nous a permis d’établir le premier intéractome d’IRT1. Parmi les protéines interagissant avec IRT1, nous avons isolé AHA2 et FRO2 qui participent toutes les deux au processus d’absorption du fer chez Arabidopsis, ainsi que la protéine à domaine SPFH appelée HIR2. HIR2 est localisée dans des microdomaines membranaires chez Arabidopsis mais sa fonction reste jusqu’à présent assez énigmatique. Cependant, chez les animaux, les protéines à domaine SPFH ont été proposées comme étant impliquées dans la formation des microdomaines membranaires; de plus certaines protéines à domaines SPFH appelées Flotillines interviennent dans des mécanismes d’endocytose chez les animaux et les plantes. Après avoir validé les interactions entre les protéines IRT1 et FRO2/AHA2/HIR2 par des approches complémentaires, nous avons analysé la dynamique intracellulaire de ces protéines par microscopie. Nos résultats suggèrent l’existence d’un complexe protéique regroupant les trois acteurs majeurs de l’homéostasie du fer chez Arabidopsis : IRT1, FRO2 et AHA2, dont la fonction pourrait être d’optimiser l’absorption du fer dans la racine. Contrairement à ce qui est observé pour IRT1, les protéines FRO2 et AHA2 ne sont pas massivement endocytées en réponse à un excès de métaux (Zn,Mn, Co) et ceci bien qu’elles puissent être présentes au sein d’un complexe contenant IRT1. Nous avons en outre montré que FRO2 et AHA2 étaient ubiquitinées, mais contrairement à IRT1, de façon indépendante de la concentration en métaux non ferreux. En utilisant des approches de génétique inverse, nous avons mis en évidence que HIR2 était impliquée dans le maintien de l’homéostasie du fer, les mutants hir2 étant extrêmement sensibles à la carence en fer. D’autre part nous avons montré que l’accumulation de la protéine IRT1 était dérégulée chez le mutant hir2 et ceci de façon post-transcriptionnelle. Nous cherchons actuellement à déterminer comment HIR2 régule la dynamique et/ou la stabilité d’IRT1 dans la cellule. HIR2 pourrait assurer le recrutement d’IRT1 et plus généralement du complexe d’acquisition du fer décrit ci-dessus dans des microdomaines membranaires spécifiques. D’autre part, nous avons également émis l’hypothèse que HIR2 pourrait être impliquée dans une voie d’endocytose d’IRT1 indépendante de la clathrine. / Iron is an essential nutrient for plant growth and development. In Arabidopsis thaliana, the transporter IRT1, which allows iron absorption through the epidermic cells of the root, is a major actor in iron nutrition. Despite of it, IRT1 also transports the non-iron metals zinc (Zn), manganese (Mn) and cobalt (Co). These metals are considered as the secondary substrates of IRT1, and therefore this transporter is considered as poorly specific. Our laboratory has recently uncover that these secondary substrates regulate IRT1 endocytosis. In order to uncover the different proteins that can be implicated in the traffic or in the regulation of IRT1 activity, we have proceeded to perform IRT1 immnopurifications, followed by mass spectrometry analyses. This approach has allowed us to produce a first interactome list of IRT1. Among the proteins that interact with IRT1, we have isolated AHA2 and FRO2, both well known in the process of iron acquisition in Arabidopsis, and also a SPFH domain containing protein known as HIR2. Although it is known that HIR2 is contained in membrane microdomains in Arabidopsis, its function is still to be determined. Nevertheless, in the animal kingdom, SPFH domain containing proteins have been proposed as implicated in the formation of membrane microdomains. This is specially the case of the specific SPFH domain containing proteins known as Flotillins, which have the ability to mediate endocytosis in animals as in plants. After validation of the interaction between IRT1 and FRO2/AHA2/HIR2 by different complementary approaches, we have microscopically analyzed the intracellular dynamics of these proteins. Our results suggest the existence of a protein complex that reunites the three major actors of iron homeostasis in Arabidopsis: IRT1, FRO2 and AHA2. We suspect that the main function of this complex is to optimize the process of iron absorption in the root. In spite of what is known for IRT1 and despite being part of a same complex, FRO2 and AHA2 are not massively endocytosed in response to a non-iron metal excess (Zn, Mn, Co). Furthermore, we have shown that FRO2 and AHA2 are ubiquitinated, although their ubiquitination is also independent of the concentration of the non-iron metals, unlike the ubiquitination of IRT1. Finally, using reverse genetic approaches, we have been able to show that HIR2 is implicated in the maintenance of the iron homeostasis. Indeed, hir2 mutants are extremely sensitive to lack of Fe, even though they present posttranslational deregulations that result in the an overaccumulation of the protein IRT1. We are currently trying to determine how HIR2 regulates the dynamics and/or the stability of IRT1 inside the cell. HIR2 could be assuring the recruiting of IRT1 (or the recruitment of the whole iron acquisition complex) into specific membrane microdomains. On the other hand, HIR2 could be implicated in a new pathway of internalization of IRT1, independent of clathrin.

No description available.

Endothelial cells (EC) are essential regulator of vascular homeostasis through the generation and release of various bioactive agents, including nitric oxide (NO). NO modulates several vascular functions such as vascular tone and permeability, through the stimulation of soluble guanylate cyclase (sGC) leading to the production of cGMP. Conversely, phosphodiesterases (PDEs) are enzymes metabolizing cyclic nucleotides (cGMP and cAMP) and are therefore major regulatory players for cGMP and cAMP signalling pathways. Although ECs are the main source of NO, little is known on the endothelial NO-cGMP signalling pathway and cellular outcomes. It was then hypothesized that a specific population of cGMP-phosphodiesterases allows ECs to stabilize cGMP levels despite the elevated production of NO. Expression of cGMP-phosphodiesterases was initially studied in resistance mesenteric arteries from mice. PDE5 and PDE6 were both found at mRNA and protein levels in native arteries but PDE6 is not found in cultured ECs. Interestingly, subcellular distributions of both enzymes were distinct. PDE5 appeared to be homogeneously distributed whilst PDE6 catalytic subunits (PDE6 and PDE6) showed a preferential staining in the perinuclear region. These results suggest that PDE6 might be involved in the regulation of cGMP microdomains. Based on these findings, a mathematical model was developed. Simulations of dynamic cGMP levels in ECs support the notion of cGMP microdomains dependent on PDE6 expression and localization. In the absence of PDE6, application of NO either as a single bolus or repetitive pulses led to a homogeneous increase in cGMP levels in ECs despite PDE5 homogeneous distribution. However, PDE6 subcellular targeting to the perinuclear membrane generated a cGMP-depleted perinuclear space. The findings from this study provide the first evidence of the expression and specific intracellular distribution of PDE6 in native endothelial cells that strongly support their involvement in the generation of cGMP microdomains / Les cellules endothéliales (CEs) participent au maintien de l’homéostasie vasculaire en générant et libérant de nombreux agents bioactifs, incluant l’oxyde nitrique (NO). Le NO module plusieurs fonctions vasculaires telles que le tonus et la perméabilité vasculaire via la stimulation de la guanylate cyclase soluble (GCs) provoquant la formation de GMPc. D’autre part, les phosphodiestérases (PDEs) sont des enzymes métabolisant les nucléotides cycliques (GMPc et AMPc) et participent donc à des étapes essentielles du contrôle des voies de signalisation du GMPc et de l’AMPc. Bien que les CEs soient la source principale de NO, la voie de signalisation NO-GMPc endothéliale et les répercussions fonctionnelles demeurent méconnues. Nous avons alors émis l’hypothèse qu’une population spécifique de PDEs ciblant le GMPc (PDEs-GMPc) permettrait aux CEs de maintenir des niveaux de GMPc faible malgré l’importante production de NO. L’expression des isoformes de PDEs-GMPc dans les artères mésentériques de souris fut initialement déterminée. PDE5 et PDE6 furent détectées tant sous la forme d’ARNm que de protéines dans les artères natives alors que PDE6 est absente de lignées de CEs en culture. La distribution intracellulaire des deux enzymes est distincte. Alors que PDE5 est distribué uniformément dans le cytoplasme des cellules endothéliales, les sousunités catalytiques de PDE6 ( et ) sont préférentiellement présentes dans la région périnucléaire. Ces résultats suggèrent que PDE6 puisse être impliqué dans le contrôle de microdomaines de GMPc. Des simulations effectuées à l’aide d’un modèle mathématique développé sur la base de ces données sont en accords avec la notion selon laquelle l’expression et la distribution subcellulaire de PDE6 sont responsables de microdomaines de GMPc dans l’endothélium. En absence de PDE6, l’ajout de NO sous forme de bolus unique ou répétée mène à une augmentation homogène de la concentration cytoplasmique en GMPc malgré la présence de PDE5. Toutefois, la présence de PDE6 à la membrane péri-nucléaire crée un espace péri-nucléaire pauvre en GMPc. Les résultats de cette étude forment les premières évidences de l’expression et de la distribution intracellulaire hétérogène de PDE6 dans les cellules endothéliales natives et suggèrent leur implication dans la génération de microdomaines.

Morphine is an opioid agonist, which can exert cardioprotective effects under certain conditions. Lipid rafts are considered important platforms for membrane organization of signaling proteins and, therefore, these structures could play a role in the effects of morphine, which acts through the opioid receptors. The aim of this thesis was to investigate the distribution of the main components of the opioid receptor and Gi/o-mediated signaling pathway in lipid rafts isolated from rat myocardium, which was affected by various doses of morphine. Because we used different isolation techniques with different solubilization agents (Triton X-100, CHAPS, cholate and sodium carbonate) for preparation of lipid rafts, it was of interest to characterize more closely these preparations. Another aim of this study was to investigate how different methods of isolating these structures affect activity of the key target enzyme of the opioid signaling pathway, i.e. adenylyl cyclase. The presence of signaling molecules of the Gi/o/AC pathway of the opioid system in membrane rafts was confirmed and the distribution of selected proteins was dependent on the type of extractant. We also observed the effect of morphine on the localization of proteins in lipid rafts. Different extractants provided different degree of...

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2004. / Vita. Bibliography: 150-177.

This work is a collection of three research articles and one review article focused on flotillins (FLOTs) and hypersensitive induced reaction proteins (HIRs) in Arabidopsis thaliana. FLOTs and HIRs are closely related membrane-associated proteins forming two subfamilies both belonging to SPFH domain superfamily. While FLOTs are present in organisms of all evolutionary lineages HIRs are plant specific proteins. The review article sums up the knowledge gained on FLOTs and HIRs from different organisms in terms of cellular localization, interaction with cellular membranes and with other proteins, and physiological functions. The research articles were targeted at three aspects of AtFLOTs and AtHIRs: involvement in response to exogenous stimuli; determination of protein interactors; and subcellular localization and dynamics. The first aspect was approached by transcription measurement of AtFLOTs and phenotypic screen of single loss-of-function mutants of AtFLOTs upon various treatments covering biotic and abiotic stress and phytohormone application. Although we observed changes in transcription none of the treatments provoked a phenotype manifestation in any of AtFLOT mutants. In the second article we focused on interactome of AtFLOT2 and performed co- immunoprecipitation followed by mass spectrometry...

Abstract The function of skeletal muscle is to generate force and produce movement. These tasks are carried out by long multinucleated cells, the skeletal myofibres. The membrane system and the cytoskeleton of these cells are uniquely organized to respond rapidly to neuronal stimuli and to achieve efficient contraction. In the present study the organization and distribution of selected protein/lipid based microdomains that reside in the plasma membrane and sarcoplasmic reticulum of isolated rat skeletal myofibres, were investigated. Aquaporin 4 (AQP4) water channels are arranged as higher order oligomers of several sizes in the sarcolemma and in the T tubules. These oligomers, however, were absent from many specialized micro- and- macrodomains. The distribution of AQP4 coincided with that of a highly organized protein assembly, the dystrophin glycoprotein complex (DGC), in the sarcolemma. A chimaeric venus-AQP4 was equally mobile in the T tubules and sarcolemma, but the anchoring mechanisms of the protein appeared to be different. In contrast to AQP4, the proteins resident in cholesterol and sphingolipid-based microdomains, known as rafts, also occupied DGC deficient areas, which surround the T tubule openings. Indeed, flotillin-1 rafts were located in the neck portions of the T tubules. The rafts defined by the influenza haemagglutinin (HA) also resided in DGC deficient areas, but at the borders of the DGC area. Importantly, of the raft proteins, only the localization of caveolin 3 (CAV3) was dependent on the cholesterol enriched lipid environment, as evidenced by cholesterol depletion experiments and localization studies on a non-raft associated variant of HA. The organization and distribution of membrane associated rough ER (RER) proteins were also analysed. Biochemical detergent extraction analyses and immunofluorescence staining indicated that the ER proteins were assembled as microdomains within the sarcoplasmic reticulum (SR). The microdomains were distributed throughout the SR network and they were capable of protein translocation. Taken together, skeletal myofibres comprise visually distinct microdomains both in the plasma membrane and in the SR. In the plasma membrane, different types of microdomains are not homogenously distributed and function in diverse locations. This may have important physiological implications concerning, among other things, local regulation of ion concentrations and cell signalling cascades. Different constraints ranging from protein-protein interactions to the surrounding lipid environment are important for dictating the observed distribution patterns. / Tiivistelmä Luustolihaksen toimintojen perustana ovat supistumiskykyiset lihassolut, joiden kalvorakenne on järjestynyt erityisellä tavalla ohjaamaan supistusta. Tässä tutkimuksessa analysoitiin proteiini- ja lipidiperustaisten mikroalueiden järjestäytymistä ja tähän vaikuttavia tekijöitä luustolihassolun solukalvolla sekä lihassolun sisäisessä kalvojärjestelmässä, sarkoplasmisessa verkossa (SR). Ensin analysoitiin vesikanavatyyppiä 4 (AQP4), joka oligomerisoituessaan muodostaa erikokoisia mikroalueita. Havaittiin, että AQP4-mikroalueita esiintyy kaikkialla solukalvolla lukuun ottamatta eräitä erilaistuneita mikro- ja makroalueita. AQP4-oligomeerien jakauma solukalvon lateraalisessa osassa, sarkolemmalla, noudatti dystrofiini-glykoproteiinikompleksin jakaumaa. Fluoresoivan venus-AQP4-proteiinin avulla osoitettiin, että proteiinin liikkuvuus oli samanlainen solun sisään ulottuvissa poikkiputkistoissa ja sarkolemmalla, mutta liikkuvuutta rajoittavat tekijät olivat erilaisia näissä solukalvon osissa. Toiseksi analysoitiin kolesteroli- ja sfingolipidipitoisia mikroalueita, kalvolauttoja. Flotilliini-1- ja influenssaviruksen hemagglutiniini (HA) -proteiinia sisältäviä lauttoja esiintyi vain poikkiputkien suuaukkojen alueella, mutta lauttojen jakauma oli erilainen. Lauttojen lipidiympäristöllä ei ollut vaikutusta proteiinien sijaintiin. Tämä osoitettiin kolesterolin poistokokeilla sekä kokeilla, joissa käytettiin mutatoitua HA-proteiinia, joka ei hakeudu kolesteroliympäristöön. Kaveoliini-3-proteiinin sijainti poikkeaa edellä mainituista, ja kolesterolin poisto vaikutti merkittävästi sijainnin määräytymiseen. Kolmanneksi analysoitiin, miten karkean endoplasmakalvoston proteiinit ovat järjestäytyneet SR:ssä. Havaittiin, että endoplasmiset kalvoproteiinit eivät ole homogeenisesti levittäytyneet SR-kalvostoon vaan muodostavat pieniä mikroalueita. Detergenttiuuttoanalyysit osoittivat lisäksi, että näissä mikroalueissa on erilainen lipidikoostumus kuin SR:ssä yleensä. Huomattavaa oli myös, että mikroalueet olivat toiminnallisia kaikkialla SR-kalvostossa. Tulosten perusteella luustolihassolujen kalvojärjestelmä sisältää mikroalueita, joiden jakautuminen vaikuttaa hyvin organisoituneelta. Erityisesti solukalvon mikroalueet esiintyvät tietyillä spesifeillä alueilla, joissa niiden voidaan olettaa toimivan mm. erilaisissa solusignalointitapahtumissa ja paikallisessa ionipitoisuuksien säätelyssä. Eräissä tapauksissa lipidiympäristöllä on merkitystä mikroalueiden sijainnin määräytymisessä, mutta proteiinien sitoutuminen solukalvo- tai solukalvon alaisiin rakenteisiin saattaa myös olla määräävä tekijä.