Добірка наукової літератури з теми "Microdomain organisation"

Cholesterol plays a key role in the molecular and mesoscopic organisation of lipid membranes and it is expected that changes in its molecular structure (e.g., through environmental factors such as oxidative stress) may affect adversely membrane properties and function. In this study, we present evidence that oxidation of cholesterol has significant effects on the mechanical properties, molecular and mesoscopic organisation and lipid–sterol interactions in condensed monolayers composed of the main species found in the inner leaflet of the erythrocyte membrane. Using a combination of experimental methods (static area compressibility, surface dilatational rheology, fluorescence microscopy, and surface sensitive X-ray techniques) and atomistic molecular dynamics simulations, we show that oxidation of cholesterol to 7-ketocholesterol leads to stiffening of the monolayer (under both static and dynamic conditions), significant changes in the monolayer microdomain organisation, disruption in the van der Waals, electrostatic and hydrophobic interactions between the sterol and the other lipid species, and the lipid membrane hydration. Surface sensitive X-ray techniques reveal that, whilst the molecular packing mode is not significantly affected by cholesterol oxidation in these condensed phases, there are subtle changes in membrane thickness and a significant decrease in the coherence length in monolayers containing 7-ketocholesterol.

The Langmuir monolayer technique and voltammetric analysis were used to investigate the properties of model lipid membranes prepared from dioleoylphosphatidylcholine (DOPC), hexadecaprenol (C80), and their mixtures. Surface pressure-molecular area isotherms, current-voltage characteristics, and membrane conductance-temperature were measured. Molecular area isobars, specific molecular areas, excess free energy of mixing, collapse pressure and collapse area were determined for lipid monolayers. Membrane conductance, activation energy of ion migration across the membrane, and membrane permeability coefficient for chloride ions were determined for lipid bilayers. Hexadecaprenol decreases the activation energy and increases membrane conductance and membrane permeability coefficient. The results of monolayer and bilayer investigations show that some electrical, transport and packing properties of lipid membranes change under the influence of hexadecaprenol. The results indicate that hexadecaprenol modulates the molecular organisation of the membrane and that the specific molecular area of polyprenol molecules depends on the relative concentration of polyprenols in membranes. We suggest that hexadecaprenol modifies lipid membranes by the formation of fluid microdomains. The results also indicate that electrical transmembrane potential can accelerate the formation of pores in lipid bilayers modified by long chain polyprenols.

The fluid mosaic model of the plasma membrane has evolved considerably since its original formulation 30 years ago. Membrane lipids do not form a homogeneous phase consisting of glycerophospholipids (GPLs) and cholesterol, but a mosaic of domains with unique biochemical compositions. Among these domains, those containing sphingolipids and cholesterol, referred to as membrane or lipid rafts, have received much attention in the past few years. Lipid rafts have unique physicochemical properties that direct their organisation into liquid-ordered phases floating in a liquid-crystalline ocean of GPLs. These domains are resistant to detergent solubilisation at 4°C and are destabilised by cholesterol- and sphingolipid-depleting agents. Lipid rafts have been morphologically characterised as small membrane patches that are tens of nanometres in diameter. Cellular and/or exogenous proteins that interact with lipid rafts can use them as transport shuttles on the cell surface. Thus, rafts act as molecular sorting machines capable of co-ordinating the spatiotemporal organisation of signal transduction pathways within selected areas (‘signalosomes’) of the plasma membrane. In addition, rafts serve as a portal of entry for various pathogens and toxins, such as human immunodeficiency virus 1 (HIV-1). In the case of HIV-1, raft microdomains mediate the lateral assemblies and the conformational changes required for fusion of HIV-1 with the host cell. Lipid rafts are also preferential sites of formation for pathological forms of the prion protein (PrPSc) and of the β-amyloid peptide associated with Alzheimer's disease. The possibility of modulating raft homeostasis, using statins and synthetic sphingolipid analogues, offers new approaches for therapeutic interventions in raft-associated diseases.

L'objectif de ce travail a été de comprendre comment les canaux ioniques sont adressés, organisés et régulés dans des domaines spécialisés de la membrane plasmique des cardiomyocytes. Parmi les partenaires des canaux, les protéines MAGUK (Membrane Associated GUanylate Kinase) sont spécialisées dans l'ancrage, l'agrégation et la formation de complexes macromoléculaires à la membrane. J'ai caractérisé pour la première fois au niveau cardiaque une de ces protéines MAGUK, la protéine CASK. CASK est localisée à la membrane latérale des cardiomyocytes et exclue des disques intercalaires, zones de conduction privilégiée dans l'axe longitudinal. À la membrane latérale, la protéine CASK est exprimée au sein du complexe costamérique dystrophine/glycoprotéines. L'inhibition de CASK entraîne l'augmentation du courant sodique INa dans les HEK293 et les myocytes cardiaques. Dans les HEK293, la microscopie à onde évanescente (TIRF) et des expériences de biotinylation ont mis en évidence que cette augmentation du courant INa est associée à une augmentation du nombre de canaux NaV1.5 à la membrane. La microscopie de conductance ionique (SICM) couplée au patch clamp en configuration cellule attachée a permis de montrer que CASK retient les canaux sodiques au niveau des crêtes et prévient leur agrégation en clusters dans les T-tubules. Enfin, l'inhibition de CASK in vivo par une stratégie reposant sur l'utilisation de virus adéno-associés (AAV) est responsable d'un allongement de la durée de dépolarisation ventriculaire et de l'apparition d'une cardiopathie dilatée<br>The aim of the thesis was to understand how ion channels are addressed, organized and regulated in specialized domains of the plasma membrane of cardiac myocytes. Among these partners, the MAGUK proteins (Membrane Associated GUanylate Kinase) are specialized in anchoring, aggregation and clustering of macromolecular complexes at the plasma membrane. In particular, characterized for the first time at the level of the hearth, one of these MAGUK proteins is the CASK protein. CASK is localized at the lateral membrane of cardiomyocytes, but excluded from intercalated disks which are privilege zones of the longitudinal axial conduction. At the lateral membrane, CASK protein is expressed among the costameric dystrophin/glycoproteins complex. CASK inhibition leads to the increase in sodium current density in HEK293 cells and in cardiomyocytes. In HEK293, evanescent wave microscopy (TIRF) and biotinylation experiments pointed out that the INa increase is associated to an increase in the number of NaV1.5 channels at the plasma membrane. Scanning ion conductance microscopy (SICM) coupled to cell-attached patch-clamp has demonstrated that CASK holds together sodium channels at the crest level and prevents their aggregation into clusters in the T-tubules. Finally, inhibition of CASK, in vivo, using an adeno-associated virus strategy resulted to an increase in duration of ventricular depolarization and to the appearance of dilated cardiomyopathy

L'invasion de l'hôte par un pathogène induit une réponse immunitaire innée qui aboutit à l'activation cellulaire, à une réaction inflammatoire et parfois à l'initiation d'une immunité acquise. Des différences fondamentales existent dans l'organisation de la réponse immune innée à un pathogène, d'une cellule , d'un organe, ou d'un individu à l'autre. L'organisation de la réponse immune innée en réponse à l'invasion bactérienne, parasitaire ou fongique nécessitent la coopération du récepteur de type Toll 2 (TLR2) avec TLR1 ou TLR6 et la formation de complexes d'activation dans les radeaux lipidiques, qui déterminent une réponse spécifique et adaptée au pathogène. Cependant, les mécanismes de rapprochement moléculaire et la régulation des voies de signalisation impliqués dans la réponse inflammatoire sont encore mal compris. L'objectif de ce travail était d'investiguer les mécanismes impliqués dans la reconnaissance et la signalisation via TLR2, ainsi que dans la variabilité du phénotype inflammatoire. Par une méthode d'analyse protéomique différentielle, nous avons étudié la composition moléculaire du complexe d'activation et les modifications post-traductionnelles en fonction du dimère de TLR2 engagé. Cette approche a permis d'identifier et caractériser le rôle de la tyrosine kinase Lyn et d'IMPDHII dans la régulation de la signalisation de TLR2. Enfin, dans une approche translationnelle nous avons étudié le rôle d'un polymorphisme du gène IRAK1, dont la protéine est en aval de la majorité des TLRs, dans la variabilité du phénotype chez des patients septiques. Ce travail introduit des perspectives nouvelles sur l'organisation, la régulation et la variabilité de la réponse immunitaire innée<br>Host invasion by micro-organisms induces an innate immune response that leads to cell activation, inflammation and potentially to the initiation of an adaptative response. Major differences are found within the organisation of innate immune responses depending on the pathogen, the invaded cell-type and the host itself. Initiation and organisation of innate immune responses facing bacteria, parasites, or fungi need the cooperation of Toll-like receptor 2 (TLR2) with TLR1 or TLR6, and multimolecular complexes in lipid rafts that determine a specific response. However, our understanding of molecular interactions within lipid rafts remains cryptic. Specifically, little is known regarding the composition of signaling complexes induced by distinct pathogens and the activation mechanisms involved in the initiation and regulation of a potent inflammatory response. The aim of this research project was to investigate the mechanisms involved in recognition and signaling downstream TLR2, and factors that account for variability of the inflammatory phenotype. For this purpose, we established a differential proteomic strategy to identiy the composition of TLR2 activation cluster and post-translationnal modifications of proteins after the recruitment of TLR2 dimers to lipid rafts. This approach enabled us to identify tyrosine-kinase Lyn and IMPDHII as essential regulators of TLR2 signaling. In a translational approach, we studied the effect of an haplotype of IRAKI gene, which codes for a key signaling protein downstream TLRs, on clinical variability in patients with septic shock. Our results provide new insights in ogranisation, regulation and variability of innate imune response

Dans le but d’approfondir les connaissances fondamentales entre la structure des polymères associatifs intramoléculaires (polysavons) et leurs propriétés physico-chimiques en milieux aqueux, trois nouvelles familles de polymères amphiphiles cationiques ont été synthétisées par deux méthodes complémentaires permettant une grande variabilité de structure. Les polymères obtenus sont des poly(méth)acrylamides en peigne avec des groupes latéraux de type ammonium quaternaire portant une chaîne alkyle de taille variable. Une étude du comportement physico-chimique de ces polymères en solution, par viscosimétrie et spectroscopie de fluorescence avec deux sondes aux caractéristiques complémentaires, montre qu’ils présentent des propriétés de polysavons qui varient progressivement avec la structure des polymères amphiphiles étudiés, notamment la longueur de la chaîne alkyle latérale, la taille de l’espaceur entre les deux sites polaires amide et ammonium quaternaire et la masse molaire moyenne en nombre. En parallèle, la tensiométrie a montré que ces polyamphiphiles ont une très faible activité à l’interface eau/air confirmant la prédominance de l’effet hydrophobe, alors que les modèles moléculaires correspondants présentent d’excellentes propriétés tensio-actives. Des films de Langmuir ont ensuite été réalisés dans le cadre de la première étude de cette importance sur des polyamphiphiles cationiques. Dans ce domaine également, la grande variabilité de structure des polymères a permis des observations originales et de dégager de nouvelles relations entre la structure du polymère et les caractéristiques des isothermes de compression obtenues<br>In order to improve the fundamental knowledge of the relationships between the chemical structure of intramolecular associative polymers (polysoaps) and their physical chemical properties in aqueous media, three new families of cationic amphiphilic polymers were obtained by complementary methods offering great structure variability. The corresponding polymers were comb poly(meth)acrylamides with pendant ammonium groups with alkyl side chains of variable lengths. A first investigation of their physical chemical behaviour in aqueous solutions, by viscometry and fluorescence spectrometry with two complementary fluorescent probes, showed that they displayed polysoap properties which varied progressively with their chemical features, in particular the length of the alkyl side chain, the size of the spacer between the two polar amide and ammonium groups and the polymer molecular weight. Tensiometry confirmed the prevailing of the hydrophobic effect by showing that these polymers displayed a very weak activity at the water/air interface although the corresponding molecular models showed excellent tensio-active properties. Langmuir’s films were eventually obtained in the first study of this importance on cationic amphiphilic polymers. Here again, the great structural variability enabled original observations and new structure/properties relationships were obtained for the corresponding compression isotherms

Several disorders including pulmonary hypertension (PH) and heart failure (HF) could lead to right ventricle (RV) hypertrophy and failure. RV failure is one of the most important prognostic factors for morbidity and mortality in these disorders. However, there is still no therapy to prevent the RV hypertrophy in PH. Treatments developed for the left ventricle (LV) failure do not improve the survival in patients with RV failure probably due to the significant differences in the chambers physiology and hemodynamic function. A better understanding of the cellular and molecular mechanisms of RV hypertrophy is needed. Our focus lies into the alterations of cellular microarchitecture that promotes functional changes in Ca2+ handling. Recently our group showed that reorganisation of the transverse-axial tubular system (TATS) in HF are of particular importance for Ca2+ mishandling and contractile impairment of failing cells. Rationale: This study aims to establish the differences in membrane organisation of Ca2+ handling between healthy RV and LV myocytes, and to investigate the remodelling of RV during disease. Specifically, the objectives are: (1) To study the membrane organisation of RV and LV myocytes by revealing the surface topography using Scanning Ion Conductance Microscopy and by studying the TATS using confocal microscopy. (2) To assess the contraction and Ca2+ transients in RV and LV myocytes. (3) To determine the spatial distribution and properties of single L-type Ca2+ channels (LTCC) in RV myocytes using “smart patch clamp” technique. (4) To describe the changes occurring in the RV and LV in the two disease rat models: PH induced by monocrotaline injection and HF induced by chronic myocardial infarction (MI). This thesis showed that in healthy samples the TATS of RV myocytes has a different organization as compared to LV. Two main Ca2+ channels for the excitation-contraction coupling: LTCC and ryanodine receptors (RyR) were studied by immunofluorescence staining. The density of LTCC was lower in RV than in LV myocytes. However, the density of RyR was similar between the chambers. Contraction duration was longer in RV than in LV myocytes. The distribution of functional LTCCs in RV myocytes was uniform along the cell surface, in contrast to LV myocytes, where LTCCs were primarily located in the T-tubules. Secondly, PH rats showed a reduction of the conduction velocity anisotropy throughout the RV as well as prolongation of the refractoriness of the tissue. The hypertrophy of RV myocytes in PH was accompanied by the reduction of the TATS organisation. The amplitude of contraction of RV PH myocytes was higher, the activation of Ca2+ transients was more desynchronised than in controls, and the rate of spontaneous Ca2+ activity was significantly elevated. Functionally, in PH the open probability (Po) of LTCC located in the T-tubules was significantly higher. On the other hand, PH LV myocytes had preserved TATS but still showed prolonged Ca2+ transients that could influence increased refractoriness of LV tissue. Thirdly, by studying RV myocytes from the MI model, a significant hypertrophy was found, accompanied by a reduction of TATS organisation. The study reports a prolongation of Ca2+ transients with more frequent local Ca2+ waves in MI versus control RV myocytes. Higher Po of LTCCs was shown in MI RV myocytes could be associated with the PKA-mediated phosphorylation. In summary, RV myocytes have a lower TATS organisation than LV myocytes probably related to the lower workload of the RV chamber. Consequently, RV myocytes present several differences with LV myocytes, including changes in the Ca2+ handling or a more uniform distribution of LTCC on the membrane. Diseases induce reduction of TATS and Ca2+ mishandling in both chambers. Due to the intrinsic differences of RV versus LV myocytes, the RV could be more prone to pathological events in early stages of the diseases, which should be investigated further.