The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.</p>

The plasma membrane of eukaryotic cells contains cholesterol and glycosphingolipids enriched nanodomains known as lipid rafts; which are believed to exist in a liquid ordered (lo) state. Methyl-beta-cyclodextrin (MBCD) is used to deplete cellular cholesterol and a widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyze this in T cells a progressive cholesterol extraction protocols was established. At 37ºC, MBCD treatment does not lead to the preferential loss of cholesterol from TX-DRMs. At 0ºC only 35% of total cholesterol could be extracted demonstrating that less than 35% of the cell’s cholesterol is found in the plasma membrane. Moreover, incubation of cells at 0ºC causes loss of plasma membrane cholesterol and an increase in cholesteryl esters. The increase in cholesterol esters upon cold exposure is linked to the cholesterol concentration induced activation of ACAT enzyme which converts cholesterol to cholesteryl esters. Cholesterol concentration specific activation of ACAT and conversion of cholesterol to cholesteryl esters during the loading of cholesterol onto T cells by MBCD was also observed. By using MBCD for progressive cholesterol depletion from T cells at 37ºC, the effect of cholesterol depletion on T cell signaling was addressed. At 10-20% cholesterol depletion levels, tyrosine phosphorylation is increased and ERK is activated. Peripheral actin polymerization, cell spreading and membrane protrusions are also triggered by limited cholesterol depletion. Upon limited cholesterol depletion aggregation of lipid rafts in the plasma membrane was observed. The aggregation of lipid rafts upon cholesterol depletion does not dependent on the signaling proteins such as Src-kinases. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press.</p>

The plasma membrane of eukaryotic cells contains cholesterol and glycosphingolipids enriched nanodomains known as lipid rafts; which are believed to exist in a liquid ordered (lo) state. Methyl-beta-cyclodextrin (MBCD) is used to deplete cellular cholesterol and a widespread assumption is that MBCD preferentially targets cholesterol in lipid rafts. To analyze this in T cells a progressive cholesterol extraction protocols was established. At 37ºC, MBCD treatment does not lead to the preferential loss of cholesterol from TX-DRMs. At 0ºC only 35% of total cholesterol could be extracted demonstrating that less than 35% of the cell’s cholesterol is found in the plasma membrane. Moreover, incubation of cells at 0ºC causes loss of plasma membrane cholesterol and an increase in cholesteryl esters. The increase in cholesterol esters upon cold exposure is linked to the cholesterol concentration induced activation of ACAT enzyme which converts cholesterol to cholesteryl esters. Cholesterol concentration specific activation of ACAT and conversion of cholesterol to cholesteryl esters during the loading of cholesterol onto T cells by MBCD was also observed. By using MBCD for progressive cholesterol depletion from T cells at 37ºC, the effect of cholesterol depletion on T cell signaling was addressed. At 10-20% cholesterol depletion levels, tyrosine phosphorylation is increased and ERK is activated. Peripheral actin polymerization, cell spreading and membrane protrusions are also triggered by limited cholesterol depletion. Upon limited cholesterol depletion aggregation of lipid rafts in the plasma membrane was observed. The aggregation of lipid rafts upon cholesterol depletion does not dependent on the signaling proteins such as Src-kinases. Upon cholesterol depletion there is an increase in overall plasma membrane order, indicative of more ordered domains forming at the expense of disordered domains. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: In press.

No description available.

The connection between T cell activation, plasma membrane order and actin filament dynamics was the main focus of this study. Laurdan and di-4-ANEPPDHQ, membrane order sensing probes, were shown to report only on lipid packing rather than being influenced by the presence of membrane-inserted peptides justifying their use in membrane order studies. These dyes were used to follow plasma membrane order in live cells at 37°C. Disrupting actin filaments had a disordering effect while stabilizing actin filaments had an ordering effect on the plasma membrane, indicating there is a basal level of ordered domains in resting cells. Lowering PI(4,5)P2 levels decreased the proportion of ordered domains strongly suggesting that the connection of actin filaments to the plasma membrane is responsible for the maintaining the level of ordered membrane domains. Membrane blebs, which are detached from the underlying actin filaments, contained a low fraction of ordered domains. Aggregation of membrane components resulted in a higher proportion of ordered plasma membrane domains and an increase in cell peripheral actin polymerization. This strongly suggests that the attachment of actin filaments to the plasma membrane induces the formation of ordered domains. Limited cholesterol depletion with methyl-beta-cyclodextrin triggered peripheral actin polymerization. Cholesterol depleted cells showed an increase in plasma membrane order as a result of actin filament accumulation underneath the membrane. Moderate cholesterol depletion also induced membrane domain aggregation and activation of T cell signaling events. The T cell receptor (TCR) aggregation caused redistribution of domains resulting in TCR patches of higher order and the bulk membrane correspondingly depleted of ordered domains. This suggests the preexistence of small ordered membrane domains in resting T cells that aggregate upon cell activation. Increased actin polymerization at the TCR aggregation sites showed that actin polymerization is strongly correlated with the changes in the distribution of ordered domains. The distribution of the TCR in resting cells and its colocalization with actin filaments is cell cycle dependent. We conclude that actin filament attachment to the plasma membrane, which is regulated via PI(4,5)P2, plays a crucial role in the formation of ordered domains. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 4: Manuscript.

Les cellules Natural Killer (NK) sont des lymphocytes du système immunitaire inné capables de tuer des cellules cibles et de produire des cytokines telles que l'interféron-&#947;. Au cours de mon travail de thèse, j'ai utilisé des approches de génétique directe et inverse dans le but d'étudier les mécanismes impliqués dans la régulation des capacités effectrices des cellules NK. La tolérance des cellules NK au soi est en partie assurée par les récepteurs inhibiteurs de surface qui sont spécifiques des molécules du complexe majeur d'histocompatibilité de classe I (CMH-I) exprimées par les cellules du soi. Cependant, des cellules NK qui ne sont pas capables de détecter l'expression du CMH-I ne sont pas autoréactives. Dans la première partie de ce travail de thèse, nous avons cherché à déterminer, chez la souris, les mécanismes de la tolérance NK, indépendante de la reconnaissance du CMH-I, qui est associée à une hyporeactivité des cellules NK. En utilisant des techniques de spectrométrie de fluorescence par corrélation à spot variable (svFCS), nous avons montré que dans les cellules NK hyporéactives les récepteurs activateurs et inhibiteurs sont confinés à la membrane plasmique par des réseaux structurés d'actine. A l'inverse, la reconnaissance par les cellules NK du CMH-I, qui « éduque » les cellules NK pour qu'elles acquièrent leurs capacités effectrices maximales, est associée une relocalisation des récepteurs activateurs au sein de nanodomaines. Ces résultats suggèrent que ce serait le confinement particulier des récepteurs activateurs à la membrane des cellules NK qui assure la tolérance au soi. / Natural killer (NK) cells are bone marrow-derived innate immune lymphocytes able to kill cellular targets and secrete cytokines such as interferon-&#947;. During my PhD work, I used reverse and forward genetic approaches to dissect the mechanisms involved in the regulation of NK cell effector capabilities at steady state. NK cell tolerance to self is partly ensured by major histocompatibility complex class I (MHC- I)-specific inhibitory receptors on NK cells, which detect MHC-I expression on self-cells and prevent NK cell activation. However, NK cells that do not detect self MHC-I are not autoreactive. In the first part of this PhD work, we sought to determine the mechanism at the basis of this MHC-I independent NK cell tolerance. Using spot variation fluorescence correlation spectroscopy (svFCS), we showed that MHC-I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC-I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We thus propose that the confinement of activating receptors at the plasma membrane is essential to ensuring self-tolerance of NK cells.

No description available.

No description available.