Au cours de ces dernières années, des études ont montré l’existence d’une compartimentation latéraledes composants de la membrane plasmique végétale, de manière analogue à ce qui avait été montréchez les animaux et les levures. L’objectif de cette thèse était d’apporter de nouveaux éléments decaractérisation de cette compartimentation (propriétés physiques de domaines particuliers, mécanismesde mise en place de ces domaines, de contrôle de leur taille, etc…) et d’étudier son rôle dans laphysiologie de la cellule végétale.Le développement d’une méthodologie de microscopie confocale spectrale couplée à l’utilisationd’une sonde environnementale a permis d’apporter la première description à l'échellesubmicrométrique de l’organisation du plasmalemme en territoires aux propriétés physiquesdifférentiées. Ces domaines coexistent au sein de la membrane plasmique de cellules en suspension,comme à celle de membranes artificielles composées de lipides modèles ou de lipides de membranescellulaires, de vésicules géantes constituées de membrane plasmique purifiée, ou de protoplastes.Cependant, les différences de l’organisation latérale observées chez ces différentes membranes ontpermis de montrer l’importance des phytostérols qui seraient, par le biais d'interactions spécifiquesavec d’autres lipides végétaux tels que les GIPCs, des composés essentiels pour la formation locale dedomaines lipidiques ordonnés. La grande diversité des lipides végétaux organiserait ainsi lacompartimentation de la membrane plasmique permettant la ségrégation dynamique des composantsmembranaires. Si les stérols augmentent de manière importante le degré de compaction de la bicouche,les protéines le diminuent. Le cytosquelette et la paroi ne semblent, quant à eux, modifier ni laprésence, ni l’organisation des domaines ordonnés de la membrane plasmique. Nous avons égalementmontré que l’organisation de ces domaines évolue transitoirement lors des étapes précoces de lacascade de signalisation induite par des réactions de défense. De fait, nous avons identifié desmodifications des propriétés physiques globales et de l’organisation fine de la membrane provoquéespar différents éliciteurs de réactions de défense, dont la cryptogéine, une protéine sécrétée parl’oomycète Phytophthora cryptogea. Nous avons montré que ces modifications sont un élémentgénérique de la signalisation de défense, sous la dépendance de phénomènes de phosphorylation, leburst oxydatif étant également une étape clé de l’augmentation du degré d’ordre observé dans lesphases précoces de cette signalisation. La cryptogéine, qui présente une aptitude singulière pour piégerles stérols, a également montré une capacité spécifique à augmenter la fluidité membranaire, ceparamètre pouvant contrôler l’intensité de la cascade de signalisation, mesurée par la production deformes actives d’oxygène.Ces résultats ouvrent de nouvelles perspectives dans la compréhension des interactions cellule-élicitineet apportent un nouvel éclairage sur le rôle des lipides végétaux dans l’organisation latérale de lamembrane plasmique végétale et positionne la dynamique membranaire comme un élément designalisation de défense des plantes. / Recent studies have shown the existence of lateral sub-compartmentalization of plant plasmamembrane similar to that of animal cells and yeasts. The aim of this thesis was to provide newelements to characterize this compartmentalization (physical properties of specific domains,mechanisms of their formation, determination of their size, etc...) and to study its role in thephysiology of plant cells.The development spectral confocal microscopy coupled with the use of an environment-sensitiveprobe enabled to obtain the first description at the submicron scale of plasma membrane organizationinto domains exhibiting various physical properties. These domains coexist at the plasma membranesurface of tobacco suspension cells as well as the membrane of vesicles composed of models lipids orcell plasma membrane lipids, purified plasma membrane vesicles, and protoplasts. However,differences in the lateral organization observed in these membranes have shown the importance ofphytosterols which are, through specific interactions with neighboring plant lipids such as GIPCs,essential for local formation of ordered domains. The huge diversity of plant lipids drives thecompartmentalization of the plasma membrane allowing the dynamic segregation of membranecomponents. Sterols greatly increase membrane order, whereas proteins tends to decrease it.Cytoskeleton and cell wall do alter neither presence nor organization of ordered domains of the plasmamembrane. We have also shown that the organization of these domains is transiently modified duringthe early stages of defense signaling cascade. In fact, we have identified changes in overall physicalproperties and fine lateral organization of the membrane caused by various elicitors of defensereactions, including cryptogein, a protein secreted by the oomycete Phytophthora cryptogea. We haveshown that these changes are a generic element of defense signaling cascade and depend onphosphorylation processes; oxidative burst being also a major actor of the control of the increase ofmembrane order observed during the very early stages of the signalling process. Cryptogein, whichexhibits the particular ability to trap sterols, also showed a specific capacity to increase membranefluidity and amplify the intensity of the signalling cascade, as measured by the production of reactiveoxygen species.These results open new perspectives in the understanding of cell-elicitin interactions and provide anew view on the central role of sterol composition in the lateral organization of plant plasmamembrane. They also identify membrane dynamics as a new player in the signalling cascade occurringduring plant defense.

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 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.