ER membrane phospholipids and surface tension control cellular lipid droplet formation

Cells convert excess energy into neutral lipids that are made in the endoplasmic reticulum (ER) bilayer. The lipids are then packaged into spherical or budded lipid droplets (LDs) covered by a phospholipid monolayer containing proteins. LDs play a key role in cellular energy metabolism and homeostasis. A key unanswered question in the life of LDs is how they bud off from the ER. Here, we tackle this question by studying the budding of artificial LDs from model membranes. We find that the bilayer phospholipid composition and surface tension are key parameters of LD budding. Phospholipids have differential LD budding aptitudes, and those inducing budding decrease the bilayer tension. We observe that decreasing tension favors the egress of neutral lipids from the bilayer and LD budding. In cells, budding conditions favor the formation of small LDs. Our discovery reveals the importance of altering ER physical chemistry for controlled cellular LD formation

Adhesion and phagocytosis of functionalized emulsion droplets

La phagocytose par les macrophages est un processus biologique essentiel au système immunitaire et joue un rôle clé dans le maintien de l’homéostasie cellulaire. Les cibles à éliminer varient en terme de tailles, des bactéries (µm) aux cellules cancéreuses ou senescentes (10 – 20 µm). La plupart des études quantitatives sur la phagocytose reposent sur l'utilisation de microparticules de polymère rigides en tant que cibles modèles pour la compréhension des paramètres qui régissent ce processus. Cependant, ces particules ne rendent pas compte de la mobilité latérale des ligands à leur surface malgré la pertinence de ce paramètre dans le contexte immunologique. Cette étude a pour but de synthétiser un matériau biomimétique qui constitue un nouveau système modèle pour l’étude de la phagocytose. Il s’agit de gouttes d’émulsions monodisperses fonctionnalisées avec des IgGs libres de diffuser sur toute la surface. Dans le cadre de cette thèse, nous avons obtenu différentes densités contrôlées de fonctionnalisation, caractérisées de façon quantitative. Ainsi, il nous a été possible de déterminer une densité minimale d’IgGs nécessaire à induire une internalisation efficace des gouttes. Pour des densités supérieures d’IgGs, ces gouttes sont efficacement et spécifiquement internalisées par phagocytose induite par les récepteurs FcR in vitro. Nous avons plus précisément cherché à approfondir la compréhension de certains aspects mécaniques. Nous montrons que, contrairement à des billes de polymères solides, l’internalisation des gouttes est efficace même pour de faibles densités d’IgGs. La phagocytose s’accompagne, pendant la phase d’adhésion à la surface du macrophage, d’une mobilité latérale des ligands en zone de contact. Il apparaît donc que la mobilité latérale des protéines à l'interface d'une cible améliore considérablement sa phagocytose par les macrophages. Ainsi, ces gouttes permettrons d’aborder de nouvelles questions biologiques pour approfondir certaines mécanismes moléculaires et/ou mécaniques.Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. The size of targets to be eliminated ranges from small particles as bacteria to large objects as cancerous or senescent cells. Most of our current quantitative knowledge on phagocytosis is based on the use of solid polymer microparticles as model targets that are well adapted to the study of phagocytosis mechanisms that do not involve any lateral mobility of the ligands, despite the relevance of this parameter in the immunological context. The aim of this study is to synthesize a biomimetic material that constitutes a new model system for the study of phagocytosis. We designed monodisperse, lateraly mobile IgG-coated emulsion droplets, with different controlled densities of IgGs, that are efficiently and specifically internalized by macrophages through in-vitro FcγR-mediated phagocytosis. The excellent control of the opsonization density allowed us to measure the minimal IgGs density required to induce an efficient internalization. We also attempted to deepen the understanding of certain mechanical aspects. We show that, contrary to solid polymeric beads, droplet uptake is high even for low IgG densities and is accompagnied by the clustering of the opsonins in the zone of contact with the macrophage during the adhesion step. Beyond the sole interest in the design of the material, our results suggest that lateral mobility of proteins at the interface of a target greatly enhances the phagocytic uptake. Thus, emulsion droplets constitute a new interesting target to investigate different biological issues and understand molecular and/or mechanical mechansims

Adhésion et phagocytose de gouttes d'émulsions fonctionnalisées

Phagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. The size of targets to be eliminated ranges from small particles as bacteria to large objects as cancerous or senescent cells. Most of our current quantitative knowledge on phagocytosis is based on the use of solid polymer microparticles as model targets that are well adapted to the study of phagocytosis mechanisms that do not involve any lateral mobility of the ligands, despite the relevance of this parameter in the immunological context. The aim of this study is to synthesize a biomimetic material that constitutes a new model system for the study of phagocytosis. We designed monodisperse, lateraly mobile IgG-coated emulsion droplets, with different controlled densities of IgGs, that are efficiently and specifically internalized by macrophages through in-vitro FcγR-mediated phagocytosis. The excellent control of the opsonization density allowed us to measure the minimal IgGs density required to induce an efficient internalization. We also attempted to deepen the understanding of certain mechanical aspects. We show that, contrary to solid polymeric beads, droplet uptake is high even for low IgG densities and is accompagnied by the clustering of the opsonins in the zone of contact with the macrophage during the adhesion step. Beyond the sole interest in the design of the material, our results suggest that lateral mobility of proteins at the interface of a target greatly enhances the phagocytic uptake. Thus, emulsion droplets constitute a new interesting target to investigate different biological issues and understand molecular and/or mechanical mechansims.La phagocytose par les macrophages est un processus biologique essentiel au système immunitaire et joue un rôle clé dans le maintien de l’homéostasie cellulaire. Les cibles à éliminer varient en terme de tailles, des bactéries (µm) aux cellules cancéreuses ou senescentes (10 – 20 µm). La plupart des études quantitatives sur la phagocytose reposent sur l'utilisation de microparticules de polymère rigides en tant que cibles modèles pour la compréhension des paramètres qui régissent ce processus. Cependant, ces particules ne rendent pas compte de la mobilité latérale des ligands à leur surface malgré la pertinence de ce paramètre dans le contexte immunologique. Cette étude a pour but de synthétiser un matériau biomimétique qui constitue un nouveau système modèle pour l’étude de la phagocytose. Il s’agit de gouttes d’émulsions monodisperses fonctionnalisées avec des IgGs libres de diffuser sur toute la surface. Dans le cadre de cette thèse, nous avons obtenu différentes densités contrôlées de fonctionnalisation, caractérisées de façon quantitative. Ainsi, il nous a été possible de déterminer une densité minimale d’IgGs nécessaire à induire une internalisation efficace des gouttes. Pour des densités supérieures d’IgGs, ces gouttes sont efficacement et spécifiquement internalisées par phagocytose induite par les récepteurs FcR in vitro. Nous avons plus précisément cherché à approfondir la compréhension de certains aspects mécaniques. Nous montrons que, contrairement à des billes de polymères solides, l’internalisation des gouttes est efficace même pour de faibles densités d’IgGs. La phagocytose s’accompagne, pendant la phase d’adhésion à la surface du macrophage, d’une mobilité latérale des ligands en zone de contact. Il apparaît donc que la mobilité latérale des protéines à l'interface d'une cible améliore considérablement sa phagocytose par les macrophages. Ainsi, ces gouttes permettrons d’aborder de nouvelles questions biologiques pour approfondir certaines mécanismes moléculaires et/ou mécaniques

Phagocytosis of immunoglobulin-coated emulsion droplets

International audiencePhagocytosis by macrophages represents a fundamental process essential for both immunity and tissue homeostasis. The size of targets to be eliminated ranges from small particles as bacteria to large objects as cancerous or senescent cells. Most of our current quantitative knowledge on phagocytosis is based on the use of solid polymer microparticles as model targets that are well adapted to the study of phagocytosis mechanisms that do not involve any lateral mobility of the ligands, despite the relevance of this parameter in the immunological context. Herein we designed monodisperse, IgG-coated emulsion droplets that are efficiently and specifically internalized by macrophages through in-vitro FcγR-mediated phagocytosis. We show that, contrary to solid polymeric beads, droplet uptake is efficient even for low IgG densities, and is accompagnied by the clustering of the opsonins in the zone of contact with the macrophage during the adhesion step. Beyond the sole interest in the design of the material, our results suggest that lateral mobility of proteins at the interface of a target greatly enhances the phagocytic uptake

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one perilipin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific perilipins present on its LDs. Despite their early discovery, exactly how perilipins target LDs and why they displace each other in a ‘hierarchical’ manner remains unclear. They all share an amino-terminal 11-mer repeat amphipathic region suggested to be involved in LD targeting. Here, we show that in vivo this domain functions as a primary highly reversible LD targeting motif in perilipins1-3 and, in vitro, we document reversible and competitive binding between a wildtype purified perilipin1 11-mer repeat peptide and a mutant with reduced binding affinity to both ‘naked’ and phospholipid coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in perilipin1 more effectively than in perilipin2, whereas in perilipin3 it weakens binding. These findings suggest that dual amphipathic helical regions mediat

Dual binding motifs underpin the hierarchical association of perilipins1-3 with lipid droplets.

Lipid droplets (LDs) in all eukaryotic cells are coated with at least one of the perilipin (Plin) family of proteins. They all regulate key intracellular lipases but do so to significantly different extents. Where more than one Plin is expressed in a cell, they associate with LDs in a hierarchical manner. In vivo, this means that lipid flux control in a particular cell or tissue type is heavily influenced by the specific Plins present on its LDs. Despite their early discovery, exactly how Plins target LDs and why they displace each other in a "hierarchical" manner remains unclear. They all share an amino-terminal 11-mer repeat (11mr) amphipathic region suggested to be involved in LD targeting. Here, we show that, in vivo, this domain functions as a primary highly reversible LD targeting motif in Plin1-3, and, in vitro, we document reversible and competitive binding between a wild-type purified Plin1 11mr peptide and a mutant with reduced binding affinity to both "naked" and phospholipid-coated oil-water interfaces. We also present data suggesting that a second carboxy-terminal 4-helix bundle domain stabilizes LD binding in Plin1 more effectively than in Plin2, whereas it weakens binding in Plin3. These findings suggest that dual amphipathic helical regions mediate LD targeting and underpin the hierarchical binding of Plin1-3 to LDs