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

Membrane Curvature Catalyzes Lipid Droplet Assembly

Lipid droplet (LD) biogenesis begins in the endoplasmic reticulum (ER) bilayer, but how the ER topology impacts this process is unclear. An early step in LD formation is nucleation, wherein free neutral lipids, mainly triacylglycerols (TGs) and sterol esters (SEs), condense into a nascent LD. How this transition occurs is poorly known. Here, we found that LDs preferably assemble at ER tubules, with higher curvature than ER sheets, independently of the LD assembly protein seipin. Indeed, the critical TG concentration required for initiating LD assembly is lower at curved versus flat membrane regions. In agreement with this finding, flat ER regions bear higher amounts of free TGs than tubular ones and present less LDs. By using an in vitro approach, we discovered that the presence of free TGs in tubules is energetically unfavorable, leading to outflow of TGs to flat membrane regions or condensation into LDs. Accordingly, in vitro LD nucleation can be achieved by the sole increase of membrane curvature. In contrast to TGs, the presence of free SEs is favored at tubules and increasing SE levels is inhibitory to the curvature-induced nucleation of TG LDs. Finally, we found that seipin is enriched at ER tubules and controls the condensation process, preventing excessive tubule-induced nucleation. The absence of seipin provokes erratic LD nucleation events determined by the abundance of ER tubules. In summary, our data indicate that membrane curvature catalyzes LD assembly.Peer reviewe

Physique de la biogenèse des corps lipidiques

Cells store excess energy in the form of neutral lipids that are synthesized and encapsulated within the inter-monolayer space of the endoplasmic reticulum (ER). Then these neutral lipids demix to form a new organelle named lipid droplets (LDs), which, surprisingly, bud off mostly toward the cytosol where they regulate metabolism and multiple biological processes. The purpose of this thesis is to investigate LD directional biogenesis through soft matter physics. We reconstituted LD formation topology by embedding artificial LDs into the inter-monolayer space of bilayer vesicles. We provide evidence that the droplet behavior in the membrane is recapitulated by the physics of three-phase wetting systems, controlled by the equilibrium of surface tensions. More precisely, slight tension asymmetries between the membrane monolayers regulate the droplet budding side. We then investigated how cells are able to impose the required tension asymmetry and found that model LDs emerge on the membrane leaflet of higher coverage resulting from the insertion of both proteins and phospholipids. In cells, continuous LD emergence on the cytosol would require a constant refill of phospholipids to the ER’s cytosolic leaflet. Consistent with this model, cells deficient in phospholipids present an increased number of LDs exposed to the ER lumen and compensate the deficiency by remodeling the ER’s shape. Our results reveal an active cooperation between phospholipids and proteins to extract LDs from the ER.LDs have several biological implications determined by proteins specifically targeted to their surface, mostly through amphipathic helix (AH) motifs. How such specificity is achieved remains elusive. Our results support that neutral lipids play a key role in determining AH binding specificity, and phospholipid packing simply modulates the amount of accessible surface. These findings help to understand how AHs bind to specific LD populations. In summary, our work offers new insights on the mechanisms regulating LDs biogenesis by using well-known soft matter physics tools.En présence de surplus d’énergie, les cellules synthétisent des lipides neutres dans la bicouche du réticulum endoplasmique (RE). Ils démixent ensuite pour former de nouvelles organelles, les corps lipidiques (CLs), qui émergent principalement dans le cytoplasme et régulent le métabolisme énergétique et de nombreux processus cellulaires. Cette thèse étudie les mécanismes de cette biogénèse directionnelle des CLs à l’aide de la physique des émulsions. Pour cela, nous avons développé un système in vitro reproduisant la topologie des CLs en formation et avons montré que le comportement du CL dans une membrane était régi par un équilibre de tension de surface comme dans un système de mouillage à trois phases. La directionnalité de l’émergence des CLs est régulée par une asymétrie de tension entre les feuillets de la membrane. Nous avons ensuite essayé de comprendre comment la cellule pouvait imposer une telle différence de tension et avons découvert que les CLs artificiels émergeaient du feuillet présentant la meilleure couverture résultant de l’insertion de protéines ou de phospholipides. In vivo, l’émergence de CLs dépeuple le feuillet cytosolique du RE de ses phospholipides qui doivent être constamment remplacés pour assurer la bonne émergence des futurs CLs dans le cytoplasme. En accord avec cette hypothèse, des cellules manquant de phospholipides présentent de plus nombreux CLs en contact avec le lumen et un RE remodelé. Nos résultats révèlent une coopération active entre phospholipides et protéines pour extraire les CLs du RE. Les fonctions cellulaires des CLs sont essentiellement déterminées par leur protéome de surface. Les protéines y sont souvent recrutées spécifiquement à l’aide d’hélices amphipathiques (HAs) selon des mécanismes peu connus. Nos résultats montrent que les lipides neutres jouent un rôle prépondérant dans le recrutement spécifique des HAs et que la densité des phopholipides module simplement l’accès des HAs aux lipides neutres. Cette découverte permet de comprendre le recrutement de certaines HAs sur des sous-populations spécifiques de CLs. Cette thèse illustre comment la physique des émulsions peut apporter une compréhension originale des mécanismes régulant la biogenèse des CLs

Physics of lipid droplet biogenesis

En présence de surplus d’énergie, les cellules synthétisent des lipides neutres dans la bicouche du réticulum endoplasmique (RE). Ils démixent ensuite pour former de nouvelles organelles, les corps lipidiques (CLs), qui émergent principalement dans le cytoplasme et régulent le métabolisme énergétique et de nombreux processus cellulaires. Cette thèse étudie les mécanismes de cette biogénèse directionnelle des CLs à l’aide de la physique des émulsions. Pour cela, nous avons développé un système in vitro reproduisant la topologie des CLs en formation et avons montré que le comportement du CL dans une membrane était régi par un équilibre de tension de surface comme dans un système de mouillage à trois phases. La directionnalité de l’émergence des CLs est régulée par une asymétrie de tension entre les feuillets de la membrane. Nous avons ensuite essayé de comprendre comment la cellule pouvait imposer une telle différence de tension et avons découvert que les CLs artificiels émergeaient du feuillet présentant la meilleure couverture résultant de l’insertion de protéines ou de phospholipides. In vivo, l’émergence de CLs dépeuple le feuillet cytosolique du RE de ses phospholipides qui doivent être constamment remplacés pour assurer la bonne émergence des futurs CLs dans le cytoplasme. En accord avec cette hypothèse, des cellules manquant de phospholipides présentent de plus nombreux CLs en contact avec le lumen et un RE remodelé. Nos résultats révèlent une coopération active entre phospholipides et protéines pour extraire les CLs du RE. Les fonctions cellulaires des CLs sont essentiellement déterminées par leur protéome de surface. Les protéines y sont souvent recrutées spécifiquement à l’aide d’hélices amphipathiques (HAs) selon des mécanismes peu connus. Nos résultats montrent que les lipides neutres jouent un rôle prépondérant dans le recrutement spécifique des HAs et que la densité des phopholipides module simplement l’accès des HAs aux lipides neutres. Cette découverte permet de comprendre le recrutement de certaines HAs sur des sous-populations spécifiques de CLs. Cette thèse illustre comment la physique des émulsions peut apporter une compréhension originale des mécanismes régulant la biogenèse des CLs.Cells store excess energy in the form of neutral lipids that are synthesized and encapsulated within the inter-monolayer space of the endoplasmic reticulum (ER). Then these neutral lipids demix to form a new organelle named lipid droplets (LDs), which, surprisingly, bud off mostly toward the cytosol where they regulate metabolism and multiple biological processes. The purpose of this thesis is to investigate LD directional biogenesis through soft matter physics. We reconstituted LD formation topology by embedding artificial LDs into the inter-monolayer space of bilayer vesicles. We provide evidence that the droplet behavior in the membrane is recapitulated by the physics of three-phase wetting systems, controlled by the equilibrium of surface tensions. More precisely, slight tension asymmetries between the membrane monolayers regulate the droplet budding side. We then investigated how cells are able to impose the required tension asymmetry and found that model LDs emerge on the membrane leaflet of higher coverage resulting from the insertion of both proteins and phospholipids. In cells, continuous LD emergence on the cytosol would require a constant refill of phospholipids to the ER’s cytosolic leaflet. Consistent with this model, cells deficient in phospholipids present an increased number of LDs exposed to the ER lumen and compensate the deficiency by remodeling the ER’s shape. Our results reveal an active cooperation between phospholipids and proteins to extract LDs from the ER.LDs have several biological implications determined by proteins specifically targeted to their surface, mostly through amphipathic helix (AH) motifs. How such specificity is achieved remains elusive. Our results support that neutral lipids play a key role in determining AH binding specificity, and phospholipid packing simply modulates the amount of accessible surface. These findings help to understand how AHs bind to specific LD populations. In summary, our work offers new insights on the mechanisms regulating LDs biogenesis by using well-known soft matter physics tools

An Asymmetry in Monolayer Tension Regulates Lipid Droplet Budding Direction

International audienceCells store excess energy in the form of neutral lipids that are synthesized and encapsulated within the endoplasmic reticulum intermonolayer space. The lipids next demix to form lipid droplets (LDs), which, surprisingly, bud off mostly toward the cytosol. This directional LD formation is critical to energy metabolism, but its mechanism remains poorly understood. Here, we reconstituted the LD formation topology by embedding artificial LDs into the intermonolayer space of bilayer vesicles. We provide experimental evidence that the droplet behavior in the membrane is recapitulated by the physics of three-phase wetting systems, dictated by the equilibrium of surface tensions. We thereupon determined that slight tension asymmetries between the membrane monolayers regulate the droplet budding side. A differential regulation of lipid or protein composition around a forming LD can generate a monolayer tension asymmetry that will determine the LD budding side. Our results offer, to our knowledge, new insights on how the proteins might regulate LD formation side by generating a monolayer tension asymmetry.Cells store excess energy in the form of neutral lipids that are synthesized and encapsulated within the endoplasmic reticulum intermonolayer space. The lipids next demix to form lipid droplets (LDs), which, surprisingly, bud off mostly toward the cytosol. This directional LD formation is critical to energy metabolism, but its mechanism remains poorly understood. Here, we reconstituted the LD formation topology by embedding artificial LDs into the intermonolayer space of bilayer vesicles. We provide experimental evidence that the droplet behavior in the membrane is recapitulated by the physics of three-phase wetting systems, dictated by the equilibrium of surface tensions. We thereupon determined that slight tension asymmetries between the membrane monolayers regulate the droplet budding side. A differential regulation of lipid or protein composition around a forming LD can generate a monolayer tension asymmetry that will determine the LD budding side. Our results offer, to our knowledge, new insights on how the proteins might regulate LD formation side by generating a monolayer tension asymmetry

Neutral lipids regulate amphipathic helix affinity for model lipid droplets

International audienceCellular lipid droplets (LDs) have a neutral lipid core shielded from the aqueous environment by a phospholipid monolayer containing proteins. These proteins define the biological functions of LDs, and most of them bear amphipathic helices (AH), which can selectively target to LDs, or to LD subsets. How such binding preference happens remains poorly understood. Here, we found that artificial LDs made of different neutral lipids but presenting equal phospholipid packing densities differentially recruit AHs. Varying the phospholipid density shifts the binding levels, but the differential recruitment is unchanged. We found that the binding level of AHs is defined by their interaction preference with neutral lipids and ability to decrease surface tension. The phospholipid packing level regulates mainly the amount of neutral lipid accessible. Therefore, it is the hydrophobic nature of the phospholipid packing voids that controls the binding level of AHs. Our data bring us a major step closer to understanding the binding selectivity of AHs to lipid membranes

Making Droplet-Embedded Vesicles to Model Cellular Lipid Droplets

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Mechanisms of protein targeting to lipid droplets: A unified cell biological and biophysical perspective

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Membrane Asymmetry Imposes Directionality on Lipid Droplet Emergence from the ER

International audienceDuring energy bursts, neutral lipids fabricated within the ER bilayer demix to form lipid droplets (LDs). LDs bud off mainly in the cytosol where they regulate metabolism and multiple biological processes. They indeed become accessible to most enzymes and can interact with other organelles. How such directional emergence is achieved remains elusive. Here, we found that this directionality is controlled by an asymmetry in monolayer surface coverage. Model LDs emerge on the membrane leaflet of higher coverage, which is improved by the insertion of proteins and phospholipids. In cells, continuous LD emergence on the cytosol would require a constant refill of phospholipids to the ER cytosolic leaflet. Consistent with this model, cells deficient in phospholipids present an increased number of LDs exposed to the ER lumen and compensate by remodeling ER shape. Our results reveal an active cooperation between phospholipids and proteins to extract LDs from ER