RESUMO
Neutral lipids (NLs) are apolar oil molecules synthesized in the endoplasmic reticulum bilayer upon diverse biological stimuli. NLs synthesized are released in the hydrophobic core of the bilayer. At a critical concentration, NLs condense by phase separation and nucleate a lipid droplet (LD). After an LD forms, a fraction of NLs can be present in the bilayer but at a concentration below that of the nucleation. Here, we study whether and how the accumulation of NLs alters a lipid bilayer's mechanical properties. In synthetic systems, we found that NLs proffer unusual bilayer stretching capacities, especially in the presence of negatively curved phospholipids. This impact becomes spectacular when an LD is contiguous with the bilayer and supplies it with NLs. The tested NLs markedly decrease the bilayer area expansion modulus and significantly increase lysis tension but had opposite effects on membrane bending rigidity. Our data unveil how NL molecules modify overall membrane mechanics, the alteration of which may be linked to pathologies or anticancer treatments targeting NLs.
Assuntos
Bicamadas Lipídicas , Fosfolipídeos , Retículo Endoplasmático , Gotículas Lipídicas , MembranasRESUMO
Tools for accessing and studying organelles remain underdeveloped. Here, we present a method by which giant organelle vesicles (GOVs) are generated by submitting cells to a hypotonic medium followed by plasma membrane breakage. By this means, GOVs ranging from 3 to over 10 µm become available for micromanipulation. GOVs are made from organelles such as the endoplasmic reticulum, endosomes, lysosomes and mitochondria, or in contact with one another such as giant mitochondria-associated ER membrane vesicles. We measure the mechanical properties of each organelle-derived GOV and find that they have distinct properties. In GOVs procured from Cos7 cells, for example, bending rigidities tend to increase from the endoplasmic reticulum to the plasma membrane. We also found that the mechanical properties of giant endoplasmic reticulum vesicles (GERVs) vary depending on their interactions with other organelles or the metabolic state of the cell. Lastly, we demonstrate GERVs' biochemical activity through their capacity to synthesize triglycerides and assemble lipid droplets. These findings underscore the potential of GOVs as valuable tools for studying the biophysics and biology of organelles.
Assuntos
Retículo Endoplasmático , Membranas Intracelulares , Animais , Chlorocebus aethiops , Células COS , Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Membrana Celular/metabolismo , Mitocôndrias/metabolismo , Organelas/metabolismo , Gotículas Lipídicas/metabolismo , Triglicerídeos/metabolismo , Humanos , Lisossomos/metabolismoRESUMO
Lipid droplets (LDs) store lipids that can be utilized during times of scarcity via autophagic and lysosomal pathways, but how LDs and autophagosomes interact remained unclear. Here, we discovered that the E2 autophagic enzyme, ATG3, localizes to the surface of certain ultra-large LDs in differentiated murine 3T3-L1 adipocytes or Huh7 human liver cells undergoing prolonged starvation. Subsequently, ATG3 lipidates microtubule-associated protein 1 light-chain 3B (LC3B) to these LDs. In vitro, ATG3 could bind alone to purified and artificial LDs to mediate this lipidation reaction. We observed that LC3B-lipidated LDs were consistently in close proximity to collections of LC3B-membranes and were lacking Plin1. This phenotype is distinct from macrolipophagy, but it required autophagy because it disappeared following ATG5 or Beclin1 knockout. Our data suggest that extended starvation triggers a noncanonical autophagy mechanism, similar to LC3B-associated phagocytosis, in which the surface of large LDs serves as an LC3B lipidation platform for autophagic processes.
Assuntos
Autofagia , Gotículas Lipídicas , Animais , Humanos , Camundongos , Autofagossomos/metabolismo , Autofagia/genética , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismoRESUMO
We present a reproducible protocol to prepare droplet-embedded vesicles (DEVs) consisting of an oil droplet embedded within a phospholipid bilayer. This model system mimics a cellular lipid droplet (LD) in physical contact with the endoplasmic reticulum (ER) bilayer. It has the advantage that the lipid composition and the biophysical properties of the droplet and the bilayer are controlled and tunable. DEVs can be used to study LD biogenesis factors and determinants of protein binding between ER and LD interfaces. For complete details on the use and execution of this protocol, please refer to Chorlay and Thiam (2020) and Santinho et al. (2020).
Assuntos
Gotículas Lipídicas/química , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/fisiologia , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Modelos Biológicos , Fosfolipídeos/metabolismoRESUMO
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.
Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Gotículas Lipídicas/metabolismo , Triglicerídeos/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HeLa , HumanosRESUMO
Seipin is an oligomeric integral endoplasmic reticulum (ER) protein involved in lipid droplet (LD) biogenesis. To study the role of seipin in LD formation, we relocalized it to the nuclear envelope and found that LDs formed at these new seipin-defined sites. The sites were characterized by uniform seipin-mediated ER-LD necks. At low seipin content, LDs only grew at seipin sites, and tiny, growth-incompetent LDs appeared in a Rab18-dependent manner. When seipin was removed from ER-LD contacts within 1 h, no lipid metabolic defects were observed, but LDs became heterogeneous in size. Studies in seipin-ablated cells and model membranes revealed that this heterogeneity arises via a biophysical ripening process, with triglycerides partitioning from smaller to larger LDs through droplet-bilayer contacts. These results suggest that seipin supports the formation of structurally uniform ER-LD contacts and facilitates the delivery of triglycerides from ER to LDs. This counteracts ripening-induced shrinkage of small LDs.