Giant organelle vesicles to uncover intracellular membrane mechanics and plasticity.

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.

Perilipin membrane integration determines lipid droplet heterogeneity in differentiating adipocytes.

The storage of fat within lipid droplets (LDs) of adipocytes is critical for whole-body health. Acute fatty acid (FA) uptake by differentiating adipocytes leads to the formation of at least two LD classes marked by distinct perilipins (PLINs). How this LD heterogeneity arises is an important yet unresolved cell biological problem. Here, we show that an unconventional integral membrane segment (iMS) targets the adipocyte specific LD surface factor PLIN1 to the endoplasmic reticulum (ER) and facilitates high-affinity binding to the first LD class. The other PLINs remain largely excluded from these LDs until FA influx recruits them to a second LD population. Preventing ER targeting turns PLIN1 into a soluble, cytoplasmic LD protein, reduces its LD affinity, and switches its LD class specificity. Conversely, moving the iMS to PLIN2 leads to ER insertion and formation of a separate LD class. Our results shed light on how differences in organelle targeting and disparities in lipid affinity of LD surface factors contribute to formation of LD heterogeneity.

ARL8B mediates lipid droplet contact and delivery to lysosomes for lipid remobilization.

Lipid droplets (LDs) play a crucial role in maintaining cellular lipid balance by storing and delivering lipids as needed. However, the intricate lipolytic pathways involved in LD turnover remain poorly described, hindering our comprehension of lipid catabolism and related disorders. Here, we show a function of the small GTPase ARL8B in mediating LD turnover in lysosomes. ARL8B-GDP localizes to LDs, while ARL8-GTP predominantly favors lysosomes. GDP binding induces a conformation with an exposed N-terminal amphipathic helix, enabling ARL8B to bind to LDs. By associating with LDs and lysosomes, and with its property to form a heterotypic complex, ARL8B mediates LD-lysosome contacts and efficient lipid transfer between these organelles. In human macrophages, this ARL8B-dependent LD turnover mechanism appears as the major lipolytic pathway. Our finding opens exciting possibilities for understanding the molecular mechanisms underlying LD degradation and its potential implications for inflammatory disorders.

ATG2A-mediated bridge-like lipid transport regulates lipid droplet accumulation.

ATG2 proteins facilitate bulk lipid transport between membranes. ATG2 is an essential autophagy protein, but ATG2 also localizes to lipid droplets (LDs), and genetic depletion of ATG2 increases LD numbers while impairing fatty acid transport from LDs to mitochondria. How ATG2 supports LD homeostasis and whether lipid transport regulates this homeostasis remains unknown. Here we demonstrate that ATG2 is preferentially recruited to phospholipid monolayers such as those surrounding LDs rather than to phospholipid bilayers. In vitro, ATG2 can drive phospholipid transport from artificial LDs with rates that correlate with the binding affinities, such that phospholipids are moved much more efficiently when one of the ATG2-interacting structures is an artificial LD. ATG2 is thought to exhibit 'bridge-like" lipid transport, with lipids flowing across the protein between membranes. We mutated key amino acids within the bridge to form a transport-dead ATG2 mutant (TD-ATG2A) which we show specifically blocks bridge-like, but not shuttle-like, lipid transport in vitro. TD-ATG2A still localizes to LDs, but is unable to rescue LD accumulation in ATG2 knockout cells. Thus, ATG2 has a natural affinity for, and an enhanced activity upon LD surfaces and uses bridge-like lipid transport to support LD dynamics in cells.

LC3 conjugation to lipid droplets.

Macroautophagy/autophagy and lipid droplet (LD) biology are intricately linked, with autophagosome-dependent degradation of LDs in response to different signals. LDs play crucial roles in forming autophagosomes possibly by providing essential lipids and serving as a supportive autophagosome assembly platform at the endoplasmic reticulum (ER)-LD interface. LDs and autophagosomes share common proteins, such as VPS13, ATG2, ZFYVE1/DFCP1, and ATG14, but their dual functions remain poorly understood. In our recent study, we found that prolonged starvation leads to ATG3 localizing to large LDs and lipidating LC3B, revealing a non-canonical autophagic role on LDs. In vitro, ATG3 associates with purified and artificial LDs, and conjugated Atg8-family proteins. In long-term starved cells, only LC3B is found on the specific large LDs, positioned near LC3B-positive membranes that undergo lysosome-mediated acidification. This implies that LD-lipidated LC3B acts as a tethering factor, connecting phagophores to LDs and promoting degradation. Our data also support the notion that certain LD surfaces may function as lipidation stations for LC3B, which may move to nearby sites of autophagosome formation. Overall, our study unveils an unknown non-canonical implication of LDs in autophagy processes.Abbreviation: ATG: autophagy-related enzyme, ATP: adenosine triphosphate, E2 enzyme: ubiquitin-conjugating enzyme, ER: endoplasmic reticulum, LD: lipid droplet, LIR motif: LC3-interacting region, MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta, PE: phosphatidylethanolamine, PLIN1: perilipin 1, PNPLA2/ATGL: patatin-like phospholipase domain containing 2, SQSTM1/p62: sequestosome 1, VSP13: vacuolar protein sorting 13, ZFYVE1/DFCP1: zinc finger, FYVE domain containing 1.

Structural insights into perilipin 3 membrane association in response to diacylglycerol accumulation.

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyze how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We find that the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG-enriched membranes. Membrane binding induces a disorder to order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG-enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.

LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.

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.

Cholesterol esters form supercooled lipid droplets whose nucleation is facilitated by triacylglycerols.

Cellular cholesterol can be metabolized to its fatty acid esters, cholesteryl esters (CEs), to be stored in lipid droplets (LDs). With triacylglycerols (TGs), CEs represent the main neutral lipids in LDs. However, while TG melts at ~4 °C, CE melts at ~44 °C, raising the question of how CE-rich LDs form in cells. Here, we show that CE forms supercooled droplets when the CE concentration in LDs is above 20% to TG and, in particular, liquid-crystalline phases when the fraction of CEs is above 90% at 37 °C. In model bilayers, CEs condense and nucleate droplets when the CE/phospholipid ratio reaches over 10-15%. This concentration is reduced by TG pre-clusters in the membrane that thereby facilitate CE nucleation. Accordingly, blocking TG synthesis in cells is sufficient to strongly dampen CE LD nucleation. Finally, CE LDs emerged at seipins, which cluster and nucleate TG LDs in the ER. However, when TG synthesis is inhibited, similar numbers of LDs are generated in the presence and absence of seipin, suggesting that seipin controls CE LD formation via its TG clustering capacity. Our data point to a unique model whereby TG pre-clusters, favorable at seipins, catalyze the nucleation of CE LDs.

Triglyceride lipolysis triggers liquid crystalline phases in lipid droplets and alters the LD proteome.

Lipid droplets (LDs) are reservoirs for triglycerides (TGs) and sterol-esters (SEs), but how these lipids are organized within LDs and influence their proteome remain unclear. Using in situ cryo-electron tomography, we show that glucose restriction triggers lipid phase transitions within LDs generating liquid crystalline lattices inside them. Mechanistically this requires TG lipolysis, which decreases the LD's TG:SE ratio, promoting SE transition to a liquid crystalline phase. Molecular dynamics simulations reveal TG depletion promotes spontaneous TG and SE demixing in LDs, additionally altering the lipid packing of the PL monolayer surface. Fluorescence imaging and proteomics further reveal that liquid crystalline phases are associated with selective remodeling of the LD proteome. Some canonical LD proteins, including Erg6, relocalize to the ER network, whereas others remain LD-associated. Model peptide LiveDrop also redistributes from LDs to the ER, suggesting liquid crystalline phases influence ER-LD interorganelle transport. Our data suggests glucose restriction drives TG mobilization, which alters the phase properties of LD lipids and selectively remodels the LD proteome.

ORP5 and ORP8 orchestrate lipid droplet biogenesis and maintenance at ER-mitochondria contact sites.

Lipid droplets (LDs) are the primary organelles of lipid storage, buffering energy fluctuations of the cell. They store neutral lipids in their core that is surrounded by a protein-decorated phospholipid monolayer. LDs arise from the endoplasmic reticulum (ER). The ER protein seipin, localizing at ER-LD junctions, controls LD nucleation and growth. However, how LD biogenesis is spatially and temporally coordinated remains elusive. Here, we show that the lipid transfer proteins ORP5 and ORP8 control LD biogenesis at mitochondria-associated ER membrane (MAM) subdomains, enriched in phosphatidic acid. We found that ORP5/8 regulates seipin recruitment to these MAM-LD contacts, and their loss impairs LD biogenesis. Importantly, the integrity of ER-mitochondria contact sites is crucial for ORP5/8 function in regulating seipin-mediated LD biogenesis. Our study uncovers an unprecedented ORP5/8 role in orchestrating LD biogenesis and maturation at MAMs and brings novel insights into the metabolic crosstalk between mitochondria, ER, and LDs at the membrane contact sites.

Impact of Cyclization and Methylation on Peptide Penetration through Droplet Interface Bilayers.

Cell-penetrating peptides enter cells via diverse mechanisms, such as endocytosis, active transport, or direct translocation. For the design of orally delivered cell-penetrating peptides, it is crucial to know the contribution of these different mechanisms. In particular, the ability of a peptide to translocate through a lipid bilayer remains a key parameter for the delivery of cargos. However, existing approaches used to assess translocation often provide discrepant results probably because they have different sensitivities to the distinct translocation mechanisms. Here, we focus on the passive permeation of a range of hydrophobic cyclic peptides inspired by somatostatin, a somatotropin release-inhibiting factor. Using droplet interface bilayers (DIB), we assess the passive membrane permeability of these peptides and study the impact of the peptide cyclization and backbone methylation on translocation rates. Cyclization systematically improved the permeability of the tested peptides while methylation did not. By studying the interaction of the peptides with the DIB interfaces, we found membrane insertion and peptide intrinsic diffusion to be two independent factors of permeability. Compared to the industrial gold standard Caco-2 and parallel artificial membrane permeability assay (PAMPA) models, DIBs provide intermediate membrane permeability values, closer to Caco-2. Even for conditions where Caco-2 and PAMPA are discrepant, the DIB approach also gives results closer to Caco-2. Thereupon, DIBs represent a robust alternative to the PAMPA approach for predicting the permeability of peptides, even if the latter present extremely small structural differences.

MOSPD2 is an endoplasmic reticulum-lipid droplet tether functioning in LD homeostasis.

Membrane contact sites between organelles are organized by protein bridges. Among the components of these contacts, the VAP family comprises ER-anchored proteins, such as MOSPD2, that function as major ER-organelle tethers. MOSPD2 distinguishes itself from the other members of the VAP family by the presence of a CRAL-TRIO domain. In this study, we show that MOSPD2 forms ER-lipid droplet (LD) contacts, thanks to its CRAL-TRIO domain. MOSPD2 ensures the attachment of the ER to LDs through a direct protein-membrane interaction. The attachment mechanism involves an amphipathic helix that has an affinity for lipid packing defects present at the surface of LDs. Remarkably, the absence of MOSPD2 markedly disturbs the assembly of lipid droplets. These data show that MOSPD2, in addition to being a general ER receptor for inter-organelle contacts, possesses an additional tethering activity and is specifically implicated in the biology of LDs via its CRAL-TRIO domain.

Hepatitis C virus core protein uses triacylglycerols to fold onto the endoplasmic reticulum membrane.

Lipid droplets (LDs) are involved in viral infections, but exactly how remains unclear. Here, we study the hepatitis C virus (HCV) whose core capsid protein binds to LDs but is also involved in the assembly of virions at the endoplasmic reticulum (ER) bilayer. We found that the amphipathic helix-containing domain of core, D2, senses triglycerides (TGs) rather than LDs per se. In the absence of LDs, D2 can bind to the ER membrane but only if TG molecules are present in the bilayer. Accordingly, the pharmacological inhibition of the diacylglycerol O-acyltransferase enzymes, mediating TG synthesis in the ER, inhibits D2 association with the bilayer. We found that TG molecules enable D2 to fold into alpha helices. Sequence analysis reveals that D2 resembles the apoE lipid-binding region. Our data support that TG in LDs promotes the folding of core, which subsequently relocalizes to contiguous ER regions. During this motion, core may carry TG molecules to these regions where HCV lipoviroparticles likely assemble. Consistent with this model, the inhibition of Arf1/COPI, which decreases LD surface accessibility to proteins and ER-LD material exchange, severely impedes the assembly of virions. Altogether, our data uncover a critical function of TG in the folding of core and HCV replication and reveals, more broadly, how TG accumulation in the ER may provoke the binding of soluble amphipathic helix-containing proteins to the ER bilayer.

Origin of gradients in lipid density and surface tension between connected lipid droplet and bilayer.

We combined theory and experiments to depict physical parameters modulating the phospholipid (PL) density and tension equilibrium between a bilayer and an oil droplet in contiguity. This situation is encountered during a neutral lipid (NL) droplet formation in the endoplasmic reticulum. We set up macroscopic and microscopic models to uncover free parameters and the origin of molecular interactions controlling the PL densities of the droplet monolayer and the bilayer. The established physical laws and predictions agreed with experiments performed with droplet-embedded vesicles. We found that the droplet monolayer is always by a few percent (∼10%) less packed with PLs than the bilayer. Such a density gradient arises from PL-NL interactions on the droplet, which are lower than PL-PL trans interactions in the bilayer, i.e., interactions between PLs belonging to different leaflets of the bilayer. Finally, despite the pseudo-surface tension for the water/PL acyl chains in the bilayer being higher than the water/NL surface tension, the droplet monolayer always has a higher surface tension than the bilayer because of its lower PL density. Thus, a PL density gradient is mandatory to maintain the mechanical and thermodynamic equilibrium of the droplet-bilayer continuity. Our study sheds light on the origin of the molecular interactions responsible for the unique surface properties of lipid droplets compared with cellular bilayer membranes.

Variability in cTBS Aftereffects Attributed to the Interaction of Stimulus Intensity With BDNF Val66Met Polymorphism.

Objective: To evaluate whether a common polymorphism (Val66Met) in the gene for brain-derived neurotrophic factor (BDNF)-a gene thought to influence plasticity-contributes to inter-individual variability in responses to continuous theta-burst stimulation (cTBS), and explore whether variability in stimulation-induced plasticity among Val66Met carriers relates to differences in stimulation intensity (SI) used to probe plasticity. Methods: Motor evoked potentials (MEPs) were collected from 33 healthy individuals (11 Val66Met) prior to cTBS (baseline) and in 10 min intervals immediately following cTBS for a total of 30 min post-cTBS (0 min post-cTBS, 10 min post-cTBS, 20 min post cTBS, and 30 min post-cTBS) of the left primary motor cortex. Analyses assessed changes in cortical excitability as a function of BDNF (Val66Val vs. Val66Met) and SI. Results: For both BDNF groups, MEP-suppression from baseline to post-cTBS time points decreased as a function of increasing SI. However, the effect of SI on MEPs was more pronounced for Val66Met vs. Val66Val carriers, whereby individuals probed with higher vs. lower SIs resulted in paradoxical cTBS aftereffects (MEP-facilitation), which persisted at least 30 min post-cTBS administration. Conclusions: cTBS aftereffects among BDNF Met allele carriers are more variable depending on the SI used to probe cortical excitability when compared to homozygous Val allele carriers, which could, to some extent, account for the inconsistency of previously reported cTBS effects. Significance: These data provide insight into the sources of cTBS response variability, which can inform how best to stratify and optimize its use in investigational and clinical contexts.

Retinyl esters form lipid droplets independently of triacylglycerol and seipin.

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.

Pre-existing bilayer stresses modulate triglyceride accumulation in the ER versus lipid droplets.

Cells store energy in the form of neutral lipids (NLs) packaged into micrometer-sized organelles named lipid droplets (LDs). These structures emerge from the endoplasmic reticulum (ER) at sites marked by the protein seipin, but the mechanisms regulating their biogenesis remain poorly understood. Using a combination of molecular simulations, yeast genetics, and fluorescence microscopy, we show that interactions between lipids' acyl-chains modulate the propensity of NLs to be stored in LDs, in turn preventing or promoting their accumulation in the ER membrane. Our data suggest that diacylglycerol, which is enriched at sites of LD formation, promotes the packaging of NLs into LDs, together with ER-abundant lipids, such as phosphatidylethanolamine. On the opposite end, short and saturated acyl-chains antagonize fat storage in LDs and promote accumulation of NLs in the ER. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.

Fat inclusions strongly alter membrane mechanics.

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.