Seipin regulates ER–lipid droplet contacts and cargo delivery

Seipin is an endoplasmic reticulum (ER) membrane protein implicated in lipid droplet (LD) biogenesis and mutated in severe congenital lipodystrophy (BSCL2). Here, we show that seipin is stably associated with nascent ER–LD contacts in human cells, typically via one mobile focal point per LD. Seipin appears critical for such contacts since ER–LD contacts were completely missing or morphologically aberrant in seipin knockout and BSCL2 patient cells. In parallel, LD mobility was increased and protein delivery from the ER to LDs to promote LD growth was decreased. Moreover, while growing LDs normally acquire lipid and protein constituents from the ER, this process was compromised in seipin‐deficient cells. In the absence of seipin, the initial synthesis of neutral lipids from exogenous fatty acid was normal, but fatty acid incorporation into neutral lipids in cells with pre‐existing LDs was impaired. Together, our data suggest that seipin helps to connect newly formed LDs to the ER and that by stabilizing ER–LD contacts seipin facilitates the incorporation of protein and lipid cargo into growing LDs in human cells.     

Born this way – Biogenesis of lipid droplets from specialized ER subdomains

Both the endoplasmic reticulum (ER) and lipid droplets (LDs) are key players in lipid handling. In addition to this functional connection, the two organelles are also tightly linked due to the fact that the ER is the birthplace of LDs. LDs have an atypical architecture, consisting of a neutral lipid core that is covered by a phospholipid monolayer. LD biogenesis starts with neutral lipid synthesis in the ER membrane and formation of small neutral lipid lenses between its leaflets, followed by budding of mature LDs toward the cytosol.Several ER proteins have been identified that are required for efficient LD formation, among them seipin, Pex30, and FIT2. Recent evidence indicates that these LD biogenesis factors might cooperate with specific lipids, thus generating ER subdomains optimized for LD assembly. Intriguingly, LD biogenesis reacts dynamically to nutrient stress, resulting in a spatial reorganization of LD formation in the ER.     

Seipin concentrates distinct neutral lipids via interactions with their acyl chain carboxyl esters

Lipid droplets (LDs) are essential for cellular lipid homeostasis by storing diverse neutral lipids (NLs), such as triacylglycerol (TAG), steryl esters (SE), and retinyl esters (RE). A proper assembly of TAG-containing LDs at the ER requires Seipin, a conserved protein often mutated in lipodystrophies. Here, we show that the yeast Seipin Sei1 and its partner Ldb16 also promote the storage of other NL in LDs. Importantly, this role of Sei1/Ldb16 is evolutionarily conserved as expression of human-Seipin restored normal SE-containing LDs in yeast Seipin mutants. As in the case of TAG, the formation of SE-containing LDs requires interactions between hydroxyl-residues in human Seipin or yeast Ldb16 with NL carboxyl esters. These findings provide a universal mechanism for Seipin-mediated LD formation and suggest a model for how Seipin distinguishes NLs from aliphatic phospholipid acyl chains in the center of the membrane bilayer.     

Structure and function of lipid droplet assembly complexes

Cells store lipids as a reservoir of metabolic energy and membrane component precursors in organelles called lipid droplets (LDs). LD formation occurs in the endoplasmic reticulum (ER) at LD assembly complexes (LDAC), consisting of an oligomeric core of seipin and accessory proteins. LDACs determine the sites of LD formation and are required for this process to occur normally. Seipin oligomers form a cage-like structure in the membrane that may serve to facilitate the phase transition of neutral lipids in the membrane to form an oil droplet within the LDAC. Modeling suggests that, as the LD grows, seipin anchors it to the ER bilayer and conformational shifts of seipin transmembrane segments open the LDAC dome toward the cytoplasm, enabling the emerging LD to egress from the ER.     

Are endoplasmic reticulum subdomains shaped by asymmetric distribution of phospholipids? Evidence from a C. elegans model system

Physical contact between organelles are widespread, in part to facilitate the shuttling of protein and lipid cargoes for cellular homeostasis. How do protein‐protein and protein‐lipid interactions shape organelle subdomains that constitute contact sites? The endoplasmic reticulum (ER) forms extensive contacts with multiple organelles, including lipid droplets (LDs) that are central to cellular fat storage and mobilization. Here, we focus on ER‐LD contacts that are highlighted by the conserved protein seipin, which promotes LD biogenesis and expansion. Seipin is enriched in ER tubules that form cage‐like structures around a subset of LDs. Such enrichment is strongly dependent on polyunsaturated and cyclopropane fatty acids. Based on these findings, we speculate on molecular events that lead to the formation of seipin‐positive peri‐LD cages in which protein movement is restricted. We hypothesize that asymmetric distribution of specific phospholipids distinguishes cage membrane tubules from the bulk ER.     

Nuclear lipid droplets form in the inner nuclear membrane in a seipin-independent manner

Nuclear lipid droplets (LDs) in hepatocytes are derived from precursors of very-low-density lipoprotein in the ER lumen, but it is not known how cells lacking the lipoprotein secretory function form nuclear LDs. Here, we show that the inner nuclear membrane (INM) of U2OS cells harbors triglyceride synthesis enzymes, including ACSL3, AGPAT2, GPAT3/GPAT4, and DGAT1/DGAT2, and generates nuclear LDs in situ. mTOR inhibition increases nuclear LDs by inducing the nuclear translocation of lipin-1 phosphatidic acid (PA) phosphatase. Seipin, a protein essential for normal cytoplasmic LD formation in the ER, is absent in the INM. Knockdown of seipin increases nuclear LDs and PA in the nucleus, whereas seipin overexpression decreases these. Seipin knockdown also up-regulates lipin-1β expression, and lipin-1 knockdown decreases the effect of seipin knockdown on nuclear LDs without affecting PA redistribution. These results indicate that seipin is not directly involved in nuclear LD formation but instead restrains it by affecting lipin-1 expression and intracellular PA distribution.     

CIDE family proteins control lipid homeostasis and the development of metabolic diseases

Dysregulation of lipid homeostasis leads to the development of metabolic disorders including obesity, diabetes, cardiovascular disease and cancer. Lipid droplets (LDs) are subcellular organelles vital in the maintenance of lipid homeostasis by coordinating lipid synthesis, lipid storage, lipid secretion and lipolysis. Under fed condition, free fatty acids (FFAs) are remodeled and esterified into neutral lipids by lipogenesis and stored in the LDs. The lipid storage capacity of LDs is controlled by its growth via local lipid synthesis or by LD fusion. During fasting, neutral lipids are hydrolyzed by lipolysis, released as FFAs and secreted to meet energy demand. C ell death‐i nducing D NA fragmentation factor alpha (DFFA)‐like e ffector (CIDE) family proteins composed of Cidea, Cideb and Cidec/Fsp27 are ER‐ and LD‐associated proteins and have emerged as important regulators of lipid homeostasis. Notably, when localized on the LDs, CIDE proteins enrich at the LD‐LD contact sites (LDCSs) and control LD fusion and growth. Here, we summarize these recent advances made on the role of CIDE proteins in the regulation of lipid metabolism with a particular focus on the molecular mechanisms underlying CIDE‐mediated LD fusion and growth.     

The seipin complex Fld1/Ldb16 stabilizes ER–lipid droplet contact sites

Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER–LD contact sites.     

The lipid droplet is an important organelle for hepatitis C virus production

The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.     

The FATP1-DGAT2 complex facilitates lipid droplet expansion at the ER-lipid droplet interface

At the subcellular level, fat storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs), which are closely associated with the endoplasmic reticulum (ER). However, the molecular mechanisms that enable ER-LD interaction and facilitate neutral lipid loading into LDs are poorly understood. In this paper, we present evidence that FATP1/acyl-CoA synthetase and DGAT2/diacylglycerol acyltransferase are components of a triglyceride synthesis complex that facilitates LD expansion. A loss of FATP1 or DGAT2 function blocked LD expansion in Caenorhabditis elegans. FATP1 preferentially associated with DGAT2, and they acted synergistically to promote LD expansion in mammalian cells. Live imaging indicated that FATP1 and DGAT2 are ER and LD resident proteins, respectively, and electron microscopy revealed FATP1 and DGAT2 foci close to the LD surface. Furthermore, DGAT2 that was retained in the ER failed to support LD expansion. We propose that the evolutionarily conserved FATP1-DGAT2 complex acts at the ER-LD interface and couples the synthesis and deposition of triglycerides into LDs both physically and functionally.     

Spatial compartmentalization of lipid droplet biogenesis

Lipid droplets (LDs) are ubiquitous organelles that store metabolic energy in the form of neutral lipids (typically triacylglycerols and steryl esters). Beyond being inert energy storage compartments, LDs are dynamic organelles that participate in numerous essential metabolic functions. Cells generate LDs de novo from distinct sub-regions at the endoplasmic reticulum (ER), but what determines sites of LD formation remains a key unanswered question. Here, we review the factors that determine LD formation at the ER, and discuss how they work together to spatially and temporally coordinate LD biogenesis. These factors include lipid synthesis enzymes, assembly proteins, and membrane structural requirements. LDs also make contact with other organelles, and these inter-organelle contacts contribute to defining sites of LD production. Finally, we highlight emerging non-canonical roles for LDs in maintaining cellular homeostasis during stress.     

Dynamics of protein and polar lipid recruitment during lipid droplet assembly in Chlamydomonas reinhardtii

In plants, neutral lipids are frequently synthesized and stored in seed tissues, where the assembly of lipid droplets (LDs) coincides with the accumulation of triacylglycerols (TAGs). In addition, photosynthetic, vegetative cells can form cytosolic LDs and much less information is known about the makeup and biogenesis of these LDs. Here we focus on Chlamydomonas reinhardtii as a reference model for LDs in a photosynthetic cell, because in this unicellular green alga LD dynamics can be readily manipulated by nitrogen availability. Nitrogen deprivation leads to cellular quiescence during which cell divisions cease and TAGs accumulate. The major lipid droplet protein (MLDP) forms a proteinaceous coat surrounding mature LDs. Reducing the amount of MLDP affects LD size and number, TAG breakdown and timely progression out of cellular quiescence following nitrogen resupply. Depending on nitrogen availability, MLDP recruits different proteins to LDs, tubulins in particular. Conversely, depolymerization of microtubules drastically alters the association of MLDP with LDs. LDs also contain select chloroplast envelope membrane proteins hinting at an origin of LDs, at least in part, from chloroplast membranes. Moreover, LD surface lipids are rich in de novo synthesized fatty acids, and are mainly composed of galactolipids which are typical components of chloroplast membranes. The composition of the LD membrane is altered in the absence of MLDP. Collectively, our results suggest a mechanism for LD formation in C. reinhardtii involving chloroplast envelope membranes by which specific proteins are recruited to LDs and a specialized polar lipid monolayer surrounding the LD is formed.     

Proteomic study and marker protein identification of Caenorhabditis elegans lipid droplets

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved across almost all species. Many metabolic syndromes are directly linked to the over-storage of neutral lipids in LDs. The study of LDs in Caenorhabditis elegans (C. elegans) has been difficult because of the lack of specific LD marker proteins. Here we report the purification and proteomic analysis of C. elegans lipid droplets for the first time. We identified 306 proteins, 63% of these proteins were previously known to be LD-proteins, suggesting a similarity between mammalian and C. elegans LDs. Using morphological and biochemical analyses, we show that short-chain dehydrogenase, DHS-3 is almost exclusively localized on C. elegans LDs, indicating that it can be used as a LD marker protein in C. elegans. These results will facilitate further mechanistic studies of LDs in this powerful genetic system, C. elegans.     

Mammalian lipid droplets: structural,pathological, immunological and anti-toxicological roles

Mammalian lipid droplets (LDs) are specialized cytosolic organelles consisting of a neutral lipid core surrounded by a membrane made up of a phospholipid monolayer and a specific population of proteins that varies according to the location and function of each LD. Over the past decade, there have been significant advances in the understanding of LD biogenesis and functions. LDs are now recognized as dynamic organelles that participate in many aspects of cellular homeostasis plus other vital functions. LD biogenesis is a complex, highly-regulated process with assembly occurring on the endoplasmic reticulum although aspects of the underpinning molecular mechanisms remain elusive. For example, it is unclear how many enzymes participate in the biosynthesis of the neutral lipid components of LDs and how this process is coordinated in response to different metabolic cues to promote or suppress LD formation and turnover. In addition to enzymes involved in the biosynthesis of neutral lipids, various scaffolding proteins play roles in coordinating LD formation. Despite their lack of ultrastructural diversity, LDs in different mammalian cell types are involved in a wide range of biological functions. These include roles in membrane homeostasis, regulation of hypoxia, neoplastic inflammatory responses, cellular oxidative status, lipid peroxidation, and protection against potentially toxic intracellular fatty acids and lipophilic xenobiotics. Herein, the roles of mammalian LDs and their associated proteins are reviewed with a particular focus on their roles in pathological, immunological and anti-toxicological processes.     

Arabidopsis lipid droplet‐associated protein (LDAP) – interacting protein (LDIP) influences lipid droplet size and neutral lipid homeostasis in both leaves and seeds

Cytoplasmic lipid droplets (LDs) are found in all types of plant cells; they are derived from the endoplasmic reticulum and function as a repository for neutral lipids, as well as serving in lipid remodelling and signalling. However, the mechanisms underlying the formation, steady‐state maintenance and turnover of plant LDs, particularly in non‐seed tissues, are relatively unknown. Previously, we showed that the LD‐associated proteins (LDAPs) are a family of plant‐specific, LD surface‐associated coat proteins that are required for proper biogenesis of LDs and neutral lipid homeostasis in vegetative tissues. Here, we screened a yeast two‐hybrid library using the Arabidopsis LDAP3 isoform as ‘bait’ in an effort to identify other novel LD protein constituents. One of the candidate LDAP3‐interacting proteins was Arabidopsis At5g16550, which is a plant‐specific protein of unknown function that we termed LDIP (LDAP‐interacting protein). Using a combination of biochemical and cellular approaches, we show that LDIP targets specifically to the LD surface, contains a discrete amphipathic α‐helical targeting sequence, and participates in both homotypic and heterotypic associations with itself and LDAP3, respectively. Analysis of LDIP T‐DNA knockdown and knockout mutants showed a decrease in LD abundance and an increase in variability of LD size in leaves, with concomitant increases in total neutral lipid content. Similar phenotypes were observed in plant seeds, which showed enlarged LDs and increases in total amounts of seed oil. Collectively, these data identify LDIP as a new player in LD biology that modulates both LD size and cellular neutral lipid homeostasis in both leaves and seeds.     

FIT2 organizes lipid droplet biogenesis with ER tubule-forming proteins and septins

Lipid droplets (LDs) are critical for lipid storage and energy metabolism. LDs form in the endoplasmic reticulum (ER). However, the molecular basis for LD biogenesis remains elusive. Here, we show that fat storage–inducing transmembrane protein 2 (FIT2) interacts with ER tubule-forming proteins Rtn4 and REEP5. The association is mainly transmembrane domain based and stimulated by oleic acid. Depletion of ER tubule-forming proteins decreases the number and size of LDs in cells and Caenorhabditis elegans, mimicking loss of FIT2. Through cytosolic loops, FIT2 binds to cytoskeletal protein septin 7, an interaction that is also required for normal LD biogenesis. Depletion of ER tubule-forming proteins or septins delays nascent LD formation. In addition, FIT2-interacting proteins are up-regulated during adipocyte differentiation, and ER tubule-forming proteins, septin 7, and FIT2 are transiently enriched at LD formation sites. Thus, FIT2-mediated nascent LD biogenesis is facilitated by ER tubule-forming proteins and septins.     

Multiple C2 domain–containing transmembrane proteins promote lipid droplet biogenesis and growth at specialized endoplasmic reticulum subdomains

Lipid droplets (LDs) are neutral lipid-containing organelles enclosed in a single monolayer of phospholipids. LD formation begins with the accumulation of neutral lipids within the bilayer of the endoplasmic reticulum (ER) membrane. It is not known how the sites of formation of nascent LDs in the ER membrane are determined. Here we show that multiple C2 domain–containing transmembrane proteins, MCTP1 and MCTP2, are at sites of LD formation in specialized ER subdomains. We show that the transmembrane domain (TMD) of these proteins is similar to a reticulon homology domain. Like reticulons, these proteins tubulate the ER membrane and favor highly curved regions of the ER. Our data indicate that the MCTP TMDs promote LD biogenesis, increasing LD number. MCTPs colocalize with seipin, a protein involved in LD biogenesis, but form more stable microdomains in the ER. The MCTP C2 domains bind charged lipids and regulate LD size, likely by mediating ER–LD contact sites. Together, our data indicate that MCTPs form microdomains within ER tubules that regulate LD biogenesis, size, and ER–LD contacts. Interestingly, MCTP punctae colocalized with other organelles as well, suggesting that these proteins may play a general role in linking tubular ER to organelle contact sites.     

Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets

Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1.     

Plin3 protects against alcoholic liver injury by facilitating lipid export from the endoplasmic reticulum

Hepatic lipid accumulation is the most common pathological characteristic of alcoholic liver disease (ALD). In mammalian cells, excess neutral lipids are stored in lipid droplets (LDs). As a member of perilipin family proteins, Plin3 was recently found to regulate the LD biogenesis. However, the roles and mechanism of Plin3 in ALD progression remain unclear. Herein, we found that alcohol stimulated Plin3 expression in both mouse livers and cultured AML12 mouse hepatic cells, which was accompanied by excess LD accumulation in hepatocytes. The elevations of Plin3 in alcohol-treated hepatocytes paralleled with the levels of both PPARα and γ, and the protein degradation of Plin3 was also reduced after alcohol exposure. Moreover, Plin3 knockdown increased cellular sensitivity to alcohol-induced apoptosis, endoplasmic reticulum (ER) stress, and inflammatory cytokines release, including TNF-α, IL-1, and IL-6β. Notably, alcohol exacerbated triglycerides (TG) accumulation in the ER and caused ER dilation in Plin3-knockdown AML12 cells. Finally, we observed that Plin3 interacted with dynein subunit Dync1i1 and mediated the colocalization of LDs and microtubules, while high concentration of alcohol disrupted microtubules and caused dispersion of excess small LDs in cytoplasm. Summarily, Plin3 promotes lipid export from the ER and reduces ER lipotoxic stress, thereby, protecting against alcoholic liver injury. Moreover, Plin3 could be an adapter protein mediating LD transport by microtubules. This study explored the roles of Plin3 in alcohol-induced hepatic injury, suggesting Plin3 as a potential target for the prevention of ALD progression.     

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.