DFCP1 associates with lipid droplets

Double FYVE‐containing protein 1 (DFCP1) is ubiquitously expressed, participates in intracellular membrane trafficking and labels omegasomes through specific interactions with phosphatidylinositol‐3‐phosphate (PI3P). Previous studies showed that subcellular DFCP1 proteins display multi‐organelle localization, including in the endoplasmic reticulum (ER), Golgi apparatus and mitochondria. However, its localization and function on lipid droplets (LDs) remain unclear. Here, we demonstrate that DFCP1 localizes to the LD upon oleic acid incubation. The ER‐targeted domain of DFCP1 is indispensable for its LD localization, which is further enhanced by double FYVE domains. Inhibition of PI3P binding at the FYVE domain through wortmannin treatment or double mutation at C654S and C770S have no effect on DFCP1's LD localization. These show that the mechanisms for DFCP1 targeting the omegasome and LDs are different. DFCP1 deficiency in MEF cells causes an increase in LD number and reduces LD size. Interestingly, DFCP1 interacts with GTP‐bound Rab18, an LD‐associated protein. Taken together, our work demonstrates the dynamic localization of DFCP1 is regulated by nutritional status in response to cellular metabolism.     

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.     

LC3, a microtubule-associated protein1A/B light chain3, is involved in cytoplasmic lipid droplet formation

The cytoplasmic lipid droplet (LD) is one of organelles that has a neutral lipid core with a single phospholipid layer. LDs are believed to be generated between the two leaflets of the endoplasmic reticulum (ER) membrane and to play various roles, such as high effective energy storage. However, it remains largely unknown how LDs are generated and grow in the cytoplasm. We have previously shown that the Atg conjugation system that is essential for autophagosome formation is involved in LD formation in hepatocytes and cardiac myocytes. We show here that LC3 itself is involved in LD formation by using RNA interference (RNAi). All cultured cell lines examined, in which the expression of LC3 was suppressed by RNAi, showed reduced LD formation. Triacylglycerol, a major component of LDs, was synthesized and degraded in LC3 mRNA-knockdown cells as well as in control cells. Interestingly, potential of the bulk protein degradation in the knockdown-cells was also evident in the control cells. These findings indicate that LC3 is involved in the LD formation regardless of the bulk degradation, and that LC3 has two pivotal roles in cellular homeostasis mediated by autophagy and lipid metabolism.     

Rab18 binds PLIN2 and ACSL3 to mediate lipid droplet dynamics

Lipid droplet (LD) is a vital organelle governing lipid homeostasis and Rab18 has been linked to lipid metabolism. However, the mechanisms of Rab18-mediated LD dynamics in myoblast cells remain elusive. Here, we report that Rab18 plays an important role in oleic acid (OA)-induced LD accumulation in mouse myoblast C2C12 cells. Rab18 was translocated from the endoplasmic reticulum (ER) to LDs during LD accumulation, which was regulated by perilipin 2 (PLIN2), a major LD protein. LD-associated Rab18 bound with the C terminus of PLIN2 and the LD localization of Rab18 was diminished when PLIN2 was depleted. Moreover, loss of function of Rab18 led to reduced triacylglycerol (TAG) level and fewer but larger LDs. In contrast, overexpression of Rab18 resulted in elevated TAG content and LD number. Furthermore, LD-associated Rab18 interacted with acyl-CoA synthetase long-chain family member 3 (ACSL3), which in turn promoted the LD localization of this protein. These data show that Rab18 interacts with PLIN2 and forms a complex with PLIN2 and ACSL3, which plays a critical role in LD accumulation and dynamics of myoblast cells.     

The MAP1-LC3 conjugation system is involved in lipid droplet formation

Lipid droplets (LDs) are ubiquitous in eukaryotic cells, while excess free fatty acids and glucose in plasma are converted to triacylglycerol (TAG) and stored as LDs. However, the mechanism for the generation and growth of LDs in cells is largely unknown. We show here that the LC3 lipidation system essential for macroautophagy is involved in LD formation. LD formation accompanied by accumulation of TAG induced by starvation was largely suppressed in the hepatocytes that cannot execute autophagy. Under starvation conditions, LDs in addition to autophagosomes were abundantly formed in the cytoplasm of these tissue cells. Moreover, LC3 was localized on the surface of LDs and LC3-II (lipidation form) was fractionated to a perilipin (LD marker)-positive lipid fraction from the starved liver. Taken together, these results indicate that the LC3 conjugation system is critically involved in lipid metabolism via LD formation.     

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.     

The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide induces large lipid droplet accumulation and highlights the effect of LAMP-2 deficiency on lipid droplet degradation

Excess accumulation of intracellular lipids leads to various diseases. Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage. LDs are hydrolyzed via cytosolic lipases (lipolysis) and also degraded in lysosomes through autophagy; namely, lipophagy. A recent study has shown the size-dependent selection of LDs by the two major catabolic pathways (lipolysis and lipophagy), and thus experimental systems that can manipulate the size of LDs are now needed. The ceramide analogue N-(1-hydroxy-3-morpholino-1-phenylpropan-2-yl)decanamide (PDMP) affects the structures and functions of lysosomes/late endosomes and the endoplasmic reticulum (ER), and alters cholesterol homeostasis. We previously reported that PDMP induces autophagy via the inhibition of mTORC1. In the present study, we found that PDMP induced the accumulation of LDs, especially that of large LDs, in mouse fibroblast (L cells). Surprisingly, the LD accumulation was relieved by PDMP in L cells deficient in lysosome-associated membrane protein-2 (LAMP-2), which is reportedly important for lipophagy. An electron microscopy analysis demonstrated that the LAMP-2 deficiency caused enlarged autophagosomes/autolysosomes in L cells, which may promote the sequestration and degradation of the PDMP-dependent large LDs. Accordingly, PDMP will be useful to explore the mechanism of LD degradation, by inducing large LDs.     

Lipid droplets and lipotoxicity during autophagy

Lipid droplets (LDs) are neutral lipid storage organelles that provide a rapidly accessible source of fatty acids (FAs) for energy during periods of nutrient deprivation. Surprisingly, lipids released by the macroautophagic/autophagic breakdown of membranous organelles are packaged and stored in new LDs during periods of prolonged starvation. Why cells would store FAs during an energy crisis was unknown. In our recent study, we demonstrated that FAs released during MTORC1-regulated autophagy are selectively channeled by DGAT1 (diacylglycerol O-acyltransferase 1) into triacylglycerol (TAG)-rich LDs. These DGAT1-dependent LDs sequester FAs and prevent the accumulation of acylcarnitines, which otherwise directly disrupt mitochondrial integrity. Our findings establish LD biogenesis as a general cellular response to periods of high autophagic flux that provide a lipid buffering system to mitigate lipotoxic cellular damage.     

Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells

Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism.     

High-throughput screening of Australian marine organism extracts for bioactive molecules affecting the cellular storage of neutral lipids

Mammalian cells store excess fatty acids as neutral lipids in specialised organelles called lipid droplets (LDs). Using a simple cell-based assay and open-source software we established a high throughput screen for LD formation in A431 cells in order to identify small bioactive molecules affecting lipid storage. Screening an n-butanol extract library from Australian marine organisms we identified 114 extracts that produced either an increase or a decrease in LD formation in fatty acid-treated A431 cells with varying degrees of cytotoxicity. We selected for further analysis a non-cytotoxic extract derived from the genus Spongia (Heterofibria). Solvent partitioning, HPLC fractionation and spectroscopic analysis (NMR, MS) identified a family of related molecules within this extract with unique structural features, a subset of which reduced LD formation. We selected one of these molecules, heterofibrin A1, for more detailed cellular analysis. Inhibition of LD biogenesis by heterofibrin A1 was observed in both A431 cells and AML12 hepatocytes. The activity of heterofibrin A1 was dose dependent with 20 μM inhibiting LD formation and triglyceride accumulation by ～50% in the presence of 50 μM oleic acid. Using a fluorescent fatty acid analogue we found that heterofibrin A1 significantly reduces the intracellular accumulation of fatty acids and results in the formation of distinct fatty acid metabolites in both cultured cells and in embryos of the zebrafish Danio rerio. In summary we have shown using readily accessible software and a relatively simple assay system that we can identify and isolate bioactive molecules from marine extracts, which affect the formation of LDs and the metabolism of fatty acids both in vitro and in vivo.     

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.     

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.     

Identification and characterization of associated with lipid droplet protein 1: A novel membrane-associated protein that resides on hepatic lipid droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.     

Lipid droplet autophagy in the yeast Saccharomyces cerevisiae

Cytosolic lipid droplets (LDs) are ubiquitous organelles in prokaryotes and eukaryotes that play a key role in cellular and organismal lipid homeostasis. Triacylglycerols (TAGs) and steryl esters, which are stored in LDs, are typically mobilized in growing cells or upon hormonal stimulation by LD-associated lipases and steryl ester hydrolases. Here we show that in the yeast Saccharomyces cerevisiae, LDs can also be turned over in vacuoles/lysosomes by a process that morphologically resembles microautophagy. A distinct set of proteins involved in LD autophagy is identified, which includes the core autophagic machinery but not Atg11 or Atg20. Thus LD autophagy is distinct from endoplasmic reticulum–autophagy, pexophagy, or mitophagy, despite the close association between these organelles. Atg15 is responsible for TAG breakdown in vacuoles and is required to support growth when de novo fatty acid synthesis is compromised. Furthermore, none of the core autophagy proteins, including Atg1 and Atg8, is required for LD formation in yeast.     

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.     

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.     

GBF1-Arf-COPI-ArfGAP-mediated Golgi-to-ER transport involved in regulation of lipid homeostasis

Eukaryotic cells store neutral lipids and cholesteryl esters in cytoplasmic lipid droplets (LDs), which are generated from the endoplasmic reticulum (ER). Accumulating lines of evidence have indicated that Golgi-to-ER-retrograde transport mediated by COPI-coated vesicles under the control of Arf small GTPases is implicated in LD formation and utilization. However, the detailed mechanism underlying the regulation of lipid homeostasis by COPI-dependent transport has been poorly understood. Here we show that LD deposition and the cellular triacylglycerol content are significantly increased by siRNA-mediated depletion of not only β-COP (a subunit of the COPI coat complex) but also GBF1 (a guanine nucleotide exchange factor for Arfs), Arf4 and Arf5 (class II Arfs), and ArfGAP1-ArfGAP3 (GTPase-activating proteins for Arfs). Although a previous proteomic study suggested the presence of COPI subunits and Arfs on LDs, we have failed to show that components of the GBF1-Arf-COPI-ArfGAP retrograde transport machinery are directly associated with and closely apposed to LDs. Furthermore, although recent studies suggested that COPI-mediated transport and GBF1 participated in delivery of adipose triglyceride lipase (ATGL) onto the LD surface, we have found that depletion of β-COP or GBF1 does not affect association of ATGL with LDs or ATGL-mediated lipolysis. On the basis of these results, we propose other mechanisms how the GBF1-Arf-COPI-ArfGAP transport machinery is implicated in the regulation of lipid homeostasis.     

Seipin collaborates with the ER membrane to control the sites of lipid droplet formation

Most cells store metabolic energy in lipid droplets (LDs). LDs are composed of a hydrophobic core, covered by a phospholipid monolayer, and functionalized by a specific set of proteins. Formation of LDs takes place in the endoplasmic reticulum (ER), where neutral lipid biosynthetic enzymes are located. Recent evidence indicate that this process is confined to specific ER subdomains, where proteins meet to initiate LD assembly. The lipodystrophy protein Seipin, is emerging as a major coordinator of LD biogenesis. Seipin forms a large oligomeric toroidal structure, which traps neutral lipids to promote LD nucleation. Here, we discuss the role of LD biogenesis factors that associate with Seipin to assemble functional LDs.     

Lipid droplet biogenesis is spatially coordinated at ER–vacuole contacts under nutritional stress

Eukaryotic cells store lipids in cytosolic organelles known as lipid droplets (LDs). Lipid droplet bud from the endoplasmic reticulum (ER), and may be harvested by the vacuole for energy during prolonged periods of starvation. How cells spatially coordinate LD production is poorly understood. Here, we demonstrate that yeast ER–vacuole contact sites (NVJs) physically expand in response to metabolic stress, and serve as sites for LD production. NVJ tether Mdm1 demarcates sites of LD budding, and interacts with fatty acyl‐CoA synthases at the NVJ periphery. Artificially expanding the NVJ through over‐expressing Mdm1 is sufficient to drive NVJ‐associated LD production, whereas ablating the NVJ induces defects in fatty acid‐to‐triglyceride production. Collectively, our data suggest a tight metabolic link between nutritional stress and LD biogenesis that is spatially coordinated at ER–vacuole contact sites.     

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.