Lipid droplets: A dynamic organelle moves into focus

Lipid droplets (LDs) were perceived as static storage deposits, which passively participate in the energy homeostasis of both cells and entire organisms. However, this view has changed recently after the realization of a complex and highly dynamic LD proteome. The proteome contains key components of the fat mobilization system and proteins that suggest LD interactions with a variety of cell organelles, including the endoplasmic reticulum, mitochondria and peroxisomes. The study of LD cell biology, including cross-talk with other organelles, the trafficking of LDs in the cell and regulatory events involving the LD coat proteins is now on the verge of leaving its infancy and unfolds that LDs are highly dynamic cellular organelles.     

Lipid droplets interact with mitochondria using SNAP23

Triglyceride-containing lipid droplets (LD) are dynamic organelles stored on demand in all cells. These droplets grow through a fusion process mediated by SNARE proteins, including SNAP23. The droplets have also been shown to be highly motile and interact with other cell organelles, including peroxisomes and the endoplasmic reticulum. We have used electron and confocal microscopy to demonstrate that LD form complexes with mitochondria in NIH 3T3 fibroblasts. Using an in vitro system of purified LD and mitochondria, we also show the formation of the LD-mitochondria complex, in which cytosolic factors are involved. Moreover, the presence of LD markers in mitochondria isolated by subcellular fractionations is demonstrated. Finally, ablation of SNAP23 using siRNA reduced complex formation and beta oxidation, which suggests that the LD-mitochondria complex is functional in the cell.     

Whole-genome RNAi screen identifies methylation-related genes influencing lipid metabolism in Caenorhabditis elegans

Lipid droplets (LDs) are highly conserved multifunctional cellular organelles and aberrant lipid storage in LDs can lead to many metabolic diseases. However, the molecular mechanisms governing lipid dynamic changes remain elusive, and the high-throughput screen of genes influencing LD morphology was limited by lacking specific LD marker proteins in the powerful genetic tool Caenorhabditis elegans. In this study, we established a new method to conduct whole-genome RNAi screen using LD resident protein DHS-3 as a LD marker, and identified 78 genes involved in significant LD morphologic changes. Among them, mthf-1, as well as a series of methylation-related genes, was found dramatically influencing lipid metabolism. SREBP-1 and SCD1 homologs in C. elegans were involved in the lipid metabolic change of mthf-1(RNAi) worms, and the regulation of ATGL-1 also contributed to it by decreasing triacylglycerol (TAG) hydrolysis. Overall, this study not only identified important genes involved in LD dynamics, but also provided a new tool for LD study using C. elegans, with implications for the study of lipid metabolic diseases.     

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.     

The (social) lives,deaths, and biophysical phases of lipid droplets

Lipid droplets (LDs) are major lipid storage organelles, sequestering energy-rich triglycerides and serving as nutrient sinks for cellular homeostasis. Observed for over a century but generally ignored, LDs are now appreciated to play key roles in organismal physiology and disease. They also form numerous functional contacts with other organelles. Here, we highlight recent studies examining LDs from distinct perspectives of their life cycle: their biogenesis, “social” life as they interact with other organelles, and deaths via lipolysis or lipophagy. We also discuss recent work showing how changes in LD lipid content alter the biophysical phases of LD lipids, and how this may fine-tune the LD protein landscape and ultimately LD function.     

Recent Advances in Understanding Proteins Involved in Lipid Droplet Formation,Growth and Fusion

Lipid droplets （LDs） were once viewed as simple, inert lipid micelles. However, they are now known to be organelles with a rich proteome involved in a myriad of cellular processes. LDs are heterogeneous in nature with different sizes and compositions of phospholipids, neutral lipids and proteins. This review takes a focused look at the roles of proteins involved in the regulation of LD formation, expansion, and morphology. The related proteins are summarized such as the fat-specific protein （Fsp27）, fat storage-inducing trans- membrane （FIT） proteins, seipin and ADP-ribosylation factor 1-coat protein complex I （Arf-COPI）. Finally, we present important challenges in LD biology for a deeper understanding of this dynamic organelle to be achieved.     

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.     

The size and phospholipid composition of lipid droplets can influence their proteome

The proteomic makeup of lipid droplets (LDs) is believed to regulate the function of LDs, which are now recognized as important cellular organelles that are associated with many human metabolic disorders. However, factors that help determine LD proteome remain to be identified and characterized. Here we analyzed the phospholipid and protein composition of LDs isolated from wild type (WT) yeast cells, and also from fld1Δ, cds1, and ino2Δ mutant cells which produce ‘supersized’ LDs. LDs of fld1Δ and WT cells exhibited similar phospholipid profiles, whereas LDs of cds1 and ino2Δ strains had a higher (cds1) or lower (ino2Δ) percentage of phosphatidylcholine than those of WT, respectively. Unexpectedly, the presence of most known LD resident proteins was greatly reduced in the LD fraction isolated from cds1 and ino2Δ, including neutral lipid hydrolases. Consistent with this result, mobilization of neutral lipids was seriously impaired in these two strains. Contrary to the reduction of LD resident proteins, the Hsp90 family molecular chaperones, Hsc82 and Hsp82, were greatly increased in the LD fractions of cds1 and ino2Δ strains without changes at the level of expression. These data demonstrate the impact of LD phopholipids and size on the makeup of LD proteome.     

Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations

Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer—the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.     

Biogenesis of lipid droplets – how cells get fatter

Abstract

Lipid droplets are discrete organelles present in most cell types and organisms including bacteria, yeast, plants, insects and animals. Long considered as passive storage deposits, recent cell biology, proteomic and lipidomic analysis show that lipid droplets are dynamic organelles involved in multiple cellular functions. They have a central function in lipid distribution to different membrane-bound organelles and serve not only as main reservoirs of neutral lipids such as triglycerides and cholesterol but in addition, contain structural proteins, proteins involved in lipid synthesis and transmembrane proteins. A detailed model for how transmembrane proteins such as SNARE proteins can exist in lipid droplets is proposed.     

HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.     

Phospholipids diffusion on the surface of model lipid droplets

Lipid droplets (LD) are organelles localized in the membrane of the Endoplasmic Reticulum (ER) that play an important role in metabolic functions. They consist of a core of neutral lipids surrounded by a monolayer of phosphoplipids and proteins resembling an oil-in-water emulsion droplet. Many studies have focused on the biophysical properties of these LDs. However, despite numerous efforts, we are lacking information on the mobility of phospholipids on the LDs surface, although they may play a key role in the protein distribution. In this article, we developed a microfluidic setup that allows the formation of a triolein–buffer interface decorated with a phospholipid monolayer. Using this setup, we measured the motility of phospholipid molecules by performing Fluorescent Recovery After Photobleaching (FRAP) experiments for different lipidic compositions. The results of the FRAP measurements reveal that the motility of phospholipids is controlled by the monolayer packing decorating the interface.     

Lipid droplets as metabolic determinants for stemness and chemoresistance in cancer

Previously regarded as simple fat storage particles, new evidence suggests that lipid droplets (LDs) are dynamic and functional organelles involved in key cellular processes such as membrane biosynthesis, lipid metabolism, cell signalling and inflammation. Indeed, an increased LD content is one of the most apparent features resulting from lipid metabolism reprogramming necessary to support the basic functions of cancer cells. LDs have been associated to different cellular processes involved in cancer progression and aggressiveness, such as tumorigenicity, invasion and metastasis, as well as chemoresistance. Interestingly, all of these processes are controlled by a subpopulation of highly aggressive tumoral cells named cancer stem cells (CSCs), suggesting that LDs may be fundamental elements for stemness in cancer. Considering the key role of CSCs on chemoresistance and disease relapse, main factors of therapy failure, the design of novel therapeutic approaches targeting these cells may be the only chance for long-term survival in cancer patients. In this sense, their biology and functional properties render LDs excellent candidates for target discovery and design of combined therapeutic strategies. In this review, we summarise the current knowledge identifying LDs and CSCs as main contributors to cancer aggressiveness, metastasis and chemoresistance.     

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.     

Microorganism lipid droplets and biofuel development

Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech ‘factories’ to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development. [BMB Reports 2013; 46(12): 575-581]     

Imaging organelle membranes in live cells at the nanoscale with lipid-based fluorescent probes

Understanding how organelles interact, exchange materials, assemble, disassemble, and evolve as a function of space, time, and environment is an exciting area at the very forefront of chemical and cell biology. Here, we bring attention to recent progress in the design and application of lipid-based tools to visualize and interrogate organelles in live cells, especially at super resolution. We highlight strategies that rely on modification of natural lipids or lipid-like small molecules ex cellula, where organelle specificity is provided by the structure of the chemically modified lipid, or in cellula using cellular machinery, where an enzyme labels the lipid in situ. We also describe recent improvements to the chemistry upon which lipid probes rely, many of which have already begun to broaden the scope of biological questions that can be addressed by imaging organelle membranes at the nanoscale.     

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.     

Lipid droplet-organelle interactions; sharing the fats

Lipid droplets (LDs) are key cellular organelles involved in lipid storage and mobilisation. While the major signalling cascades and many of the regulators of lipolysis have been identified, the cellular interactions involved in lipid mobilisation and release remain largely undefined. In non-adipocytes, LDs are small, mobile and interact with other cellular compartments. In contrast, adipocytes primarily contain very large, immotile LDs. The striking morphological differences between LDs in adipocytes and non-adipocytes suggest that key differences must exist in the manner in which LDs in different cell types interact with other organelles. Recent studies have highlighted the complexity of LD interactions, which can be both homotypic, with each other, and heterotypic, with other organelles. The molecules involved in these interactions are also now emerging, including Rab proteins, key regulators of membrane traffic, and caveolin, an integral membrane protein providing a functional link between the cell surface and LDs. Here we summarise recent insights into the cell biology of the LD particularly focussing on the homotypic and heterotypic interactions in both adipocytes and non-adipocytes. We speculate that these interactions may involve inter-organelle membrane contact sites or a hemi-fusion type mechanism to facilitate lipid transfer.