Emerging role of lipid droplets in host/pathogen interactions

Lipid droplets (LDs) are highly dynamic cell organelles involved in energy homeostasis and membrane trafficking. Here, we review how select pathogens interact with LDs. Several RNA viruses use host LDs at different steps of their life cycle. Some intracellular bacteria and parasites usurp host LDs or encode their own lipid biosynthesis machinery, thus allowing production of LDs independently of their host. Although many mechanistic details of host/pathogen LD interactions are unknown, a picture emerges in which the unique cellular architecture and energy stored in LDs are important in the replication of diverse pathogens.     

Quantitative analysis of lipid droplet fusion: inefficient steady state fusion but rapid stimulation by chemical fusogens

Lipid droplets (LDs) are dynamic cytoplasmic organelles containing neutral lipids and bounded by a phospholipid monolayer. Previous studies have suggested that LDs can undergo constitutive homotypic fusion, a process linked to the inhibitory effects of fatty acids on glucose transporter trafficking. Using strict quantitative criteria for LD fusion together with refined light microscopic methods and real-time analysis, we now show that LDs in diverse cell types show low constitutive fusogenic activity under normal growth conditions. To investigate the possible modulation of LD fusion, we screened for agents that can trigger fusion. A number of pharmacological agents caused homotypic fusion of lipid droplets in a variety of cell types. This provided a novel cell system to study rapid regulated fusion between homotypic phospholipid monolayers. LD fusion involved an initial step in which the two adjacent membranes became continuous (<10 s), followed by the slower merging (100 s) of the neutral lipid cores to produce a single spherical LD. These fusion events were accompanied by changes to the LD surface organization. Measurements of LDs undergoing homotypic fusion showed that fused LDs maintained their initial volume, with a corresponding decrease in surface area suggesting rapid removal of membrane from the fused LD. This study provides estimates for the level of constitutive LD fusion in cells and questions the role of LD fusion in vivo. In addition, it highlights the extent of LD restructuring which occurs when homotypic LD fusion is triggered in a variety of cell types.     

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.     

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.     

Regulated localization of Rab18 to lipid droplets: effects of lipolytic stimulation and inhibition of lipid droplet catabolism

Rab GTPases are crucial regulators of membrane traffic. Here we have examined a possible association of Rab proteins with lipid droplets (LDs), neutral lipid-containing organelles surrounded by a phospholipid monolayer, also known as lipid bodies, which have been traditionally considered relatively inert storage organelles. Although we found close apposition between LDs and endosomal compartments labeled by expressed Rab5, Rab7, or Rab11 constructs, there was no detectable labeling of the LD surface itself by these Rab proteins. In contrast, GFP-Rab18 localized to LDs and immunoelectron microscopy showed direct association with the monolayer surface. Green fluorescent protein (GFP)-Rab18-labeled LDs underwent oscillatory movements in a localized area as well as sporadic, rapid, saltatory movements both in the periphery of the cell and toward the perinuclear region. In both adipocytes and non-adipocyte cell lines Rab18 localized to a subset of LDs. To gain insights into this specific localization, Rab18 was co-expressed with Cav3DGV, a truncation mutant of caveolin-3 shown to inhibit the catabolism and motility of lipid droplets. GFP-Rab18 and mRFP-Cav3DGV labeled mutually exclusive subpopulations of LDs. Moreover, in 3T3-L1 adipocytes, stimulation of lipolysis increased the localization of Rab18 to LDs, an effect reversed by beta-adrenergic antagonists. These results show that a Rab protein localizes directly to the monolayer surface of LDs. In addition, association with the LD surface was increased following stimulation of lipolysis and inhibited by a caveolin mutant suggesting that recruitment of Rab18 is regulated by the metabolic state of individual LDs.     

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.     

Controlling the size of lipid droplets: lipid and protein factors

Recent advances have transformed our understanding of lipid droplets (LDs). Once regarded as inert lipid storage granules, LDs are now recognized as multi-functional organelles that affect many aspects of cell biology and metabolism. However, fundamental questions concerning the biogenesis and growth of LDs remain unanswered. Recent studies have uncovered novel modes of LD growth (including rapid/homotypic as well as slow/atypical LD fusion), and identified key proteins (e.g. Fsp27, seipin, FITM2 and perilipin 1) and lipids (e.g. phosphatidylcholine and phosphatidic acid) that regulate the size of LDs. Phospholipids appear to have an evolutionarily conserved role in LD growth. Protein factors may regulate LD expansion directly and/or indirectly through modulating the level and composition of phospholipids on LD surface.     

Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites

Lipid droplets (LDs) are dynamic cellular organelles that control many biological processes. However, molecular components determining LD growth are poorly understood. Genetic analysis has indicated that Fsp27, an LD-associated protein, is important in controlling LD size and lipid storage in adipocytes. In this paper, we demonstrate that Fsp27 is focally enriched at the LD-LD contacting site (LDCS). Photobleaching revealed the occurrence of lipid exchange between contacted LDs in wild-type adipocytes and Fsp27-overexpressing cells but not Fsp27-deficient adipocytes. Furthermore, live-cell imaging revealed a unique Fsp27-mediated LD growth process involving a directional net lipid transfer from the smaller to larger LDs at LDCSs, which is in accordance with the biophysical analysis of the internal pressure difference between the contacting LD pair. Thus, we have uncovered a novel molecular mechanism of LD growth mediated by Fsp27.     

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.     

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.     

Postlipolytic insulin-dependent remodeling of micro lipid droplets in adipocytes

Despite the lipolysis-lipogenesis cycle being a fundamental process in adipocyte biology, very little is known about the morphological changes that occur during this process. The remodeling of lipid droplets to form micro lipid droplets (mLDs) is a striking feature of lipolysis in adipocytes, but once lipolysis ceases, the cell must regain its basal morphology. We characterized mLD formation in cultured adipocytes, and in primary adipocytes isolated from mouse epididymal fat pads, in response to acute activation of lipolysis. Using real-time quantitative imaging and electron tomography, we show that formation of mLDs in cultured adipocytes occurs throughout the cell to increase total LD surface area by ~30% but does not involve detectable fission from large LDs. Peripheral mLDs are monolayered structures with a neutral lipid core and are sites of active lipolysis. Electron tomography reveals preferential association of mLDs with the endoplasmic reticulum. Treatment with insulin and fatty acids results in the reformation of macroLDs and return to the basal state. Insulin-dependent reformation of large LDs involves two distinct processes: microtubule-dependent homotypic fusion of mLDs and expansion of individual mLDs. We identify a physiologically important role for LD fusion that is involved in a reversible lipolytic cycle in adipocytes.     

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.     

Lipid droplet proteins and metabolic diseases

Lipid droplets (LDs) are ubiquitous cellular organelles for lipid storage which are composed of a neutral lipid core bounded by a protein decorated phospholipid monolayer. Although lipid storage is their most obvious function, LDs are far from inert as they participate in maintaining lipid homeostasis through lipid synthesis, metabolism, and transportation. Furthermore, they are involved in cell signaling and other molecular events closely associated with human disease such as dyslipidemia, obesity, lipodystrophy, diabetes, fatty liver, atherosclerosis, and others. The last decade has seen a great increase in the attention paid to LD biology. Regardless, many fundamental features of LD biology remain obscure. In this review, we will discuss key aspects of LD biology including their biogenesis, growth and regression. We will also summarize the current knowledge about the role LDs play in human disease, especially from the perspective of the dynamics of the associated proteins. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.     

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.     

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 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.     

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.     

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.     

Lipid Droplets and Peroxisomes: Key Players in Cellular Lipid Homeostasis or A Matter of Fat—Store ’em Up or Burn ’em Down

Lipid droplets (LDs) and peroxisomes are central players in cellular lipid homeostasis: some of their main functions are to control the metabolic flux and availability of fatty acids (LDs and peroxisomes) as well as of sterols (LDs). Both fatty acids and sterols serve multiple functions in the cell—as membrane stabilizers affecting membrane fluidity, as crucial structural elements of membrane-forming phospholipids and sphingolipids, as protein modifiers and signaling molecules, and last but not least, as a rich carbon and energy source. In addition, peroxisomes harbor enzymes of the malic acid shunt, which is indispensable to regenerate oxaloacetate for gluconeogenesis, thus allowing yeast cells to generate sugars from fatty acids or nonfermentable carbon sources. Therefore, failure of LD and peroxisome biogenesis and function are likely to lead to deregulated lipid fluxes and disrupted energy homeostasis with detrimental consequences for the cell. These pathological consequences of LD and peroxisome failure have indeed sparked great biomedical interest in understanding the biogenesis of these organelles, their functional roles in lipid homeostasis, interaction with cellular metabolism and other organelles, as well as their regulation, turnover, and inheritance. These questions are particularly burning in view of the pandemic development of lipid-associated disorders worldwide.WORK for the past five decades on the yeast Saccharomyces cerevisiae has contributed fundamental insight into peroxisome biogenesis and function that is also relevant for mammalian cells. While LD research in yeast is still in its infancy and looks back to a much shorter history—the previous edition of YeastBook did not even mention LDs as an “organelle”—combined biochemical, cell biological, lipidomic, and proteomic studies in recent years have already contributed significant insight into LD biogenesis and function.