RESUMO
Lipid droplets (LDs) are intracellular organelles that dynamically regulate lipids and energy homeostasis in the cell. LDs can grow through either local lipid synthesis or LD fusion. However, how lipids involving in LD fusion for LD growth is largely unknown. Here, we show that genetic mutation of acox-3 (acyl-CoA oxidase), maoc-1 (enoyl-CoA hydratase), dhs-28 (3-hydroxylacyl-CoA dehydrogenase), and daf-22 (3-ketoacyl-CoA thiolase), all involved in the peroxisomal ß-oxidation pathway in Caenorhabditis elegans, led to rapid fusion of adjacent LDs to form giant LDs (gLDs). Mechanistically, we show that dysfunction of peroxisomal ß-oxidation results in the accumulation of long-chain fatty acid-CoA and phosphocholine, which may activate the sterol-binding protein 1/sterol regulatory element-binding protein to promote gLD formation. Furthermore, we found that inactivation of either FAT-2 (delta-12 desaturase) or FAT-3 and FAT-1 (delta-15 desaturase and delta-6 desaturase, respectively) to block the biosynthesis of polyunsaturated fatty acids (PUFAs) with three or more double bonds (n≥3-PUFAs) fully repressed the formation of gLDs; in contrast, dietary supplementation of n≥3-PUFAs or phosphocholine bearing these PUFAs led to recovery of the formation of gLDs in peroxisomal ß-oxidation-defective worms lacking PUFA biosynthesis. Thus, we conclude that n≥3-PUFAs, distinct from other well-known lipids and proteins, promote rapid LD fusion leading to LD growth.
Assuntos
Caenorhabditis elegans , Ácidos Graxos Ômega-3 , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Coenzima A/metabolismo , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Insaturados/metabolismo , Gotículas Lipídicas/metabolismo , Fosforilcolina/metabolismo , Esteróis/metabolismoRESUMO
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. Cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (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.
Assuntos
Proteínas Reguladoras de Apoptose , Doenças Metabólicas , Proteínas Reguladoras de Apoptose/metabolismo , Homeostase , Humanos , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Doenças Metabólicas/metabolismoRESUMO
Nuclear receptors play important roles in regulating fat metabolism and energy production in humans. The regulatory functions and endogenous ligands of many nuclear receptors are still unidentified, however. Here, we report that CYP-37A1 (ortholog of human cytochrome P450 CYP4V2), EMB-8 (ortholog of human P450 oxidoreductase POR), and DAF-12 (homolog of human nuclear receptors VDR/LXR) constitute a hormone synthesis and nuclear receptor pathway in Caenorhabditis elegans This pathway specifically regulates the thermosensitive fusion of fat-storing lipid droplets. CYP-37A1, together with EMB-8, synthesizes a lipophilic hormone not identical to Δ7-dafachronic acid, which represses the fusion-promoting function of DAF-12. CYP-37A1 also negatively regulates thermotolerance and lifespan at high temperature in a DAF-12-dependent manner. Human CYP4V2 can substitute for CYP-37A1 in C. elegans This finding suggests the existence of a conserved CYP4V2-POR-nuclear receptor pathway that functions in converting multilocular lipid droplets to unilocular ones in human cells; misregulation of this pathway may lead to pathogenic fat storage.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Colestenos/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Temperatura Alta , Gotículas Lipídicas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Sistema Enzimático do Citocromo P-450/genética , Humanos , Receptores Citoplasmáticos e Nucleares/genéticaRESUMO
Biomolecular droplets formed through phase separation have a tendency to fuse. The speed with which fusion occurs is a direct indicator of condensate liquidity, which is key to both cellular functions and diseases. Using a dual-trap optical tweezers setup, we found the fusion speeds of four types of droplets to differ by two orders of magnitude. The order of fusion speed correlates with the fluorescence of thioflavinâ T, which in turn reflects the macromolecular packing density inside droplets. Unstructured protein or polymer chains pack loosely and readily rearrange, leading to fast fusion. In contrast, structured protein domains pack more closely and have to break extensive contacts before rearrangement, corresponding to slower fusion. This molecular interpretation for disparate fusion speeds provides mechanistic insight into the assembly and aging of biomolecular droplets.
Assuntos
Benzotiazóis/química , Pinças Ópticas , Fluorescência , Tamanho da PartículaRESUMO
Lipid droplets (LDs) are intracellular organelles and a central site for lipid synthesis, storage, and mobilization. The size of LDs reflects the dynamic regulation of lipid metabolism in cells. Previously, we found that cell death-inducing DFFA-like effector C (CIDEC) mediates LD fusion and growth by lipid transfer through LD-LD contact sites in adipocytes and hepatocytes. The CIDE-N domains of CIDEC molecules form homodimers, whereas the CIDE-C domain plays an important role in LD targeting and enrichment. Here, using targeted protein deletions and GFP expression coupled with fluorescence microscopy, we identified a polybasic RKKR motif in the linker region that connects the CIDE-N and CIDE-C domains of CIDEC and functions as a regulatory motif for LD fusion. We found that deletion of the linker region or mutation of the RKKR motif increases the formation of supersized LDs compared with LD formation in cells with WT CIDEC. This enhanced LD fusion activity required the interaction between CIDE-N domains. Mechanistically, we found that the RKKR motif interacts with acidic phospholipids via electrostatic attraction. Loss of this motif disrupted the protein-lipid interaction, resulting in enhanced lipid droplet fusion activity and thus formation of larger LDs. In summary, we have uncovered a CIDEC domain that regulates LD fusion activity, a finding that provides insights into the inhibitory regulation of LD fusion through CIDEC-lipid interactions.
Assuntos
Gotículas Lipídicas/metabolismo , Fosfolipídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Células 3T3 , Motivos de Aminoácidos , Animais , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Fenótipo , Ligação ProteicaRESUMO
Milk fat comprises membrane-coated droplets of neutral lipid, which constitute the predominant source of lipids for survival of the suckling neonate. From the perspective of the dairy industry, they are the basis for the manufacture of butter and essential ingredients in the production of cheese, yogurt, and specialty dairy produce. To provide mechanistic insight into the assembly and secretion of lipid droplets during lactation, we developed novel intravital imaging techniques using transgenic mice, which express fluorescently tagged marker proteins. The number 4 mammary glands were surgically prepared under a deep plane of anesthesia and the exposed glands positioned as a skin flap with intact vascular supply on the stage of a laser-scanning confocal microscope. Lipid droplets were stained by prior exposure of the glands to hydrophobic fluorescent BODIPY (boron-dipyrromethene) dyes and their formation and secretion monitored by time-lapse subcellular microscopy over periods of 1 to 2 h. Droplets were transported to the cell apex by directed (superdiffusive) motion at relatively slow and intermittent rates (0-2 µm/min). Regardless of size, droplets grew by numerous fusion events during transport and as they were budding from the cell enveloped by apical membranes. Surprisingly, droplet secretion was not constitutive but required an injection of oxytocin to induce contraction of the myoepithelium with subsequent release of droplets into luminal spaces. These novel results are discussed in the context of the current paradigm for milk fat synthesis and secretion and as a template for future innovations in the dairy industry.
Assuntos
Metabolismo dos Lipídeos , Leite/metabolismo , Animais , Membrana Celular , Feminino , Microscopia Intravital , Lactação/metabolismo , Gotículas Lipídicas , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Ocitocina/metabolismoRESUMO
Using high-resolution mass spectrometry, we have studied the synthesis of isoquinoline in a charged electrospray droplet and the complexation between cytochrome c and maltose in a fused droplet to investigate the feasibility of droplets to drive reactions (both covalent and noncovalent interactions) at a faster rate than that observed in conventional bulk solution. In both the cases we found marked acceleration of reaction, by a factor of a million or more in the former and a factor of a thousand or more in the latter. We believe that carrying out reactions in microdroplets (about 1-15 µm in diameter corresponding to 0·5 pl - 2 nl) is a general method for increasing reaction rates. The mechanism is not presently established but droplet evaporation and droplet confinement of reagents appear to be two important factors among others. In the case of fused water droplets, evaporation has been shown to be almost negligible during the flight time from where droplet fusion occurs and the droplets enter the heated capillary inlet of the mass spectrometer. This suggests that (1) evaporation is not responsible for the acceleration process in aqueous droplet fusion and (2) the droplet-air interface may play a significant role in accelerating the reaction. We argue that this 'microdroplet chemistry' could be a remarkable alternative to accelerate slow and difficult reactions, and in conjunction with mass spectrometry, it may provide a new arena to study chemical and biochemical reactions in a confined environment.
Assuntos
Espectrometria de Massas/métodos , 2,6-Dicloroindofenol/química , Aceleração , Aerossóis , Animais , Ácido Ascórbico/química , Fenômenos Biofísicos , Citocromos c/química , Cavalos , Substâncias Macromoleculares , Maltose/química , Miocárdio/metabolismo , Soluções , Solventes/química , Espectrometria de Massas por Ionização por Electrospray , Água/químicaRESUMO
Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Fígado Gorduroso/etiologia , Hepatócitos/metabolismo , Gotículas Lipídicas/patologia , Obesidade/patologia , Proteínas/metabolismo , Animais , Proteínas Reguladoras de Apoptose/antagonistas & inibidores , Proteínas Reguladoras de Apoptose/genética , Linhagem Celular , Células Cultivadas , Privação de Alimentos , Hepatócitos/citologia , Hepatócitos/patologia , Hepatócitos/ultraestrutura , Humanos , Gotículas Lipídicas/ultraestrutura , Fusão de Membrana , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Camundongos Obesos , Obesidade/metabolismo , Obesidade/fisiopatologia , Biogênese de Organelas , Tamanho das Organelas , Perilipina-2 , Transporte Proteico , Proteínas/antagonistas & inibidores , Proteínas/genética , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Cell death-inducing DFF45-like effector (CIDE) family proteins including Cidea, Cideb and Cidec/Fsp27 are expressed in many different tissues and are known as lipid droplet (LD)-and ER-associated proteins. Systematic analyses using genetically modified animal models have demonstrated that CIDE proteins play important roles in regulating various aspects of lipid homeostasis, including lipid storage, lipolysis and lipid secretion. Recent research in ours and other laboratories has revealed that CIDE proteins are crucial regulators of LD fusion and growth in the adipose tissue, liver, skin and mammary glands. CIDE-mediated LD fusion and growth is different from other membrane fusions in that it requires CIDE proteins to be enriched and clustered at the LD-LD contact sites (LDCS). The enriched CIDE proteins then allow the recruitment of other proteins to the LDCS and the formation of potential fusion pores. Neutral lipids in the smaller LDs of the contacted pair are transferred to the larger LDs, owing to the internal pressure difference, thus resulting in the fusion and growth of the LDs. This review summarizes the physiological roles of CIDE proteins in controlling lipid homeostasis, insulin sensitivity and the development of metabolic diseases including obesity, diabetes and fatty liver, with a particular focus on the role of CIDE proteins in controlling LD fusion and growth. This article is part of a Special Issue entitled: Recent Advances in Lipid Droplet Biology edited by Rosalind Coleman and Matthijs Hesselink.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Fusão de Membrana , Animais , Proteínas Reguladoras de Apoptose/genética , Diabetes Mellitus/genética , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Resistência à Insulina , Gotículas Lipídicas/patologia , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologiaRESUMO
The cell death-inducing DFF45-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec/Fsp27, regulate various aspects of lipid homeostasis, including lipid storage, lipolysis, and lipid secretion. This review focuses on the physiological roles of CIDE proteins based on studies on knockout mouse models and human patients bearing CIDE mutations. The primary cellular function of CIDE proteins is to localize to lipid droplets (LDs) and to control LD fusion and growth across different cell types. We propose a four-step process of LD fusion, characterized by (a) the recruitment of CIDE proteins to the LD surface and CIDE movement, (b) the enrichment and condensate formation of CIDE proteins to form LD fusion plates at LD-LD contact sites, (c) lipid transfer through lipid-permeable passageways within the fusion plates, and (d) the completion of LD fusion. Lastly, we outline CIDE-interacting proteins as regulatory factors, as well as their contribution in LD fusion.
Assuntos
Proteínas Reguladoras de Apoptose , Gotículas Lipídicas , Animais , Humanos , Gotículas Lipídicas/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Reguladoras de Apoptose/genética , Metabolismo dos LipídeosRESUMO
Droplet-based microfluidics is a powerful tool for producing monodispersed micrometer-sized droplets with controlled sizes and shapes; thus, it has been widely applied in diverse fields from fundamental science to industries. Toward a simpler method for fabricating microparticles with front-back asymmetry in their shapes, we studied anisotropic gelation of alginate droplets, which occurs inside a flow-focusing microfluidic device. In the proposed method, sodium alginate (NaAlg) aqueous phase fused with a calcium chloride (CaCl2) emulsion dispersed in the organic phase just before the aqueous phase breaks up into the droplets. The fused droplet with a front-back asymmetric shape was generated, and the asymmetric shape was kept after geometrical confinement by a narrow microchannel was removed. The shape of the fused droplet depended on the size of prefused NaAlg aqueous phase and a CaCl2 emulsion, and the front-back asymmetry appeared in the case of the smaller emulsion size. The analysis of the velocity field inside and around the droplet revealed that the stagnation point at the tip of the aqueous phase also played an important role. The proposed mechanism will be potentially applicable as a novel fabrication technique of microparticles with asymmetric shapes.
RESUMO
Membrane contact between intracellular organelles is important in mediating organelle communication. However, the assembly of molecular machinery at membrane contact site and its internal organization correlating with its functional activity remain unclear. Here, we demonstrate that a gel-like condensation of Cidec, a crucial protein for obesity development by facilitating lipid droplet (LD) fusion, occurs at the LD-LD contact site (LDCS) through phase separation. The homomeric interaction between the multivalent N terminus of Cidec is sufficient to promote its phase separation both in vivo and in vitro. Interestingly, Cidec condensation at LDCSs generates highly plastic and lipid-permeable fusion plates that are geometrically constrained by donor LDs. In addition, Cidec condensates are distributed unevenly in the fusion plate generating stochastic sub-compartments that may represent unique lipid passageways during LD fusion. We have thus uncovered the organization and functional significance of geometry-constrained Cidec phase separation in mediating LD fusion and lipid homeostasis.
Assuntos
Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Lipídeos , Obesidade/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Diferenciação Celular/fisiologia , Homeostase/fisiologia , Humanos , CamundongosRESUMO
Fat-Specific Protein 27 (FSP27) belongs to a small group of vertebrate proteins containing a Cell-death Inducing DNA fragmentation factor-α-like Effector (CIDE)-C domain and is involved in lipid droplet (LD) accumulation and energy homeostasis. FSP27 is predominantly expressed in white and brown adipose tissues, as well as liver, and plays a key role in mediating LD-LD fusion. No orthologs have been identified in invertebrates or plants. In this study, we tested the function of mouse FSP27 in stably-transformed Arabidopsis thaliana leaves and seeds, as well as through transient expression in Nicotiana tabacum suspension-cultured cells and N. benthamiana leaves. Confocal microscopic analysis of plant cells revealed that, similar to ectopic expression in mammalian cells, FSP27 produced in plants 1) correctly localized to LDs, 2) accumulated at LD-LD contact sites, and 3) induced an increase in the number and size of LDs and also promoted LD clustering and fusion. Furthermore, FSP27 increased oil content in transgenic A. thaliana seeds. Given that plant oils have uses in human and animal nutrition as well as industrial uses such as biofuels and bioplastics, our results suggest that ectopic expression of FSP27 in plants represents a potential strategy for increasing oil content and energy density in bioenergy or oilseed crops.
Assuntos
Arabidopsis/genética , Diacilglicerol O-Aciltransferase/genética , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos/genética , Nicotiana/genética , Proteínas/genética , Animais , Arabidopsis/metabolismo , Clonagem Molecular , Diacilglicerol O-Aciltransferase/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Gotículas Lipídicas/ultraestrutura , Fusão de Membrana , Camundongos , Tamanho das Organelas , Células Vegetais/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sementes/genética , Sementes/metabolismo , Nicotiana/metabolismoRESUMO
Lipid droplets (LDs) are central organelles in maintaining lipid homeostasis. Defective LD growth often results in the development of metabolic disorders. LD fusion and growth mediated by cell death-inducing DNA fragmentation factor alpha (DFFA)-like effector (CIDE) family proteins are crucial for various biological processes including unilocular LD formation in the adipocytes, lipid storage in the liver, milk lipid secretion in the mammary epithelia cells, and lipid secretion in the skin sebocytes. Previous methodology by Gong et al. (2011) first reported a lipid-exchange rate assay to evaluate the fusion ability of each LD pair in the cells mediated by CIDE family proteins and their regulators, but photobleaching issue remains a problem and a detailed procedure was not provided. Here, we provide an improved and detailed protocol for the lipid-exchange rate measurement. The three key steps for this assay are cell preparation, image acquisition, and data analysis. The images of the fluorescence recovery are acquired after photobleaching followed by the measurement of the intensity changes in the LD pair. The difference in fluorescent intensity is used to obtain the lipid exchange rate between the LDs. The accuracy and repetitiveness of the calculated exchange rates are assured with three-cycle of photobleaching process and the linear criteria in data fitting. With this quantitative assay, we are able to identify the functional roles of the key proteins and the effects of their mutants on LD fusion.
RESUMO
Phase separation has emerged as a new paradigm currently revolutionizing our understanding of cell biology and intracellular organization. Disordered protein domains have recently been demonstrated as integral drivers of phase separation into condensed liquids with emergent material properties. Using in vitro model systems employing purified protein components is necessary to interrogate the molecular mechanisms underlying phase separation; however, these systems pose many experimental challenges. In this chapter we describe general strategies for purifying, handling, imaging, and characterizing the phase behavior of disordered proteins. We further outline methods for the purification of the model P granule protein LAF-1, the construction of phase diagrams, and the quantification of liquid droplet fusion or coalescence.
Assuntos
Proteínas Intrinsicamente Desordenadas/química , Microscopia/métodos , Transição de Fase , Animais , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/química , Grânulos Citoplasmáticos/química , Desenho de Equipamento , Proteínas Intrinsicamente Desordenadas/isolamento & purificação , Microscopia/instrumentação , Concentração Osmolar , RNA Helicases/químicaRESUMO
We describe a droplet microfluidics method to screen for multiple mutations of a same oncogene in a single experiment using passive droplet fusion. Genomic DNA from H1573 cell-line was screened for the presence of the six common mutations of the KRAS oncogene as well as wild-type sequences with a detection efficiency of 98 %. Furthermore, the mutant allelic fraction of the cell-line was also assessed correctly showing that the technique is quantitative.
Assuntos
Análise Mutacional de DNA/métodos , Genes ras/genética , Técnicas Analíticas Microfluídicas/métodos , Microfluídica/métodos , Mutação , Biomarcadores Tumorais , Linhagem Celular Tumoral , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Microfluídica/instrumentação , Reação em Cadeia da Polimerase Multiplex/métodos , Neoplasias/diagnóstico , Neoplasias/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Estatística como Assunto/métodosRESUMO
Lipid droplets (LDs) are highly dynamic cellular organelles found in most eukaryotic cell types. In white adipocytes, LDs grow into a characteristic unilocular morphology that is well suited for its specialized role as an efficient energy storage organelle. Overexpansion of LDs in white adipocytes results in the development of obesity and insulin resistance. Besides its central role in lipid storage and mobilization, LDs play crucial roles in various cellular processes including virus packaging, host defense, protein storage, and degradation. CIDE proteins, in particular Fsp27, initiates a unique LD fusion process in adipocytes by clustering and enriching at LD contact site and promoting neutral lipid exchange and transfer between contacted LDs. Here, we summarize our approaches to quantitatively measure intracellular LD size and neutral lipid exchange between LDs. Utilization of these methods has greatly facilitated our understanding of molecular pathways governing LD growth in adipocytes.