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White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPAR[gamma] in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity., Introduction Obesity is associated with various complications such as insulin resistance, type 2 diabetes, dyslipidemia, and atherosclerosis (1), (2). It results from an imbalance between energy intake and energy expenditure [...]

Phosphatidylinositol 3,5-bisphosphate ( PtdIns(3,5) P2) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5) P2 using freeze-fracture electron microscopy. GST-ATG18-4× FLAG was used to label PtdIns(3,5) P2 and its binding to phosphatidylinositol 3-phosphate ( PtdIns(3)P) was blocked by an excess of the p40phox PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5) P2 was concentrated in intramembrane particle ( IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5) P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5) P2 metabolism is defective. In HeLa cells, PtdIns(3,5) P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5) P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5) P2-rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy. [ABSTRACT FROM AUTHOR]

Summary Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca 2+ homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics. [ABSTRACT FROM AUTHOR]

Nuclear lipid droplets (LDs) in hepatocytes are derived from precursors of very-low-density lipoprotein in the ER lumen, but it is not known how cells lacking the lipoprotein secretory function form nuclear LDs. Here, we show that the inner nuclear membrane (INM) of U2OS cells harbors triglyceride synthesis enzymes, including ACSL3, AGPAT2, GPAT3/GPAT4, and DGAT1/DGAT2, and generates nuclear LDs in situ. mTOR inhibition increases nuclear LDs by inducing the nuclear translocation of lipin-1 phosphatidic acid (PA) phosphatase. Seipin, a protein essential for normal cytoplasmic LD formation in the ER, is absent in the INM. Knockdown of seipin increases nuclear LDs and PA in the nucleus, whereas seipin overexpression decreases these. Seipin knockdown also up-regulates lipin-1β expression, and lipin-1 knockdown decreases the effect of seipin knockdown on nuclear LDs without affecting PA redistribution. These results indicate that seipin is not directly involved in nuclear LD formation but instead restrains it by affecting lipin-1 expression and intracellular PA distribution., (© 2020 Sołtysik et al.)

Phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P 2 ] is a phosphoinositide that plays important roles in signal transduction, endocytosis, and cell migration among others. The intracellular distribution of PtdIns(3,4)P 2 has mainly been studied by observing the distribution of GFP-tagged PtdIns(3,4)P 2 -binding protein domains in live cells and by labeling with anti-PtdIns(3,4)P 2 antibody in fixed cell samples, but these methods only offer low spatial resolution results and may have pitfalls. In the present study, we developed an electron microscopic method to observe the PtdIns(3,4)P 2 distribution using the SDS-treated freeze-fracture replica labeling method. The recombinant GST-tagged pleckstrin homology (PH) domain of TAPP1 was used as the binding probe, and its binding to PtdIns(3,4)P 2 in the freeze-fracture replica was confirmed by using liposomes containing different phosphoinositides and by the lack of labeling by a mutant probe, in which one amino acid in the PH domain was substituted. The method was applied to NIH3T3 cell samples and showed that the increase of PtdIns(3,4)P 2 in cells treated with hydrogen peroxide occurs in the cytoplasmic leaflet of the plasma membrane, except in the caveolar membrane. The present method can define the distribution of PtdIns(3,4)P 2 at a high spatial resolution and will facilitate our understanding of the physiological function of this less studied phosphoinositide.

Niemann-Pick type C is a storage disease caused by dysfunction of NPC proteins, which transport cholesterol from the lumen of lysosomes to the limiting membrane of that compartment. Using freeze fracture electron microscopy, we show here that the yeast NPC orthologs, Ncr1p and Npc2p, are essential for formation and expansion of raft-like domains in the vacuolar (lysosome) membrane, both in stationary phase and in acute nitrogen starvation. Moreover, the expanded raft-like domains engulf lipid droplets by a microautophagic mechanism. We also found that the multivesicular body pathway plays a crucial role in microautophagy in acute nitrogen starvation by delivering sterol to the vacuole. These data show that NPC proteins promote microautophagy in stationary phase and under nitrogen starvation conditions, likely by increasing sterol in the limiting membrane of the vacuole.

Lipid droplets (LDs) in the nucleus of hepatocyte-derived cell lines were found to be associated with premyelocytic leukemia (PML) nuclear bodies (NBs) and type I nucleoplasmic reticulum (NR) or the extension of the inner nuclear membrane. Knockdown of PML isoform II (PML-II) caused a significant decrease in both nuclear LDs and type I NR, whereas overexpression of PML-II increased both. Notably, these effects were evident only in limited types of cells, in which a moderate number of nuclear LDs exist intrinsically, and PML-II was targeted not only at PML NBs, but also at the nuclear envelope, excluding lamins and SUN proteins. Knockdown of SUN proteins induced a significant increase in the type I NR and nuclear LDs, but these effects were cancelled by simultaneous knockdown of PML-II. Nuclear LDs harbored diacylglycerol O-acyltransferase 2 and CTP:phosphocholine cytidylyltransferase α and incorporated newly synthesized lipid esters. These results corroborated that PML-II plays a critical role in generating nuclear LDs in specific cell types., (© 2016 Ohsaki et al.)

Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1-coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf-GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs., (© 2015 Suzuki et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

The lipid droplet (LD) is different from other cellular organelles in that most of its volume is made of lipid esters and its surface is lined by a phospholipid monolayer. This uniquely lipid-dominant structure poses a problem for electron microscopy (EM) because the aldehydes commonly used as a fixative do not react with most lipids. To circumvent this difficulty and utilize the high resolving power of EM, many methods have been developed. In this chapter, we discuss methods that have been used and/or are potentially useful to study LDs. The methods include conventional EM to observe the LD core, cryoelectron microscopy to observe the LD surface, freeze-substitution, immunoelectron microscopy (pre-embedding, post-embedding, and cryosectioning methods), and freeze-fracture. Each method has strong and weak points and therefore some caution is necessary in interpreting the obtained results. In combination with methods of other disciplines, the electron microscopic techniques should contribute significantly to solving the remaining questions on LDs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Apolipoprotein B-100 (ApoB) is the principal component of very low density lipoprotein. Poorly lipidated nascent ApoB is extracted from the Sec61 translocon and degraded by proteasomes. ApoB lipidated in the endoplasmic reticulum (ER) lumen is also subjected to proteasomal degradation, but where and how it dislocates to the cytoplasm remain unknown. In the present study, we demonstrate that ApoB after lipidation is dislocated to the cytoplasmic surface of lipid droplets (LDs) and accumulates as ubiquitinated ApoB in Huh7 cells. Depletion of UBXD8, which is almost confined to LDs in this cell type, decreases recruitment of p97 to LDs and causes an increase of both ubiquitinated ApoB on the LD surface and lipidated ApoB in the ER lumen. In contrast, abrogation of Derlin-1 function induces an accumulation of lipidated ApoB in the ER lumen but does not increase ubiquitinated ApoB on the LD surface. UBXD8 and Derlin-1 bind with each other and with lipidated ApoB and show colocalization around LDs. These results indicate that ApoB after lipidation is dislocated from the ER lumen to the LD surface for proteasomal degradation and that Derlin-1 and UBXD8 are engaged in the predislocation and postdislocation steps, respectively.

An important goal of membrane biology is to define the local heterogeneity of membrane lipid composition. Here we describe a quantitative electron microscopic method that enables the localization of specific membrane lipids at the nanometer scale. The method involves freezing cells rapidly to halt the molecular motion, physically stabilizing membrane molecules in the freeze-fracture replica by the deposition of evaporated platinum and carbon layers and labeling with specific probes for electron microscopic observation. Lipids in both the outer and inner membrane leaflets can thus be labeled, and their distributions can be analyzed quantitatively by statistical methods. A major advantage of this method is that it does not require the expression of artificial probes. Therefore, this method can be applied to any cell in vitro or in vivo, and the whole procedure can be completed in 1-2 d.

Apolipoprotein B-100 (ApoB) is a major component of very-low-density lipoproteins, and is deposited in a region around lipid droplets (LDs) called the ;ApoB-crescent'. The ApoB-crescent is thought to be related to ApoB degradation because it drastically increases when proteasome or autophagy is inhibited. In the present study, we found that ApoB-crescents were significantly reduced when ApoB lipidation was suppressed by either the inhibition or knockdown of the microsomal triglyceride-transfer protein. By contrast, ApoB-crescents increased under conditions that are presumed to cause lipidated ApoB abnormalities in secretory compartments. By electron microscopic analyses, we identified the ApoB-crescent as a thin cholesterol-rich ER cistern fused to an LD, and - topologically - this structure is equivalent to a lipid-ester globule between the two leaflets of the ER membrane. ApoB localized in the thin cisternal lumen, and its binding to LDs was resistant to alkaline treatment. Overexpression of ADRP or TIP47 suppressed the increase in the number of ApoB-crescents, whereas knockdown of these proteins had the opposite effect. From these results, we inferred that the ApoB-crescent is formed by an LD that is arrested in the ER membrane by tight binding of lipidated ApoB to its luminal surface. We suggest that ApoB processing and LD formation are closely linked.

Purpose: To investigate the effect of light stimulation on lipid droplets (LDs) and LD proteins in the retinal pigment epithelium (RPE)., Methods: Dark-adapted mouse eyes were exposed to intense flashes of light, and ARPE-19 cells were treated with all-trans-retinol. The two specimens were labeled with BODIPY493/503 for LDs and with antibodies for three LD proteins: adipocyte differentiation-related protein (ADRP), TIP47, and Rab18. The labeling intensity in fluorescence microscopy was quantified by image analysis. Localization of mutated TIP47 was also examined. Immunoelectron microscopy was performed for ADRP in mouse RPE. Expression of TIP47 in ARPE-19 cells was knocked down by RNA interference (RNAi), and its effect on retinyl ester storage was measured by HPLC., Results: Both flashes of light on mouse eyes and all-trans-retinol on ARPE-19 cells caused rapid translocation of TIP47 from the cytosol to LDs, whereas ADRP distributed constitutively in LDs. The density of LDs did not show visible changes by any treatment. The localization of TIP47 to LDs was abolished when either the amino-terminal or the carboxyl-terminal half of the molecule was deleted, but was enhanced by a short deletion in the carboxyl terminus. Manipulation of TIP47 expression by RNAi or cDNA transfection did not affect the retinyl ester amounts in ARPE-19 cells significantly., Conclusions: All-trans-retinol generated by photobleaching in the retina induces rapid translocation of TIP47 to LDs in the RPE.

Presence of microdomains has been postulated in the cell membrane, but two-dimensional distribution of lipid molecules has been difficult to determine in the submicrometer scale. In the present paper, we examined the distribution of gangliosides GM1 and GM3, putative raft molecules in the cell membrane, by immunoelectron microscopy using quick-frozen and freeze-fractured specimens. This method physically immobilized molecules in situ and thus minimized the possibility of artifactual perturbation. By point pattern analysis of immunogold labeling, GM1 was shown to make clusters of <100 nm in diameter in normal mouse fibroblasts. GM1-null fibroblasts were not labeled, but developed a similar clustered pattern when GM1 was administered. On cholesterol depletion or chilling, the clustering of both endogenous and exogenously-loaded GM1 decreased significantly, but the distribution showed marked regional heterogeneity in the cells. GM3 also showed cholesterol-dependent clustering, and although clusters of GM1 and GM3 were found to occasionally coincide, these aggregates were separated in most cases, suggesting the presence of heterogeneous microdomains. The present method enabled to capture the molecular distribution of lipids in the cell membrane, and demonstrated that GM1 and GM3 form clusters that are susceptible to cholesterol depletion and chilling.

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways.

Lipid droplets (LDs) are organelles that store neutral lipids, but their regulatory mechanism is not well understood. In the present study, we identified Rab18 as an LD component of HepG2 cells by proteomic analysis, and confirmed its localization by immunohistochemistry and western blotting. Wild-type and dominant-active Rab18 localized to LDs but the dominant-negative form did not. Endogenous Rab18 coexisted with adipocyte differentiation-related protein (ADRP) in LDs, but the labeling intensity of the two proteins showed clear reciprocity. Consistent with this observation, overexpression of Rab18 induced a decrease in the amounts of ADRP in LDs in HepG2 and BALB/c 3T3 cells. Furthermore, Rab18 overexpression caused close apposition of LDs to membrane cisternae connected to the rough ER. Two other procedures that decrease ADRP, i.e. RNA interference and brefeldin A treatment, induced the same morphological change, indicating that decrease in ADRP was the cause of the LD-ER apposition. In accordance with similar structures found between ER and other organelles, we propose that the ER membrane apposed to LDs should be named the LD-associated membrane, or LAM. The present results suggested that Rab18 regulates LAM formation, which is likely to be involved in mobilizing lipid esters stored in LDs.