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 PPARγ 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.

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.Key words: Lipid droplet, Mass spectrometry, Rab18, Endoplasmic reticulum, Membrane apposition SummaryRab18 localizes to lipid droplets and induces their close apposition to the endoplasmic reticulum-derived membrane

Lipid droplets are depots of neutral lipids that exist virtually in any kind of cell. Recent studies have revealed that the lipid droplet is not a mere lipid blob, but a major contributor not only to lipid homeostasis but also to diverse cellular functions. Because of the unique structure as well as the functional importance in relation to obesity, steatosis, and other prevailing diseases, the lipid droplet is now reborn as a brand new organelle, attracting interests from researchers of many disciplines.

PML-II plays a critical role in generating nuclear lipid droplets, which are associated with promyelocytic leukemia nuclear bodies as well as with the extension of the inner nuclear membrane.

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.DOI: http://dx.doi.org/10.7554/eLife.25960.001

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. INTRODUCTIONLipid droplets (CLDs) consist of a neutral lipid core with a surrounding phospholipid monolayer (Murphy and Vance, 1999;Tauchi-Sato et al., 2002). CLDs are prominent in adipose cells and steroidogenic cells, but they also exist in other cell types. With the exception of a few cell types, CLDs have been considered as inert excess lipid deposits. However, recent studies have shown that a variety of proteins are localized in CLDs, suggesting that they may play more active functional roles than previously thought. In addition to PAT family proteins, enzymes involved in eicosanoid formation, enzymes for cholesterol synthesis, signaling proteins, caveolins, and Rab proteins have been reported in CLDs (Ozeki et al., 2005, and references therein). Proteomic studies identified many more proteins of both known and unknown functions in the CLD-rich fraction (Brasaemle et al., 2004;Fujimoto et al., 2004;Liu et al., 2004;Umlauf et al., 2004). The presence of functional proteins implies that CLDs are not mere lipid storage vessels but may be involved in various cellular activities.In hepatocytes, the triglycerides in CLDs are thought to be used for the synthesis of very low-density lipoprotein (VLDL) (Gibbons et al., 2000). Enzyme activities that hydrolyze and reesterify neutral lipids are found in CLDs and/or endoplasmic reticulum (ER), but the detailed mechanism by which the CLD content is mobilized and incorporated into lipoprotein particles is still unclear (Murphy, 2001). Apolipoprotein B (ApoB)-100, the primary protein component of VLDL, is assembled with lipids in the ER lumen, and further lipidation and maturation of the lipoprotein particles occur in the ER or the pre-Go...

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. INTRODUCTIONMicrodomains enriched with cholesterol and sphingolipids, or rafts, have been postulated to exist in the cell membrane (Simons and Ikonen, 1997). Domains showing a liquid-ordered state have been visualized in model membranes (Korlach et al., 1999;Dietrich et al., 2001a), but whether similar domains exist in the biological membranes of living cells, and what their basic properties would be, including size, life span and dynamics, are still under debate (Simons and Ikonen, 1997;Edidin, 2003;Munro, 2003;Kusumi et al., 2004;Mayor and Rao, 2004;Mukherjee and Maxfield, 2004). Recent results, obtained by single-particle tracking and fluorescent resonance energy transfer experiments, have suggested that rafts in normal unstimulated cells are extremely small and may last for Ͻ1 ms (Kenworthy et al., 2004;Kusumi et al., 2004;Sharma et al., 2004). Detergent-resistant membranes have often been regarded as an in vitro correlate of rafts, but detergents themselves have been found to cause domain formation artificially (Heerklotz, 2002). These results have thus posed questions regarding the true existence of rafts in living, nonstimulated cells. In addition, although microscopic identification of rafts has been attempted in a number of studies, putative raft molecules generally show diffuse distribution in the cell membrane without any concentration at the resolution of light microscopy. This result has been interpreted in several different ways, i.e., that rafts do not in fact exist, rafts are too small to be resolved by light microscopy, rafts occupy the majority of the membrane, or rafts do exist but may be disrupted by experimental procedures.GM1 has been generally re...

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.