Adiposome and Artificial Lipid Droplets

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Construction of Nano-Droplet/Adiposome and Artificial Lipid Droplets Yang Wang, Xiao-Ming Zhou, Xuejing Ma, Yalan Du, Lemin Zheng, and Pingsheng Liu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.5b06852 • Publication Date (Web): 24 Feb 2016 Downloaded from http://pubs.acs.org on February 26, 2016

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Construction

of

Nano-Droplet/Adiposome

and

Artificial Lipid Droplets Yang Wang1, Xiao-Ming Zhou1, Xuejing Ma1,2, Yalan Du1,3, Lemin Zheng4, and Pingsheng Liu1,2* 1

National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China 2

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University of Chinese Academy of Sciences, Beijing, 100049, China

Department of Histology and Embryology, University of South China, Hengyang, Hunan, 421001, China 4

The Institute of Cardiovascular Sciences and Institute of Systems

Biomedicine, School of Basic Medical Sciences, and Key Laboratory of Molecular Cardiovascular Sciences of Ministry of Education, Peking University Health Science Center, Beijing 100191, China *Correspondence to: Pingsheng Liu, Email: [email protected], Tel.: +86-10-64888517, Fax: +8610-64888517

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Abstract

The lipid droplet (LD) is a cellular organelle that consists of a neutral lipid core with a monolayer-phospholipid membrane and associated proteins. Recent LD studies demonstrate its importance in metabolic diseases and biofuel development. However, the mechanisms governing its formation and dynamics remain elusive. Therefore, we developed an in vitro system to facilitate the elucidation of these mechanisms. We generated sphere-shaped structures with a neutral lipid core and a monolayer-phospholipid membrane by mechanically mixing neutral lipids and phospholipids followed by a two-step purification. We named the nano-droplet “adiposome”. We then recruited LD structure-like/resident proteins to the adiposome, including the bacterial MLDS, C. elegans MDT-28/PLIN-1, or mammalian perilipin-2. In addition, adipose triglyceride lipase (ATGL) and apolipoprotein A1 (apo A-I) were recruited to adiposome. We termed the functional protein-coated adiposomes, Artificial Lipid Droplets (ALDs). Using this experimental system, different proteins can be recruited to build ALDs for some biological goals and potential usage in drug delivery.

Keywords: lipid droplet, nano-droplet, adiposome, artificial lipid droplet

The lipid droplet (LD) is a cellular organelle consisting of a neutral lipid core, a monolayerphospholipid membrane, and many membrane proteins. The functions of the organelle have been determined: 1) synthesis, storage, metabolism, and transportation of lipids, 2) storage and degradation of proteins, 3) modification and production of lipid signaling molecules and hormones, 4) interaction with other cellular organelles.1-3 Many human diseases, especially metabolic diseases, have been associated with the organelle.4 The functions of LDs and the roles


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they play in the health disorders are defined by their protein and lipid content. In the last decade there has been substantial effort exerted to isolate LDs and elucidate their composition through proteomic and lipidomic studies. One of the efforts was a proteomic study of Chinese hamster ovary cells (CHO K2) which identified the Rab GTPases and other membrane trafficking proteins on LDs, thus broadening our understanding of this organelle beyond lipid synthesis.5 Additional LD proteomic analyses of CHO K2 cells, Huh7 hepatocytes,6 3T3 L1 adipocytes,7 A431 epithelial cells,8 and many other proteomic studies9 have illuminated the multiple functions of this organelle. Although the spherical shape of LDs is probably not regulated, they do contain a set of proteins that are considered to be structure-like or resident proteins. The first, perilipin, was identified in mammals by Londos’ laboratory in 1991.10 Adipocyte differentiation-related protein (ADRP) was identified one year later in Serrero’s laboratory.11 The third, Tip47 (tail-interacting protein of 47 kDa), was localized on LDs by Brasaemle’s group in 2001.12 Two conserved domains were identified in all three proteins leading to the classification of perilipin, ADRP, and Tip47 as PAT family proteins.13 Two more proteins, S3-12 and OXPAT, were later found to share sequence similarity with the PAT domains.14, 15 All five proteins were eventually renamed as perilipins 1-5.16 Based on sequence similarity, two more proteins, LSD1 and LSD2, were identified in Drosophila.17 The proteins DHS-3 and MDT-28/PLIN-1 were identified in C. elegans as LD marker, or structure-like/resident proteins.18-20 In yeast LDs, the protein Erg6p is the most abundant protein21 and LDP1 is involved in large LD formation.22 MLDP was identified as a major LD protein in green algae.23 MLDS is one of the most abundant proteins in LDs of the bacteria Rhodococcus sp. RHA1 (RHA1) and Rhodococcus opacus PD630 (PD630).24, 25


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The functions of some LD surface proteins have been identified. For example, perilipin-1 protects LD triacylglycerol (TAG) from hydrolysis but phosphorylated perilipin-1 recruits hormone sensitive lipase (HSL) to promote TAG degradation.26 Fsp27 associates with perilipin-1 to mediate LD fusion.27 Arf1 and COP1 can be recruited to LDs28 and induce small LDs to bud off from larger ones.29 LD-localized Rab proteins are involved in the interaction between LDs and other cellular organelles. For example, Rab18 regulates interactions between LDs and the endoplasmic reticulum (ER)30 and Rab5 serves a similar function for contact between LDs and early endosomes.31 Rab8, coupled with Fsp27, mediates LD fusion.32 SNARE proteins also play an important role in LD fusion33 and the interaction between LDs and mitochondria.34 Besides LD-associated proteins, some membrane phospholipids have also been found to play roles in LD dynamics. An increase in LD-associated phosphatidic acid (PA) induces an increase in LD size.35 Phosphatidylcholine (PC) homeostasis, mediated by CTP:phosphocholine cytidylyltransferase (CCT), is involved in controlling growth and fusion of LDs.36 The neutral lipids of the organelle have not been well investigated in the involvement of LD formation and dynamics, including the newly identified LD neutral lipid, ether lipid.37 Although many individual observations regarding the functions or phenomena associated with LD proteins have accumulated, we still lack a comprehensive understanding of how LDs form, the functions they serve, and the mechanisms of their regulation. It is the complexities of their composition and interactions that interferes with our ability to dissect these mechanisms. One approach to address these difficulties is to study isolated LDs in simplified experimental settings. Unfortunately, proteomic and other compositional studies have found that isolated LDs almost invariably contain fragments of ER, mitochondria, and other cellular organelles, which severely limits the conclusions that can be drawn from in vitro experiments. Similar confounding issues


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confronted the study of other cellular organelles. Fortunately, the use of phospholipid-bilayer liposomes provides an invaluable model system to study those organelles. Both integral and peripheral membrane proteins have been successfully reconstituted into liposomes and studies of the functions of these proteins have been carried out on these artificial vesicles. Therefore, a structure with neutral lipid core covered by monolayer phospholipids should be a good experimental system for LD research. Previously, several groups have tried to establish such structure. Dr. Huang and his colleagues constructed a structure, which they termed an artificial oil body (AOB), by mixing TAG, phospholipids, and the plant LD protein oleosin two decades ago.38 Using the AOB, his group and others were able to make some progress in plant LD studies. Recently, a similar structure lacking proteins was generated by Dr. Walther’s group and was termed a lipid emulsion,36 which they used to examine CCT enzymatic activity. Using lipid emulsions, Dr. Yang’s group discovered the role of PA in controlling LD size.35 Nevertheless, no experimental system has been developed yet which models LDs as closely as liposomes have for bilayer organelles. Here we describe the generation of artificial LDs which are more pure, can be reproducibly generated, and are closer to native LDs in terms of structure and composition than previous models. To avoid the formation of complicated lipid/protein structures, we first generated sphere-shaped structures with a neutral lipid core and a phospholipid-monolayer membrane by mechanically mixing neutral lipids and phospholipids followed by a purification step to remove multi-layered phospholipid structures as well as non-spherical complexes. In analogy to the liposome, we named this monolayer phospholipid membrane-bound-nano-droplet structure “adiposome”. We then recruited LD structure-like/resident proteins to the adiposome. We designated the functional protein-coated adiposomes as “artificial lipid droplets”. Using this


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experimental system, distinct mixtures of lipids and proteins can be reconstituted into a functional platform to be used for biological and medical goals.

Results Construction and characterization of adiposomes To generate a structure with a neutral lipid core and a monolayer-phospholipid membrane that mimics LDs in living organisms, we used phospholipids and neutral lipids such as TAG to construct a sphere structure (Fig. 1Aa). Briefly, we coated phospholipids onto the inner surface of an eppendorf tube, added aqueous buffer and neutral lipids, and then vortexed the sample until it formed a milky, homogenous solution (Fig. 1Ab, Materials and Methods), which resembled preparations of LDs purified from natural sources.39 This preparation was examined by light and electron microscopy (EM). Although some sphere-shaped structures containing neutral lipids (as marked by neutral lipid fluorescent dye) were found, many other types of structures were also present in the solution (Fig. 1B, Original preparation). To remove the structures lacking neutral lipids or otherwise not resembling LDs, the solution was centrifuged at high speed. The pellet and solution (Fig. 1Ab, first red arrow) were removed and examined by microscopy. The pellet consisted of many membranous structures, a few of which were spherical (Fig. 1B, Pellet fraction). The pellet-removed sample was suspended in buffer and centrifuged at low speed. The top, white fraction was removed and examined, which was enriched in large spherical structures, but also contained other non-regular structures (Fig. 1Ab, second red arrow and Fig. 1B, Upper fraction). The fraction underneath the floating white band was collected and analyzed morphologically. The images from light microscope (DIC) showed that near all structures in the fraction were spherical (Fig. 1C, DIC), and all were Nile red positive (Fig. 1C, Nile red).


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Importantly, the sizes of Nile red-labeled structures were same as those sphere-shaped structures in DIC picture, suggesting that they were all contained neutral lipid cores. These structures were then positively stained, visualized under EM (Fig. 1D, Adiposome) and compared with liposomes, artificial vesicles with double-layer phospholipid membrane (Fig. 1D, Liposome). There were clear morphological differences in the appearance between liposomes and the neutral lipid core structures. We termed these artificial lipid structures “adiposomes”, in analogy to liposomes, and plan to use them to mimic LDs in in vitro assays. To further characterize adiposomes and determine if they were bounded by monolayerphospholipid membrane, we examined them by transmission EM (Fig. 2Aa) and Cryo-EM (Fig. 2Ab). Micrographs from both EM techniques revealed structures consistent with a phospholipid monolayer, which were distinct from the bilayer membranes of mitochondria (Fig. 2A, Mitochondria). We previously developed an equation to determine purity of isolated LDs based on the ratio of neutral lipid and phospholipid, which is distinct for a monolayer bound structure.39 We applied this approach to the adiposomes to determine if their composition was consistent with a bounded phospholipid monolayer. The average size of the adiposomes was about 189 nm as measured by dynamic light scattering (DLS) (Fig. 2B). Using our derived equation, the ratio of DOPC to TAG + DOPC (v/v) should be approximately 6.6%. We extracted lipids from newly constructed adiposomes and separated these lipids using thin layer chromatography (TLC) (Fig. 2Ca). The lipids on TLC plate were visualized by iodine vapor and the lipid spots were analyzed using ImageJ (Fig. 2Cb). The ratio of DOPC/total lipids was 7.1±1.2%, similar to the calculated value, suggesting the presence of a single layer membrane structure on the adiposomes.


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The adiposomes were then compared with isolated LDs from mouse liver and brown adipose tissue (BAT), as well as bacteria. We isolated LDs from RHA1, an oleaginous bacterium,24 mouse liver, and mouse BAT.40 Adiposomes and the isolated LDs all appeared milky solution (Fig. 2D, left panel) and we used DLS to measure their average size. BAT LDs had the largest average diameter at 1848 nm, while the RHA1 LDs were much smaller with an average diameter of 493 nm. The adiposome was smaller, but still similar to RHA1 LD with an average size of 189 nm. The sizes estimated by DLS were also confirmed by the confocal microscopy observation in DIC mode (Fig. 2D, middle panel). After staining with neutral lipid dye, all LDs and the adiposomes showed sphere-shaped structures, which suggests all of them contain a neutral lipid core (Fig. 2D, right panel).

Effectors of adiposome formation and stability To optimize conditions for adiposome generation, we systematically varied two major factors, vortex time and phospholipid to neutral lipid ratio, and measured optical density (OD) for adiposome yield and applied DLS for adiposome size. Adiposome yield increased with vortex time, as seen by photograph (Fig. 3Aa) and by OD value (Fig. 3Ab). In contrast, adiposome size decreased with increasing vortex time, reaching a minimum plateau at approximately 2 min (Fig. 3Ac). The ratio of DOPC to TAG also influenced adiposome yield and size. We found that a 2:5 (DOPC:TAG) ratio produced the highest yield of adiposomes (Fig. 3Ba). Adiposome size was reduced when the ratio of DOPC and TAG was increased, reaching a minimum plateau at a ratio of 1:5 (DOPC:TAG) (Fig. 3Bb). For all additional experiments we standardized production with a 4 min vortex time and a 2:5 (phospholipid:neutral lipid) ratio.


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In animal cells there are many types of LDs differing in phospholipid and neutral lipid composition. We next constructed adiposomes with differing lipid compositions to model this diversity. First, we altered fatty acid compositions replacing phospholipids with unsaturated oleic acid (OA) with saturated stearic acid (SA) in the form of 1,2-dioctadecanoyl-sn-glycero-3phosphocholine (DSPC). When the DSPC ratio was increased adiposome yield was reduced dramatically (Fig. 3Ca). In contrast to yield, adiposome size was dramatically increased with DSPC incorporation (Fig. 3Cb). Next we altered phospholipid head groups, replacing DOPC with 1,2-di-(9Z-octadecenoyl)-snglycero-3-phosphoethanolamine (DOPE). The incorporation of DOPE did not affect the yield until the DOPC:DOPE ratio reached 1:2 (Fig. 3Da). However, increasing DOPE content resulted in significantly larger adiposomes (Fig. 3Db). In addition, we included cholesteryl esters (CE), a common neutral lipid found in LDs from several cell types and tissues such as CHO K237 and liver. Increasing cholesteryl oleate (CO) content from 0 to 20% had no impact on adiposome yield or size (Fig. 3Ea and 3Eb). However, further increases in CO content from 20% to 33% resulted in a significant reduction in adiposome yield (Fig. 3Ea) and adiposome size was reduced slightly (Fig. 3Eb). These results indicate that lipid composition can greatly influence adiposome yield and size. Finally, we determined stability of adiposomes. Adiposomes were incubated at either room temperature or 4ºC and examined over time by OD and size, as measured by DLS. During a 7 day incubation there was no significant change in adiposome concentration or size (Fig. 3F), and there were no obvious changes in morphology, as determined by microscopy (Fig. S1), suggesting that adiposomes are relatively stable.


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Reconstitution of artificial lipid droplets by recruiting LD structure-like/resident proteins LDs have been found to be highly dynamic and are involved in myriad physiological functions and interactions, mediated by their diverse protein complement.2 The proteins on LDs can be roughly divided into two categories: structure-like/resident proteins and dynamic proteins. The structure-like/resident proteins are thought to be preferentially localized to LDs and are involved in control of LD size and mediate localization of dynamic proteins. For example, phosphorylation of perilipin-1 facilitates the movement of HSL to LDs. Therefore, for adiposomes to be useful in dissecting the mechanisms of LD functions, they must be populated with structure-like/resident proteins. We termed adiposomes with recruited LD proteins, artificial lipid droplets (ALDs) (Fig. 4A). To do so, we chose three different LD structure-like/resident proteins derived from diverse organisms from bacteria to mammals, including MLDS, the major LD protein in RHA1,24 MDT-28/PLIN-1, the most abundant protein in C. elegans LDs,19 and perilipin-2, the major LD protein in non-adipose LDs of mammals.11 To visualize the distribution of the three recruited structure-like/resident proteins on ALDs, we generated three fusion proteins with GFP tags on their C-termini. The fusion proteins were incubated with adiposomes for 1 h, and the mixture was then centrifuged and the underlying solution containing unbound proteins was removed. The neutral lipids of ALDs were stained using LipidTOX red, and the ALDs were viewed under a confocal microscope. The fluorescent proteins on ALDs appeared as ringed structures, suggesting an even distribution over the ALD surface (Fig. S2). Occasionally, large aggregations of fluorescent proteins were observed. Free GFP was not detected on ALDs as shown in Supplementary Figure 2. To improve on the resolution obtainable with the confocal microscope, we visualized the ALDs under a DeltaVision


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OMX (SIM) microscope. The proteins still appeared to be evenly distributed over the ALDs at this higher resolution (Fig. 4B). Recombinant versions of these three proteins without the GFP tag were also expressed and purified (For human perilipin-2 expression and purification, see Fig. S3 and Supporting Methods). 5 µg each protein were incubated with 50 µl adiposome solution at room temperature for 1 h and the unbound proteins were removed as described. The adiposomes containing the recruited proteins were washed with 100 µl Buffer B to remove non-specifically bound proteins. As shown in Supplementary Figure 4, above 50% of the input of each of the three major structure-like/resident proteins was recruited to adiposomes (lanes 2, 5, and 8). In contrast, an equivalent mass of the plasma lipid carrier protein BSA was incubated with adiposomes but was barely detectable in the adiposome fraction (Fig. S4, lane 12), demonstrating the specificity of structure-like/resident protein recruitment. Then a series dose of tagged perilipin-2 was incubated with same amount of adiposomes. Figure 4C showed that adiposome-associated perilipin-2 reached a plateau, suggesting the saturation of tagged perilipin-2 binding. To build on the success recruiting structure-like/resident proteins to form ALDs, we next tried to recruit functional proteins to adiposomes. Since adipose triglyceride lipase (ATGL) is the key enzyme in TAG hydrolysis, we generated recombinant ATGL and incubated it with 30 µl adiposome solution for 1 h. When the concentration of SMT3-ATGL incubated with adiposomes was increased, the amount of SMT3-ATGL recruited reached a plateau at an input of 0.244 µg/µl (Fig. 5A, lane 5), demonstrating that recruitment was saturable. We then determined if temperature plays a role in the binding. After 1 h incubation at both room temperature and 4°C, roughly one third of the 10 µg SMT3-ATGL was located on ALDs (Fig. S5).


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Multiple proteins exist on natural LDs at the same time including structure-like/resident proteins and dynamic proteins. Recruiting both types of proteins on adiposomes not only models natural LDs better but also facilitates us to study how structure-like/resident proteins affect the function of dynamic proteins. We incubated 2.5 µg SMT3-tagged perilipin-2 and 2.5 µg SMT3tagged ATGL respectively or together with adiposomes. The ALDs were washed as described after 1 h incubation. Both proteins could be recruited to adiposomes with similar quantity when incubated with adiposomes respectively (Fig. S6), suggesting that ALDs with both structurelike/resident proteins and functional dynamic proteins can be constructed. Plasma lipoprotein particles, such as low density lipoprotein (LDL) and high density lipoprotein (HDL), have a structure similar to LDs in that they have a neutral lipid core bounded by a phospholipid-monolayer membrane. Therefore, we determined whether the HDL protein apolipoprotein A1 (apo A-I) could be recruited to adiposomes. After 1 h incubation with 9 µg of purified apo A-I, half of the input protein was detected on the adiposomes (Fig. 5B lane 1), suggesting that the adiposomes may also be useful in the study of HDL and other lipoproteins. Together, these data demonstrated that we have established an in vitro model for the study of LDs and lipoproteins.

Discussion The potential for insight into human metabolic disorders has fueled significant recent investment and progress in LD research. A simplified in vitro model system is critical to assist in dissecting the functions and regulation of LDs in their many cellular roles. Previously, several groups have made similar structures for their in vitro experiments.29, 35, 36 Drs. Huang and Tzen were pioneers with their construction of artificial oil bodies, accomplished by mixing plant oil


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and plant oil body proteins such as oleosin, caleosin, and steroleosin.38 However, these proteins, with their 60 amino acid hydrophobic sequence that inserts in the TAG core, are specific to plants and are not useful for experiments in animal cells. Several laboratories adapted this method in their LD research, mixing neutral lipids, phospholipids, and proteins. However, in our hands the method produced many non-spherical structures in the mixture (Fig. 1B). When we used

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C labeled DOPC to trace the yield of phospholipids, about 81% of total phospholipids

were in the pellet fraction and 11% in the upper fraction. Only 4% phospholipids were remained in final adiposome fraction (Fig. S7). Experiments in our laboratory using sonication to mix neutral lipids and phospholipids produced similar non-spherical and morphologically complex forms which are not representative of cellular LDs (Fig. S8). Our efforts at optimizing these methods through the manipulation of lipid composition, vortex times, time, and temperature did not eliminate the generation of non-spherical contaminants. Therefore, we developed a two-step purification procedure to remove the contamination and obtain a more homogenous preparation of spherical, neutral lipid-cored structures we termed adiposomes (Fig. 1C). With the exception of some plant proteins, LD structure-like/resident proteins, such as perilipin-2, MDT-28/PLIN-1, and MLDS, are peripheral membrane proteins, and thus must be recruited to pre-formed adiposomes. This recruitment process likely resembles the physiological path these proteins follow in the cell. The hydrophobic nature of the LD structure-like/resident proteins used in this study makes them hard to express in and purify from bacteria since they tend to form highly aggregated masses. For example, perilipin-2 has previously not been successfully produced as a recombinant protein. To overcome the difficulty we fused perilipin-2 with a highly hydrophilic protein. Using this strategy, along with some optimization of the protein purification, we successfully expressed


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and purified the fusion protein (Fig. S3). Since cleavage of the hydrophilic carrier protein from perilipin-2 resulted in the formation of big aggregates, we performed all experiments with the fusion protein. Following recruitment of the fusion protein to the adiposomes, we cleaved the hydrophilic protein by enzymatic digestion. The recruited free perilipin-2 remained localized to adiposomes and did not form obvious aggregates (Fig. 4B). Many functional proteins associated with LDs have significant hydrophobic regions and would likely present similar issues for expression and purification. Therefore, the two step approach we developed for perilipin-2 may be similarly useful to recruit these proteins to form ALDs with specific functions. In the absence of LDs, perilipin-2 always aggregates. As a result it is almost impossible to obtain enough soluble perilipin-2 for crystallization and structural analysis. However, perilipin-2 maintains a physiological conformation when it associates with LDs. Therefore, it may be possible to analyze perilipin-2 structure following recruitment to adiposomes. If successful, this approach could be applied to other hydrophobic LD proteins. Our ability to recruit ATGL to adiposomes provides a window into the experimental possibilities ALDs open up. ATGL spontaneously associates with adiposomes, presumably through interaction with the phospholipid monolayer. Through this experimental system, it is now possible to determine if this recruitment is sensitive to phospholipid composition. Furthermore, we can now determine if ATGL’s association with adiposomes can be modulated through the presence of structure-like/resident proteins such as perilipin-2, perilipin-1, or CGI58. Since both perilipin-2 and ATGL were successfully recruited to adiposome, this system is suitable to dissect the regulatory mechanisms controlling ATGL localization and activation, including phosphorylation, the effect of G0S2, and the presence of CGI-58. The ability to dissect


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these mechanisms of regulation will accelerate our understanding of the basic biology underlying lipolysis disorders and other related human metabolic diseases. Apolipoprotein A1 can be recruited to adiposome, which makes a possibility to utilize adiposome mimic HDL and other lipoprotein particles in blood, suggesting that adiposome can be applied as nano drug delivery carrier. Since its hydrophobic core occupied most of the volume, the capacity for hydrophobic drug should be much higher than liposome and micelle. Compared to lipid emulsion, it has much less contaminations of other structures such as liposome or micelle, which not only raises its drug delivery capacity but also makes it more stable. The adiposome only consists of naturally existed materials such as TAG and phospholipids, which has higher biocompatibility than the artificial nano polymer materials. The construction process also hardly leaves organic solvents. Not like solid lipid droplets (SLD), the TAG is liquid under room temperature and the drug stability will not be disturbed by the crystallization process. Most importantly, LD and lipoprotein particle proteins can be recruited to adiposome, making a possibility to target certain cells or tissues. Adiposome is a promising nano drug delivery system that needs to be developed.

Conclusion This study establishes a reliable and reproducible method to build stable structures termed adiposomes, in analogy to the phospholipid-bilayer liposome. Similar to liposomes, there is a great deal of flexibility to construct adiposomes with defined neutral and polar lipid compositions. Importantly, it is simple to recruit structure-like/resident and functional proteins to adiposomes to form what we term artificial lipid droplets (ALDs). The entire process is simple and can be completed within 4 h without special equipment. Finally, the adiposomes that are


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generated by this method are stable, greatly facilitating downstream experimentation. Therefore, adiposomes and ALDs have the required characteristics to support future in vitro LD functional assays.

Materials and Methods Materials 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (DOPC), 1,2-di-(9Z-octadecenoyl)-snglycero-3-phosphoethanolamine (DOPE) and 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (DSPC) were purchased from Avanti. Triacylglycerol (TAG) was extracted from rat fat pad in the laboratory. Cholesteryl oleate (CO) was purchased from Alfa Aesar. Nile red was from Sigma-Aldrich. LipidTOX red (H34476) and Colloidal Blue staining kit were obtained from Invitrogen. The Vortex-Genie 1 Touch Mixer was from Scientific Industries, Inc. Anti-perilipin2 was purchased from Abcam (ab108323). Percoll was purchased from GE Healthcare. 25% glutaraldehyde solution (EM grade) and uranyl acetate were obtained from Electron Microscopy Sciences (Hatfield, USA). Osmium tetraoxide (EM grade) was from Nakalai Tesque (Kyoto, Japan). Quetol 812 was purchased from Nisshin EM (Tokyo, Japan). Tannic acid was from Sigma. C57BL/6 mice were purchased from Vital River Laboratories (Beijing).

Construction of adiposomes 2 mg of total phospholipids in chloroform or other solvents was added to a 1.5 ml micocentrifuge tube, and the solvent was dried under a stream of N2. 100 µl of Buffer B (20 mM HEPES, 100 mM KCl, 2 mM MgCl2, pH 7.4) was added and then 5 µg of total neutral lipids was then added in the buffer. The tube contained lipids and buffer was then vortexed for 24 cycles of


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10 seconds on and 10 seconds off. Alternatively, the tubes were sonicated in a water bath sonicator at 200 W for 24 cycles of 10 seconds on and 10 seconds off. After the above processes, the milky lipid mixture was centrifuged at 20,000g. The fraction containing adiposomes formed a floating white band at the top of the tube. The underlying solution and pellet were removed. 100 µl of Buffer B was added to the fraction containing adiposomes which were resuspended by vortexing. The sample was centrifuged again at 20,000g for 5 min and the solution underneath of white band and pellet, if present, were removed. Following two cycles of this procedure no pellet formed upon centrifugation. The white band containing adiposomes was resuspended in 100 µl Buffer B again and then the sample was centrifuged at 1,000g for 5 min. The milky solution underneath a floating white band was collected, transferred to a new microcentrifuge tube, and centrifuged at 1,000g for 5 min again. The adiposomes underneath the floating white band was then collected for further morphological, biochemical, and functional analyses. The adiposome sizes were determined by dynamic light scattering (DLS, Delsa Nano C Particle Analyzer, Beckman) and the adiposome concentration was measured by optical density at 600 nm (OD600) using an Eppendorf Biophotometer.

Isolation of lipid droplets LDs were purified from different tissues and organisms by methods previously reported.39, 40 The RHA1 were cultivated in 1 L mineral salt medium. The bacteria were collected, washed twice with Buffer A (25 mM tricine, 25 mM sucrose, pH 7.8) containing 0.5 mM phenylmethanesulfonyl fluoride (PMSF), and were incubated on ice for 20 min. The cells were then ruptured in a high-pressure cell press (JNBIO JN-3000 PLUS) under a pressure of 1,200 bar. Cell debris was removed by centrifugation at 3,000g for 10 min. The supernatant (8 ml) was


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loaded in a SW40 tube and was overlaid with 3 ml of Buffer B. The LD fraction was collected from the top after centrifugation at 38,000 rpm for 1 h at 4°C. The LDs were washed with Buffer B by centrifugation at 20,000g at 4°C three times until no pellet was visible. For purification of LDs from liver and brown adipose tissue (BAT), tissues were obtained from sacrificed C57BL/6 male mice. The tissues were rinsed in ice-cold PBS, sliced into pieces in Buffer A with 0.5 mM PMSF, and then homogenized gently through a 200 µm mesh. The homogenate was centrifuged at 500g to remove tissue debris and nuclei of cells, generating a post-nuclear supernatant (PNS). The liver PNS was centrifuged in SW40 tubes with Buffer B overlaid on top at 8,000g for 20 min. For BAT, the PNS was centrifuged at 2,000g for 3 min. The LDs in the top layer was collected and washed three times by Buffer B.

Isolation of mitochondria from mouse brown adipose tissue Mitochondria were purified as previously reported.40 Briefly, BAT of one male C57BL/6 mouse was isolated and a PNS fraction was obtained. The PNS was centrifuged at 8,000g for 10 min at 4°C to obtain a mitochondrial membrane fraction. The fraction was washed with Buffer B twice and was resuspended in 400 µl Buffer B. 3 ml of 50% and 8 ml of 25% Percoll was loaded in a SW40 tube and the suspension was carefully overlaid on top. The sample was centrifuged at 18,000 rpm for 45 min at 4°C, and the interface between the 25% and 50% Percoll step gradient was collected as a purified mitochondria fraction. Percoll was removed and the mitochondria were washed twice with Buffer B by centrifugation at 20,000g for 10 min at 4°C.

Preparation of liposome


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Unilamellar liposomes of PC/PE (3:7, w/w) were prepared using a Mini-extruder set with 100 nm polycarbonate filters (Avanti) as described previously.41 In brief, aqueous suspensions of lipids were placed in the syringe at room temperature and extruded through the orifice to produce liposomes with size of approximately 100 nm.

Fluorescence Microscopy Adiposomes or purified LDs were incubated with Nile red (1 µg/ml) or LipidTOX red (1:1,000 dilution) for 30 minutes at room temperature. A drop containing 6 µl of adiposomes or LDs was applied to a slide and mixed with 2 µl mounting media and covered by a coverslip. Fluorescence images were obtained using a Zeiss M2 fluorescence microscope, Olympus FV1000 confocal microscope, or DeltaVision OMX V3 super-resolution microscope.

Positive Staining TEM For positive staining TEM, 8 µl of purified adiposomes or liposomes were loaded onto glowdischarged carbon film coated grids for one minute followed by blotting with filter paper to remove extra sample. The sample was then fixed with 1% osmium tetroxide for 10 min and rinsed with deionized water. The sample was stained with 0.1% tannic acid for 5 min and 2% uranyl acetate for 5 min, successively, and was rinsed with deionized water. Micrographs were recorded on a CM120-FEG (FEI) microscope operating at 100 kV.

Ultrathin Section The purified adiposomes were quickly mixed with melted 3% agarose (low melting point agarose, Sigma) and solidified on ice. The solidified samples were cut into blocks of


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approximately 1 mm3. For mitochondria, the mouse BAT tissue was also prepared as small blocks for subsequent operation. The resulting blocks of adiposomes and BAT tissue were fixed with 2.5% glutaraldehyde in 0.1 M PB (pH 7.4) for 30 min and then with 1% osmium tetroxide in 0.1 M PB (pH 7.4) for 1 h at room temperature. The fixed blocks were washed with deionized water, were dehydrated with ethanol, and then infiltrated and embedded with Epon (Embed 812) which was polymerized at 60°C for 24 h. 70 nm ultrathin sections were prepared using a Leica EM UC6 Ultramicrotome. The BAT tissue ultrathin sections were then additionally stained with 4% uranyl acetate for 15 min and lead citrate for 5 min respectively at room temperature. All Micrographs were recorded on a CM120-FEG (FEI) transmission electron microscope operating at 100 kV.

Cryo-EM For cryo-EM, 4 µl of fresh adiposome and mitochondria were applied to grids and blotted for 3 s in 100% humidity using a FEI Vitrobot Mark IV and were then vitrified by quickly plunging into liquid ethane pre-cooled with liquid nitrogen. Micrographs were recorded using a Gatan UltraScan4000 (model 895) 16-megapixel CCD in an FEI Titan Krios cryo-electron microscope operating at 300 kV.

Lipid analysis by thin layer chromatography Total adiposome lipids and other fractions were extracted twice with a mixture of chloroform: methanol: buffer B (1:1:1, v/v/v). For lipid droplets, total lipids were extracted using chloroform: acetone (7:3, v/v). The organic phase was collected and dried under N2. The dried lipids were dissolved in 100 µl chloroform and then applied to a silica gel plate. The samples were


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developed in a solvent system of hexane: diethyl ether: acetic acid (80:20:1, v/v/v) to separate neutral lipids. After air drying, the plate was developed in a second solvent system of chloroform: methanol: acetic acid: H2O (75:13:9:3, v/v/v/v) to separate phospholipids. The samples were visualized under saturate iodine vapor and quantified by gray scale scanning with ImageJ.

Radioisotope assay for phospholipid analysis For phospholipid content analysis of each fraction during adiposome construction, 2 mg cold DOPC was mixed with trace amount of 14C-DOPC (0850, ARC; the final radioactivity was about 17,760 dpm). The mixture was then dried by N2 and vortexed with 5 mg TAG and 100 µl Buffer B to construct adiposome. Each fraction during the construction and purification was collected and then mixed thoroughly with scintillation cocktail (PerkinElmer, 1200-439). The radioactivity was measured by Wallac 1450 Microbeta JET. The ratio of 14C-DOPC content in each fraction to the original fraction was calculated.

Expression and purification of proteins Standard molecular cloning techniques were used to fuse genes of perilipin-2 and GFP, MDT28/PLIN-1 and GFP, MLDS and GFP. Wild type perilipin-2 and ATGL were cloned into pET28a-SMT3 expression vector and expressed with N-terminal 6×-his tag and SMT3 domain. Wild type MDT-28/PLIN-1 and MLDS were cloned into pGEX-6p-1 expression vector and expressed with N-terminal GST tag. Perilipin-2-GFP, MDT-28/PLIN-1-GFP and MLDS-GFP were cloned into pET28a vector and expressed with N-terminal 6×-his tag. The detailed


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purification protocol is provided in the supplemental information (SI). Ulp1 was a kind gift of Dr. Sarah Perret’s Lab. All fusion proteins, except SMT3-ATGL, were expressed in BL21 (DE3) E. coli in 2× YT media. SMT3-ATGL was expressed in Rosetta E. coli in 2× YT media. The cells were grown to an OD600 of 0.6, induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and grown at 16ºC for 24 h. The cells were harvested by centrifugation at 4,000 rpm for 20 min (Sigma 3K30) and resuspended in 50 mM Tris-HCl, 150 mM NaCl (pH7.4). For SMT3-ATGL, buffer was supplemented with 5% glycerol. Cells were then lysed in a high-pressure cell press (JNBIO JN-3000 PLUS) followed by centrifugation to prepare a clarified lysate. Cell lysates were centrifuged at 30,000g for 50 min to remove insoluble materials. The supernatants containing soluble SMT3-perilipin-2, SMT3-ATGL, perilipin-2-GFP, MLDS-GFP and MDT28/PLIN-1-GFP proteins were applied to nickel affinity chromatography resin (Chelating Sepharose Fast Flow, Amersham Biosciences) and purified according to manufacturer's guidelines. The supernatant containing soluble GST-MDT-28/PLIN-1 and GST-MLDS were applied to GST affinity chromatography resin (Glutathione sepharose 4B, GE) and purified according to manufacturer's guidelines. Imidazole and GSH used for eluting proteins from affinity resins were removed from the purified proteins by buffer exchange using Amicon centrifugal concentrators (Millipore). The purity of the proteins was estimated by SDS-PAGE.

Recruitment of proteins to adiposome Defined quantities of purified proteins were added to adiposome preparations to a final volume of 100 µl. The mixture was gently vortexed and then incubated on ice or at room temperature for 1 h or longer. For SMT3-perilipin-2, 25 ng of Ulp1 was incubated along with SMT3-perilipin-2


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to cleave the SMT3 tag if needed. The adiposome was centrifuged at 20,000g for 5 min and the solution was removed for analysis. The remaining adiposome was then resuspended in 100 µl Buffer B and then centrifuged again. The wash procedure was repeated three times to remove non-specific bound proteins. The washed adiposome was then observed by microscopy or for protein analysis.

Acknowledgment The authors thank Dr. John Zehmer for his critical reading and useful suggestions. The authors also thank Congyan Zhang and Huimin Na for providing constructions for MLDS and MDT28/PLIN-1 expression, Wei Ji and Wei Zhao for making Cryo-EM samples and taking the pictures, Dr. Fan Wu for providing liposome samples, Shuoguo Li (Center for Biological Imaging, IBP, CAS) for her help of taking and analyzing SIM images, Shuyan Zhang for making the thin-section samples and taking EM photos, Yanxia Jia (Center for Biological Imaging, IBP, CAS) for assistance with EM and Hongjie Zhang for suggestion to radioisotope experiments. This work was supported by grant 2011CBA00906 from the Ministry of Science and Technology of China and grants from the National Natural Science Foundation of China (No. 31000365, No. 61273228, No. 81270932, No. U1402225). References 1. Martin, S.; Parton, R. G. Lipid Droplets: A Unified View of a Dynamic Organelle. Nat. Rev. Mol. Cell Biol. 2006, 7, 373-378. 2. Zehmer, J. K.; Huang, Y.; Peng, G.; Pu, J.; Anderson, R. G.; Liu, P. A Role for Lipid Droplets in Inter-Membrane Lipid Traffic. Proteomics 2009, 9, 914-921. 3. Farese, R. V., Jr.; Walther, T. C. Lipid Droplets Finally Get a Little R-E-S-P-E-C-T. Cell 2009, 139, 855-860. 4. Greenberg, A. S.; Coleman, R. A.; Kraemer, F. B.; McManaman, J. L.; Obin, M. S.; Puri, V.; Yan, Q. W.; Miyoshi, H.; Mashek, D. G. The Role of Lipid Droplets in Metabolic Disease in


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Rodents and Humans. J. Clin. Invest. 2011, 121, 2102-2110. 5. Liu, P. S.; Ying, Y. S.; Zhao, Y. M.; Mundy, D. I.; Zhu, M. F.; Anderson, R. G. W. Chinese Hamster Ovary K2 Cell Lipid Droplets Appear to Be Metabolic Organelles Involved in Membrane Traffic. J. Biol. Chem. 2004, 279, 3787-3792. 6. Fujimoto, Y.; Itabe, H.; Sakai, J.; Makita, M.; Noda, J.; Mori, M.; Higashi, Y.; Kojima, S.; Takano, T. Identification of Major Proteins in the Lipid Droplet-Enriched Fraction Isolated from the Human Hepatocyte Cell Line HuH7. Biochim. Biophys. Acta 2004, 1644, 47-59. 7. Brasaemle, D. L.; Dolios, G.; Shapiro, L.; Wang, R. Proteomic Analysis of Proteins Associated with Lipid Droplets of Basal and Lipolytically Stimulated 3T3-L1 Adipocytes. J. Biol. Chem. 2004, 279, 46835-46842. 8. Umlauf, E.; Csaszar, E.; Moertelmaier, M.; Schuetz, G. J.; Parton, R. G.; Prohaska, R. Association of Stomatin with Lipid Bodies. J. Biol. Chem. 2004, 279, 23699-23709. 9. Yang, L.; Ding, Y.; Chen, Y.; Zhang, S.; Huo, C.; Wang, Y.; Yu, J.; Zhang, P.; Na, H.; Zhang, H.; Ma, Y.; Liu, P. The Proteomics of Lipid Droplets: Structure, Dynamics, and Functions of the Organelle Conserved from Bacteria to Humans. J. Lipid Res. 2012, 53, 1245-1253. 10. Greenberg, A. S.; Egan, J. J.; Wek, S. A.; Garty, N. B.; Blanchette-Mackie, E. J.; Londos, C. Perilipin, a Major Hormonally Regulated Adipocyte-Specific Phosphoprotein Associated with the Periphery of Lipid Storage Droplets. J. Biol. Chem. 1991, 266, 11341-11346. 11. Jiang, H. P.; Serrero, G. Isolation and Characterization of a Full-Length cDNA Coding for an Adipose Differentiation-Related Protein. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 7856-7860. 12. Wolins, N. E.; Rubin, B.; Brasaemle, D. L. TIP47 Associates with Lipid Droplets. J. Biol. Chem. 2001, 276, 5101-5108. 13. Miura, S.; Gan, J. W.; Brzostowski, J.; Parisi, M. J.; Schultz, C. J.; Londos, C.; Oliver, B.; Kimmel, A. R. Functional Conservation for Lipid Storage Droplet Association Among Perilipin, ADRP, and TIP47 (PAT)-Related Proteins in Mammals, Drosophila, and Dictyostelium. J. Biol. Chem. 2002, 277, 32253-32257. 14. Wolins, N. E.; Skinner, J. R.; Schoenfish, M. J.; Tzekov, A.; Bensch, K. G.; Bickel, P. E. Adipocyte Protein S3-12 Coats Nascent Lipid Droplets. J. Biol. Chem. 2003, 278, 37713-37721. 15. Yamaguchi, T.; Matsushita, S.; Motojima, K.; Hirose, F.; Osumi, T. MLDP, a Novel PAT Family Protein Localized to Lipid Droplets and Enriched in the Heart, Is Regulated by Peroxisome Proliferator-Activated Receptor Alpha. J. Biol. Chem. 2006, 281, 14232-14240. 16. Kimmel, A. R.; Brasaemle, D. L.; McAndrews-Hill, M.; Sztalryd, C.; Londos, C. Adoption of PERILIPIN as a Unifying Nomenclature for the Mammalian PAT-Family of Intracellular Lipid Storage Droplet Proteins. J. Lipid Res. 2010, 51, 468-471. 17. Beller, M.; Riedel, D.; Jansch, L.; Dieterich, G.; Wehland, J.; Jackle, H.; Kuhnlein, R. P. Characterization of the Drosophila Lipid Droplet Subproteome. Mol. Cell. Proteomics 2006, 5, 1082-1094. 18. Zhang, P.; Na, H.; Liu, Z.; Zhang, S.; Xue, P.; Chen, Y.; Pu, J.; Peng, G.; Huang, X.; Yang, F.; Xie, Z.; Xu, T.; Xu, P.; Ou, G.; Zhang, S. O.; Liu, P. Proteomic Study and Marker Protein Identification of Caenorhabditis elegans Lipid Droplets. Mol. Cell. Proteomics 2012, 11, 317-328. 19. Na, H.; Zhang, P.; Chen, Y.; Zhu, X.; Liu, Y.; Liu, Y.; Xie, K.; Xu, N.; Yang, F.; Yu, Y.; Cichello, S.; Mak, H. Y.; Wang, M. C.; Zhang, H.; Liu, P. Identification of Lipid Droplet Structure-Like/Resident Proteins in Caenorhabditis elegans. Biochim. Biophys. Acta 2015, 1853, 2481–2491. 20. Chughtai, A. A.; Kassak, F.; Kostrouchova, M.; Novotny, J. P.; Krause, M. W.; Saudek,


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V.; Kostrouch, Z.; Kostrouchova, M. Perilipin-Related Protein Regulates Lipid Metabolism in C. elegans. PeerJ 2015, 3, e1213. 21. Athenstaedt, K.; Zweytick, D.; Jandrositz, A.; Kohlwein, S. D.; Daum, G. Identification and Characterization of Major Lipid Particle Proteins of the Yeast Saccharomyces cerevisiae. J. Bacteriol. 1999, 181, 6441-6448. 22. Zhu, Z.; Ding, Y.; Gong, Z.; Yang, L.; Zhang, S.; Zhang, C.; Lin, X.; Shen, H.; Zou, H.; Xie, Z.; Yang, F.; Zhao, X.; Liu, P.; Zhao, Z. K. Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides. Eukaryotic Cell 2015, 14, 252-264. 23. Moellering, E. R.; Benning, C. RNA Interference Silencing of a Major Lipid Droplet Protein Affects Lipid Droplet Size in Chlamydomonas reinhardtii. Eukaryotic Cell 2010, 9, 97106. 24. Ding, Y.; Yang, L.; Zhang, S.; Wang, Y.; Du, Y.; Pu, J.; Peng, G.; Chen, Y.; Zhang, H.; Yu, J.; Hang, H.; Wu, P.; Yang, F.; Yang, H.; Steinbuchel, A.; Liu, P. Identification of the Major Functional Proteins of Prokaryotic Lipid Droplets. J. Lipid Res. 2012, 53, 399-411. 25. Chen, Y.; Ding, Y.; Yang, L.; Yu, J.; Liu, G.; Wang, X.; Zhang, S.; Yu, D.; Song, L.; Zhang, H.; Zhang, C.; Huo, L.; Huo, C.; Wang, Y.; Du, Y.; Zhang, H.; Zhang, P.; Na, H.; Xu, S.; Zhu, Y.; Xie, Z.; He, T.; Zhang, Y.; Wang, G.; Fan, Z.; Yang, F.; Liu, H.; Wang, X.; Zhang, X.; Zhang, M. Q.; Li, Y.; Steinbuchel, A.; Fujimoto, T.; Cichello, S.; Yu, J.; Liu, P. Integrated Omics Study Delineates the Dynamics of Lipid Droplets in Rhodococcus opacus PD630. Nucleic Acids Res. 2014, 42, 1052-1064. 26. Miyoshi, H.; Souza, S. C.; Zhang, H. H.; Strissel, K. J.; Christoffolete, M. A.; Kovsan, J.; Rudich, A.; Kraemer, F. B.; Bianco, A. C.; Obin, M. S.; Greenberg, A. S. Perilipin Promotes Hormone-Sensitive Lipase-Mediated Adipocyte Lipolysis via Phosphorylation-Dependent and Independent Mechanisms. J. Biol. Chem. 2006, 281, 15837-15844. 27. Sun, Z.; Gong, J.; Wu, H.; Xu, W.; Wu, L.; Xu, D.; Gao, J.; Wu, J. W.; Yang, H.; Yang, M.; Li, P. Perilipin1 Promotes Unilocular Lipid Droplet Formation Through the Activation of Fsp27 in Adipocytes. Nat. Commun. 2013, 4, 1594. 28. Bartz, R.; Zehmer, J. K.; Zhu, M.; Chen, Y.; Serrero, G.; Zhao, Y.; Liu, P. Dynamic Activity of Lipid Droplets: Protein Phosphorylation and GTP-Mediated Protein Translocation. J. Proteome Res. 2007, 6, 3256-3265. 29. Thiam, A. R.; Antonny, B.; Wang, J.; Delacotte, J.; Wilfling, F.; Walther, T. C.; Beck, R.; Rothman, J. E.; Pincet, F. COPI Buds 60-nm Lipid Droplets from Reconstituted WaterPhospholipid-Triacylglyceride Interfaces, Suggesting a Tension Clamp Function. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 13244-13249. 30. Ozeki, S.; Cheng, J.; Tauchi-Sato, K.; Hatano, N.; Taniguchi, H.; Fujimoto, T. Rab18 Localizes to Lipid Droplets and Induces Their Close Apposition to the Endoplasmic ReticulumDerived Membrane. J. Cell Sci. 2005, 118, 2601-2611. 31. Liu, P. S.; Bartz, R.; Zehmer, J. K.; Ying, Y. S.; Zhu, M.; Serrero, G.; Anderson, R. G. W. Rab-Regulated Interaction of Early Endosomes with Lipid Droplets. Biochim. Biophys. Acta, Mol. Cell Res. 2007, 1773, 784-793. 32. Wu, L.; Xu, D.; Zhou, L.; Xie, B.; Yu, L.; Yang, H.; Huang, L.; Ye, J.; Deng, H.; Yuan, Y. A.; Chen, S.; Li, P. Rab8a-AS160-MSS4 Regulatory Circuit Controls Lipid Droplet Fusion and Growth. Dev. Cell 2014, 30, 378-393. 33. Bostrom, P.; Andersson, L.; Rutberg, M.; Perman, J.; Lidberg, U.; Johansson, B. R.; Fernandez-Rodriguez, J.; Ericson, J.; Nilsson, T.; Boren, J.; Olofsson, S. O. SNARE Proteins Mediate Fusion Between Cytosolic Lipid Droplets and Are Implicated in Insulin Sensitivity. Nat.


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Cell Biol. 2007, 9, 1286-1293. 34. Jagerstrom, S.; Polesie, S.; Wickstrom, Y.; Johansson, B. R.; Schroder, H. D.; Hojlund, K.; Bostrom, P. Lipid Droplets Interact with Mitochondria Using SNAP23. Cell Biol. Int. 2009, 33, 934-940. 35. Fei, W. H.; Shui, G. H.; Zhang, Y. X.; Krahmer, N.; Ferguson, C.; Kapterian, T. S.; Lin, R. C.; Dawes, I. W.; Brown, A. J.; Li, P.; Huang, X.; Parton, R. G.; Wenk, M. R.; Walther, T. C.; Yang, H. Y. A Role for Phosphatidic Acid in the Formation of "Supersized" Lipid Droplets. PLoS Genet. 2011, 7. 36. Krahmer, N.; Guo, Y.; Wilfling, F.; Hilger, M.; Lingrell, S.; Heger, K.; Newman, H. W.; Schmidt-Supprian, M.; Vance, D. E.; Mann, M.; Farese, R. V.; Walther, T. C. Phosphatidylcholine Synthesis for Lipid Droplet Expansion Is Mediated by Localized Activation of CTP:Phosphocholine Cytidylyltransferase. Cell Metab. 2011, 14, 504-515. 37. Bartz, R.; Li, W. H.; Venables, B.; Zehmer, J. K.; Roth, M. R.; Welti, R.; Anderson, R. G. W.; Liu, P. S.; Chapman, K. D. Lipidomics Reveals That Adiposomes Store Ether Lipids and Mediate Phospholipid Traffic. J. Lipid Res. 2007, 48, 837-847. 38. Tzen, J. T. C.; Huang, A. H. C. Surface-Structure and Properties of Plant Seed Oil Bodies. J. Cell Biol. 1992, 117, 327-335. 39. Ding, Y. F.; Zhang, S. Y.; Yang, L.; Na, H. M.; Zhang, P.; Zhang, H. N.; Wang, Y.; Chen, Y.; Yu, J. H.; Huo, C. X.; Xu, S. M.; Garaiova, M.; Cong, Y. S.; Liu, P. S. Isolating Lipid Droplets from Multiple Species. Nat. Protoc. 2013, 8, 43-51. 40. Yu, J.; Zhang, S.; Cui, L.; Wang, W.; Na, H.; Zhu, X.; Li, L.; Xu, G.; Yang, F.; Christian, M.; Liu, P. Lipid Droplet Remodeling and Interaction with Mitochondria in Mouse Brown Adipose Tissue During Cold Treatment. Biochim. Biophys. Acta 2015, 1853, 918-928. 41. Nakatogawa, H.; Ichimura, Y.; Ohsumi, Y. Atg8, a Ubiquitin-Like Protein Required for Autophagosome Formation, Mediates Membrane Tethering and Hemifusion. Cell 2007, 130, 165-178.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at http://pubs.acs.org.


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Figure 1. Adiposomes were generated by vortexing followed by a two-step centrifugation-based purification. A. Flowchart depicting adiposome construction. a. Adiposome was constructed by phospholipids and neutral lipids such as triacylglycerol (TAG). b. Briefly, 2 mg phospholipid was dried on the inner surface of an EP tube, and then 100 µl Buffer B and 5 mg TAG were added. The mixture was vortexed for 24 cycles, 10 s on/10 s off. The pellet and solution were removed after centrifugation and the floating adiposome fraction was resuspended in 100 µl Buffer B. The adiposome-containing fraction was then centrifuged again at a speed of 1,000g for 5 min. The floating upper fraction was removed and the adiposome was collected. Blue arrow stands for vortex. Red arrows show the fractions being removed. B. The morphology of fractions during adiposome construction. a-b. Each fraction was suspended in 100 µl Buffer B and stained by Nile red and then imaged by DIC (a) and fluorescence microscopy (b). Arrows show the non-spherical structures. Bar=10 µm. c. The fractions were imaged by TEM after positive staining. Bar=1 µm. C. The morphology of purified adiposomes. The purified adiposomes were treated as described above for imaging by DIC (a) and fluorescence microscopy (b). Bar=10 µm. D. The adiposomes and liposomes were positive stained and observed under TEM. Bar= 500 nm. 190x190mm (300 x 300 DPI)


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Figure 2. Adiposome characteristics and comparison with organelles and liposomes. A. Morphology of adiposomes and brown adipose tissue (BAT) mitochondria. The adiposomes and mitochondria were observed under TEM by ultra-thin section (a) and by cryo-EM (b). Bar= 500 nm. B. Adiposome size. Adiposome diameter was determined by dynamic light scattering (DLS). Polydispersity Index=0.085. C. TLC analysis of DOPC/total lipids ratio of fractions during adiposome construction. a. Ten percent of the total extracted lipids were subjected to thin layer chromatography (TLC) and were stained by iodine. TAG: triacylglycerol; DAG: diacylglycerol; DOPC: 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine. b. The ratio of DOPC to total lipids of fractions was measured. The grey scale was analyzed by ImageJ. n=3, average±S.D. D. Adiposome and LD morphology. Adiposomes and LDs from mouse liver, BAT and RHA1 were purified and observed by DIC and fluorescence microscopy (stained by Nile red). Bar=5 µm. 150x190mm (300 x 300 DPI)


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Figure 3. The influence of various factors on adiposome formation, size and stability. A. Adiposome morphology (a), concentration (b) and size (c) following different vortex times. B. Concentration (a) and size (b) of adiposomes generated with different ratios of DOPC and TAG. DOPC: 1,2-di-(9Z-octadecenoyl)sn-glycero-3-phosphocholine; TAG: triacylglycerol. C. Concentration (a) and size (b) of adiposomes made with different ratios of DOPC and DSPC (1,2-dioctadecanoyl-sn-glycero-3-phosphocholine). D. Concentration (a) and size (b) of adiposomes made with different ratios of DOPC and DOPE (1,2-di-(9Z-octadecenoyl)-snglycero-3-phosphoethanolamine). E. Concentration (a) and size (b) of adiposomes made with different ratios of TAG and CO (cholesteryl oleate). F. Concentration (a) and size (b) of adiposomes incubated at 4°C or room temperature for 7 days. 204x190mm (300 x 300 DPI)


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Figure 4. Generation of artificial lipid droplets (ALDs) through recruitment of LD resident/structural-like proteins. A. A scheme of ALD Construction. The ALDs were generated by recruitment of lipid droplet (LD) resident/structural-like proteins. B. The adiposome could recruit LD resident/structural-like protein-GFP fusion proteins. Ten micrograms of MLDS-GFP, MDT-28-GFP, human perilipin-2-GFP or free GFP were incubated with adiposomes. Fluorescence was imaged using a DeltaVision OMX (SIM) microscope following three washes and LipidTOX red staining. Bar=5 µm. C. The perilipin-2 recruited on adiposome can be saturated. Mouse SMT3-perilipin-2 with a series of concentration was incubated with adiposome and the protein on adiposome was determined by SDS-PAGE with Colloidal Blue staining (a) or Western blot with anti-perilipin-2 (b). SMT3-perilipin-2 concentration from lane 1-8: 0.089, 0.173, 0.326, 0.462, 0.583, 0.791, 0.963, 1.108 µg/µl. 163x190mm (300 x 300 DPI)


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Figure 5. Recruitment of ATGL and apo A-I to adiposomes. A. A series of doses of SMT3-ATGL was incubated with adiposomes and the non-specific bound proteins were removed by three washes. The adiposomeassociated ATGL was analyzed by silver staining (a) or Western blot (b). SMT3-ATGL concentration from lane 1-7: 0.091，0.132，0.171，0.209，0.244，0.278，0.310 µg/µl. B. 9 µg purified human apo A-I, the major protein of HDL, were incubated with adiposomes at 4°C for 1 h and the non-specific bound proteins were removed by three washes. The adiposome-associated apo A-I was analyzed by silver staining. 229x173mm (300 x 300 DPI)


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Artificial Lipid Droplet ACS Paragon Plus Environment

Adiposome and Artificial Lipid Droplets

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