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1 Vol May 28 doi:1.138/nature6928 Funtional genomi sreen reveals genes involved in lipid-droplet formation and utilization Yi Guo 1,4, Toias C.Walther 1,5, Meghana Rao 4, Nio Stuurman 2, Gohta Goshima 2 {, Koji Terayama 4, Jinny S. Wong 4, Ronald D. Vale 2,6, Peter Walter 1,6 & Roert V. Farese Jr 1,3,4 Eukaryoti ells store neutral lipids in ytoplasmi lipid droplets 1,2 enlosed in a monolayer of phospholipids and assoiated proteins 3,4. These dynami organelles 5 serve as the prinipal reservoirs for storing ellular energy and for the uilding loks for memrane lipids. Exessive lipid aumulation in ells is a entral feature of oesity, diaetes and atheroslerosis, yet remarkaly little is known aout lipid-droplet ell iology. Here we show, y means of a genome-wide RNA interferene (RNAi) sreen in Drosophila S2 ells that aout 1.5% of all genes funtion in lipid-droplet formation and regulation. The phenotypes of the gene knokdowns sorted into five distint phenotypi lasses. Genes enoding enzymes of phospholipid iosynthesis proved to e determinants of lipid-droplet size and numer, suggesting that the phospholipid omposition of the monolayer profoundly affets droplet morphology and lipid utilization. A suset of the Arf1 COPI vesiular transport proteins also regulated droplet morphology and lipid utilization, therey identifying a previously unreognized funtion for this mahinery. These phenotypes are onserved in mammalian ells, suggesting that insights from these studies are likely to e entral to our understanding of human diseases involving exessive lipid storage. We studied lipid-droplet formation in Drosophila Shneider 2 (S2) ells, a proven system for funtional genomi studies with effiient gene inativation y RNAi 6. We indued lipid-droplet formation y inuation with 1 mm oleate for 24 h. Staining with 4,4-difluoro- 1,3,5,7,8-pentamethyl-4-ora-3a,4a-diaza-s-indaene (493/ 53) showed that droplet size, numer and overall volume were inreased (Fig. 1a); ellular triaylglyerol ontent inreased sevenfold (Fig. 1). We onfirmed that -stained fluoresent signals orresponded to lipid droplets with a red fluoresent protein mcherry 7 fused with lipid storage droplet-1 (LSD1), whih loalizes exlusively to the surfae of lipid droplets 3 (not shown). Imaging this proess y time-lapse mirosopy of laelled ells after oleate addition (Supplementary Movie 1) showed that droplet formation ourred in steps (Fig. 1). First, inreased numers of small droplets formed in dispersed loations throughout the ell. Next, droplets inreased in size and finally aggregated into one or several large lusters, resemling grapes. Eletron mirosopy onfirmed the tight lustering of the droplets, whih were often near the nuleus (Supplementary Fig. 3). To unravel the moleular mehanisms governing this progression of hanges during lipid-droplet formation, we performed a genomewide RNAi sreen in S2 ells (Fig. 2a). Images were aquired and examined y two independent oservers, who sored them for alterations in droplet numer, size and dispersion. The same data were analysed omputationally (Supplementary Methods). From visual sreening, oth oservers identified 847 andidate genes with altered lipid-droplet morphology. To verify these genes and to minimize the misidentifiation of genes from off-target effets of RNAi treatments 8,9, RNAi experiments for these genes were repeated with a seond, distint set of doule-stranded (ds)rnas 1. Visual analyses identified 132 genes whose knokdown onsistently and repeatedly altered lipid-droplet morphology (Supplementary Tale 1) and an additional 48 genes for whih knokdown phenotypes were sored in two of three rounds (Supplementary Tale 2). Computational analysis onfirmed 86 of these 18 genes and added 47 genes with altered lipid-droplet morphology (Supplementary Tale 3). Thus, we identified 227 genes (aout 1.5% of the genome) that we onlude, with high onfidene, affet lipid-droplet morphology. However, the high stringeny of our riteria may have aused us to miss some genes involved in lipid-droplet morphology. a + Time (min) Phase ontrast Merge Figure 1 inreases the formation of lipid droplets in Drosophila S2 ells. a, S2 ells inuated for 24 h without (upper) or with (lower) 1 mm oleate. Staining with, phase-ontrast image, and overlay are shown., -loaded ells have an inreased triaylglyerol (TG) ontent. Cells were inuated as in a and their TG ontents were measured. Results are means and s.d. for three experiments; P,.5., Lipid-droplet formation ours in steps. Cells treated as in a were stained with. Single ells were followed y four-dimensional onfoal time-lapse mirosopy. Representative maximum projetions of three-dimensional staks at the indiated times are shown. Sale ars, 3 mm. TG (nmol per mg of protein) Department of Biohemistry and Biophysis, 2 Department of Cellular and Moleular Pharmaology, and 3 Department of Mediine, University of California, San Franiso, California 94158, USA. 4 Gladstone Institute of Cardiovasular Disease, San Franiso, California 94158, USA. 5 Max-Plank Institute of Biohemistry, D Martinsried, Germany. 6 Howard Hughes Medial Institute, University of California, San Franiso, California , USA. {Present address: Institute for Advaned Researh, Nagoya University, Nagoya, Japan. These authors ontriuted equally to this work. 657

2 NATURE Vol May 28 The 132 genes with striking phenotypes were ategorized into five distint phenotypi lasses (Fig. 2 and Supplementary Tale 1), whih were validated for seleted knokdowns y eletron mirosopy (Supplementary Fig. 3). Class I genes showed redued numers of droplets and inluded midway (enoding a diaylglyerol ayltransferase), suunits of the proteasome and the splieosome, and several unharaterized open reading frames. Class II genes gave smaller, more dispersed, droplets and inluded suunits of the COP9 signallosome omplex, dynein, and RNA polymerase II suunits. Class III genes showed more dispersed droplets of slightly larger size and were memers of the Arf1 COPI vesiular transport mahinery. Class IV genes yielded highly ondensed lusters of droplets and inluded memers of the translational mahinery. Class V genes ontained one or a few very large droplets and inluded an orthologue of sterol regulatory element inding-protein (SREBP), a master transriptional regulator of lipid metaolism, and SREBP leavage ativating protein (SCAP) 11. In Drosophila, the SREBP pathway is sensitive to and regulates phospholipid iosynthesis 12. This lass also inluded and Ct2, whih enode isoforms of phosphoholine ytidylyltransferase, the enzyme that atalyses the ratelimiting step in phosphatidylholine synthesis 13, and CG221, whih is predited to have holine kinase ativity that phosphorylates and ativates holine 14. Thus, most lass V genes were linked diretly or indiretly to phospholipid iosynthesis. To further explore how phospholipid metaolism regulates lipiddroplet formation, we haraterized the and Ct2 knokdowns. Larger droplets in Ct knokdowns ould arise from a failure to form new droplets, foring newly synthesized neutral lipids into a few large droplets, or from the fusion of independently formed droplets. To distinguish etween these possiilities, we oserved the dynamis of lipid-droplet formation y time-lapse mirosopy (Supplementary Movie 2) and found evidene that the droplets fuse (Fig. 3a and Supplementary Fig. 2). We then examined where CCT proteins at. In untreated ells, mcherry-tagged loalized exlusively to the nuleus (Fig. 3), similar to mammalian ytidylyltransferase-a (CT-a) 13,15. After treatment with oleate, a signifiant portion of loalized to the lipiddroplet surfae. By ontrast, similarly tagged Ct2 loalized to the ytoplasm ut was also onentrated on droplet surfaes after treatment with oleate. This marked transloation of CCT enzymes to the droplet surfae may serve to provide adequate phosphatidylholine to the phospholipid monolayers of growing lipid droplets. If so, the ratio of surfae phospholipids to ore neutral lipids may regulate lipid-droplet morphology: when phospholipids are limiting (as in or Ct2 knokdowns), fusion is indued to derease the surfae-to-volume ratio of droplets. In fat, the ontent a h 4 h 8.5 h 9.5 h 1 h 11 h mcherry Merge CCT1 CCT1 + oleate CCT2 a treatment 3 days 1 day treatment Fix, stain, image Visual soring Repeat CCT2 + oleate Numer Size Dispersion Examples I Fewer mdy Proteasome Splieosome Class II III IV V Smaller Dispersed Cdk9 COP9 Dynein RNA PolII /fewer /larger Dispersed garz COPI /larger Condensed RpLPO eifs Fewer Larger Ct2 HLH16 SCAP CG221 FAS Figure 2 Genome-wide sreen identified genes regulating the formation of lipid droplets. a, Outline for strategy to sreen for genes involved in lipiddroplet iogenesis. See the text for details., Genes involved in the sreen for lipid-droplet iogenesis fall into distint phenotypi lasses. The 132 most striking phenotypes were lassified aording to lipid-droplet numer, size and dispersion. From this lassifiation, five major lasses emerged; a graphi representation (top), an example image (middle) and some gene examples (ottom) are shown. Sale ar, 3 mm. 658 PC (nmol per mg of protein) 2 1 mdy TG (nmol per mg of protein) mdy Figure 3 Phosphatidylholine ontent regulates the size and aundane of lipid droplets. a, Lipid-droplet formation indued y oleate in knokdown ells (Fig. 1a). Single ells were followed y time-lapse onfoal mirosopy. Representative projetions revealed that droplets first proliferate normally (upper) and then fuse (lower). Examples of fusion are indiated (red and green arrows)., CCT enzymes loalize to the surfae of droplets after indution with oleate. and Ct2 were transiently expressed in S2 ells as amino-terminal mcherry-tagged fusion proteins and were stained and imaged efore or after indution. staining, mcherry fluoresene, and merge are shown. Sale ar, 3 mm., knokdown ells have less phosphatidylholine (PC) and more triaylglyerol (TG). S2 ells were treated with s as indiated, loaded with oleate (as in Fig. 1a) and lysed. PC (upper) and TG (lower) levels in the extrat were measured. Results are means and s.d. for three independent experiments., P,.1 versus ontrol RNAi.

3 NATURE Vol May 28 of phosphatidylholine in ells with knokdown was dereased y aout 6% (Fig. 3, upper panel), and the triaylglyerol ontent was inreased y aout 4% (Fig. 3, lower panel). The inrease in triaylglyerol may reflet ompensatory hannelling of diaylglyerol into neutral lipids. A dereased phosphatidylholine ontent would inrease the relative amount of phosphatidylethanolamine in the droplet monolayer (as oserved in flies laking (ref. 16)), whih itself may diretly promote droplet fusion 17. Our results suggest a model in whih phosphatidylholine availaility is a ruial regulator of lipid-droplet size and numer. We also investigated lass III genes, whose knokdowns showed slightly larger and more dispersed droplets. All lass III genes were memers of the Arf1 COPI mahinery, inluding, enoding an Arf1 family memer, a gene enoding guanine nuleoside exhange fator (GEF), garz, and genes enoding omponents of the COPI oat. Similar effets were otained y inuating ells with refeldin A, a speifi inhiitor of Arf1 exhange fators (Supplementary Fig. 5), and y expressing a dominant-negative version of, enoding the T31N mutant, analogously to dominantnegative mutants for Ras or Ran 18 (Fig. 4). To test the speifiity of this phenotype, we separately repeated RNAi knokdowns with s for Drosophila genes enoding six ARF proteins, three GEFs, two GTPase-ativating proteins, and all COPI suunits. We also tested other oat proteins, suh as lathrin suunits and omponents of the COPII oat (Fig. 4a). Only Arf79f, garz and six of eight memers of the COPI oat (a-, -, 9-, d-, - and f-cop) exhiited the lass III phenotype, indiating that our sreen identifies a highly speifi suset of vesiular transport omponents. Arf12F knokdown gave a partial phenotype. This funtion of the Arf1 COPI mahinery in lipid-droplet formation seems to have een evolutionarily onserved; similar phenotypes were found in yeast and human ells (Supplementary Fig. 4). We next sought to determine whether ats diretly on lipid droplets. ARF proteins exhange rapidly etween ative (GTPound) and inative (GDP-ound) forms, making it diffiult to loalize only the ative form. However, (T31N) inds its exhange fator tightly, and the distriution of the exhange fator is predited to reflet the loalization of ative ARF protein. Expressed (T31N) appeared diffusely in the ytosol ut was enrihed at the droplet surfae (Fig. 4). Thus, may at at the lipid-droplet surfae where, as for other ARF proteins, it interats with its GEF (presumaly enoded y garz) and reruits COPI a No Arf12F Arf51F Arf72A Arf84F CG656 (T31N) ARFs garz se71 CG31158 ArfGAP1 Gap69C ARF ARF GEFs GAPs COPI suunits a-cop -Cop Cop d-cop g-cop z-cop e-cop -like Cop sar1 se12 se24 se13 Ch Cl COPII suunits Clathrin Phase ontrast Merge Before After (T31N) d + Day Day 1 e Glyerol (µg per mg of protein) (Q71L) Transgene Figure 4 ArfI COPI omplex memers funtion in the formation of lipid droplets. a, Knokdowns of, the GEF gene gartenzwerg (garz) and speifi suunits of the COPI oat affeted lipid-droplet formation similarly. Representative images are shown. For desriptions of ontrols see Supplementary Information. GAP, GTPase-ativating protein., (T31N) loalizes to the droplet surfae and auses a similar phenotype to knokdown. (T31N) expressed as a aroxyterminal mcherry-tagged fusion protein in S2 ells was oserved y onfoal mirosopy after loading with oleate and staining with. A representative onfoal midsetion is shown for (top left), mcherry fluoresene (top right) and a merge (ottom right)., (T31N) mcherry loalizes to the droplet surfae. The photoleahed region (6 min) is indiated y a red irle. Arrows indiate the assoiation of (T31N) with the surfae of droplets. d,, and doule knokdowns lead to dereased lipolysis. S2 ells were treated with s for three days as indiated, loaded with 1 mm oleate for one day, and imaged y onfoal mirosopy after staining with (day, left panels). Representative onfoal midsetions are shown. was removed from the medium and the ells were starved for one day in serumfree medium to indue lipolysis (day 1, right). Sale ars, 3 mm., gene enoding a long-hain-fatty-aid-coa ligase (CG8732). e,, and doule knokdowns lead to dereased glyerol release to the medium. A transgene enoding (Q71L) leads to inreased release of glyerol. Experiments were as in d, and the glyerol released was measured. Results are means and s.d. for three independent experiments., P,.5, P,.1 versus ontrol RNAi (left) and versus ontrol transgene (right). 659

4 NATURE Vol May 28 omponents. A reent in vitro study showed that Arf1 and several suunits of the COPI omplex are reruited from the ytosol to purified lipid droplets in the presene of GTP-S (ref. 4). Although the reruitment of Arf1 to lipid droplets was reported to ativate phospholipase D (ref. 19), we found no effet of phospholipase D knokdown on lipid-droplet formation in Drosophila ells (not shown). The well-estalished funtions of lass III genes in Arf1 COPImediated vesiular transport 2,21 impliate this mahinery in a similar udding mehanism at the surfae of lipid droplets, possily to promote the udding-off of droplets during lipid moilization. Lipolysis is assoiated with the reak-up of larger droplets into smaller ones, presumaly to provide more surfae area for lipases 22. We examined the effet of the knokdown on lipolysis y induing lipiddroplet formation (Fig. 4d) and then induing lipid moilization y inuation with serum-free medium laking oleate. After 24 h, ontrol ells had few droplets. By ontrast, many droplets remained when was inativated. In addition, muh less glyerol, a produt of lipolysis, was released y ells laking the Arf1 COPI mahinery (Fig. 4e). Supporting a funtion of the Arf1 COPI mahinery in lipolysis, more glyerol was released y ells expressing a dominant-ative form of enoding (Q71L). These data indiate that the Arf1 COPI mahinery is required for effiient lipolysis. Our data agree with a report showing that lipolysis in murine adipoytes is aompanied y a refeldin A-sensitive proess that is required for the moilization of holesterol from storage pools in droplets 23. Inreased droplet surfae area during lipolysis would require more phospholipids in the surrounding monolayer. Beause knokdown limits phosphatidylholine amounts, we tested its effet on lipolysis. As predited, knokdown markedly dereased the effiieny of lipolysis, as seen y lipid-droplet staining (Fig. 4d) and glyerol release (Fig. 4e). The effets of knokdowns of and on lipolysis were additive, suggesting that these genes funtion independently. Arf1 COPI omplexes mediate retrograde vesiular traffiking of memranes and proteins from the Golgi apparatus to the endoplasmi retiulum 21 and are also involved in vesiular transport proesses from the trans-golgi network and endosomes 21. Notaly, the memers of the Arf1 COPI omplex we identified were reently found in a Drosophila sreen for genes involved in protein seretion and Golgi organization 24. Although the primary defet in lass III knokdowns is as yet unknown, the phenotype on lipid-droplet formation is not likely to e an indiret onsequene of inhiition of protein seretion. The effets are highly speifi and are not oserved with knokdowns of other proteins mediating seretory transport (endoplasmi retiulum transloation, COPII and lathrin). In addition, is reruited to the lipid-droplet surfae, as shown y us and others 4, where it is presumaly ativated y the loading of GTP on its exhange fator. Our study provides an initial systemati examination of the Drosophila genome to identify genes involved in lipid-droplet formation and utilization. Many genes that we identified sort to distint lasses of morphologial hanges, with eah lass ontaining funtionally related proteins. These lasses potentially link diverse proesses, suh as protein synthesis and degradation, the ell yle, and organelle movement, with lipid-droplet iology. The variety of genes identified lends support to the emerging view of lipid droplets as dynami organelles that are funtionally onneted to a variety of organelles and ellular proesses, inluding the repliation of intraellular pathogens suh as Chlamydia trahomatis and hepatitis C 25,26. Many omponents of these proesses are likely to e highly onserved aross speies. Our studies in S2 ells may therefore e diretly relevant to ellular lipid storage in general, holding the promise of identifying pathways and mehanisms entral to human diseases involving exessive lipid storage and to the engineering of ellular 66 lipid storage in organisms for the improved prodution of oils and iofuels. METHODS SUMMARY RNAi-mediated genomi sreen. RNAi sreening with University of California, San Franiso (UCSF), DmRNAi liraries versions 1 and 2 (15,683 genes) was as desried 1. Examination of seleted genes demonstrated 8 9% mrna knokdown (Supplementary Fig. 1); previous studies showed protein levels dereased y 8% (refs 27, 28). S2 ells ( ells ml 21 ) were treated with 1 mg of in 96-well plates for three days, and 1 mm olei aid was added for 24 h. Cells were fixed for 1 h in 4% paraformaldehyde/pbs and stained for 1 h with 493/53 in PBS. Images were otained with IC1 (Bekman) or ImageXpress Miro (Moleular Devies) automated mirosopes and a 43,.95 numerial aperture PlanApo dry ojetive lens (Nikon). Six image fields (4 6 ells per well) were aquired per RNAi experiment. The primary visual sreen was validated y treating S2 ells with an independent set of RNAs 8 1. Details of the visual and omputational sreening proedures are given in Supplementary Information. For all gene knokdowns, the penetrane of the phenotypes was high, affeting more than 7 8% of the ells. Primers used for the original sreen an e found at dropletsreen, and primers for seondary validation an e found at mpims.iohem.mpg.de/en/rg/lipidrophe/asatz_1.html. Protein loalization. mcherry was from R. Tsien 7. mcherry, mcherry Ct2 and mcherry expression vetors (atin promoter) were made with the Gateway system (Invitrogen). (T31N) mcherry and (Q71L) mcherry were generated y QuikChange II mutagenesis (Stratagene). Lipid measurements. Cells were ultured in six-well plates (16 mg of s per well) for three days; 1 mm oleate was then added for 24 h. Cells were lysed in 5 mm Tris-HCl ph 7.4,.25 M surose; samples (25 mg of protein) were assayed for lipids. Phosphatidylholine was quantified with a olorimetri method 29. Triaylglyerol ontent was measured from extrated lipids y thinlayer hromatography 3. Lipolysis studies. Cells were ultured with 1 mm oleate for 24 h. To stimulate lipolysis, oleate was removed and ells were ultured in serum-free medium for 24 h. Aliquots were analysed for glyerol (Sigma-Aldrih glyerol olorimetri assay) and protein (Bio-Rad D C protein assay) ontent. Reeived 4 Feruary; aepted 25 Marh 28. Pulished online 13 April Bartz, R. et al. Lipidomis reveals that adiposomes store ether lipids and mediate phospholipid traffi. J. Lipid Res. 48, (27). 2. Brown, D. A. Lipid droplets: proteins floating on a pool of fat. Curr. Biol. 11, R446 R449 (21). 3. Miura, S. et al. 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