lseigadgot,adi nihda ii rpe junctions droplet lipid at Ha Lucken-Ardjomande enriched Safa droplet is lipid and promotes growth, that and protein clustering brain-specific a is GRAF1a ARTICLE RESEARCH ß 4602 2014 August 8 Accepted 2013; December 11 attributed. properly Received is work original the Attribution that Commons provided Creative medium the any of distribution in terms use, reproduction the unrestricted and under permits distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is This [email protected]) ([email protected]; correspondence for *Authors CB2 Cambridge Avenue, Crick Francis UK. Biology, 0QH, Molecular of Laboratory MRC aggregation where site the be to proposed of are and (Go 2007) LD al., patients Moran, et Alzheimer’s 2003), Opstad of 1999; al., brains al., the et resting et (Barba under Zoula Re tumours LDs 2007; 2011; brain of in amount al., rises low 2007). content et a Welte, (Etschmaier has 2011; brain al., conditions et the Suzuki though lipids and 2011; polar Even triglycerides of Parton, mainly monolayer and core, a (Fujimoto lipid organelles. by neutral surrounded storage a cholesterol, lipid esterified of cytoplasmic made are are They (LDs) droplets Lipid INTRODUCTION protein BAR droplet, Lipid ARHGAP26, GRAF1, WORDS: KEY volume GRAF1 droplet not lipid in did total accumulated the mice interface that phenotype, abnormal GRAF1-knockout the gross any Although at acids. show droplets. fatty concentrated with lipid GRAF1a overloaded between conditions, cells lipid these of promoted chase Under perturbed the GRAF1a severely following and of lipolysis mobility Overexpression droplet inhibited GRAF1a-specific domain. clustering, BAR droplet a PH a primary regions, a required oleic-acid-fed membrane-binding and two in localization and droplets segment Exclusive hydrophobic lipid cells. on glial found Endogenous neonates. was of brains GRAF1a the as in known also found enriched is was We (GRAF1 ARHGAP26), isoform brain. GRAF1 the longest the in GRAF1a, that homeostasis their the associated low controlling about are at known mechanisms and is present little tumours However, Normally in diseases. accumulate neurodegenerative types. with they cell brain, all the in in levels found are droplets Lipid ABSTRACT at cd a eue oprdt GRAF1 to compared reduced was acids fatty ihmmrn cltn A oan lyarl ndroplet in role a play proteins domains that BAR suggest homeostasis. sculpting and cells, membrane non-adipocyte with lipid in to growth contributing mechanisms droplet the into insights additional provide h motneo D ntebani lohglgtdb the is by mutation highlighted whose also proteins is spartin, brain and the seipin in that LDs observation of importance The 04 ulse yTeCmayo ilgssLd|Junlo elSine(04 2,40–69doi:10.1242/jcs.147694 4602–4619 127, (2014) Science Cell of Journal | Ltd Biologists of Company The by Published 2014. a sncenocr nPrisnsptet Cl ta. 2002). al., et (Cole patients Parkinson’s in occurs -synuclein ´ ye l,19) D r lorpre oacmlt in accumulate to reported also are LDs 1997). al., et my 2 / 2 rmr lauo nuainwith incubation upon glia primary se* vneVli,HlnE oi,Ade .MKni n avyT McMahon* T. Harvey and McKenzie N. Andrew Jolin, E. Helen Vallis, Yvonne ¨sler*, ´ e-ao n Asuncion and mez-Ramos +/+ el.Teeresults These cells. soitdwt eea iess ugsigte r fulfilling are they suggesting instead. diseases, roles domain important are several they proline-rich However, with functions. their a associated about SH3 known has is C-terminal little al., a OPHN1 Relatively et possess whereas Taylor GRAF3 2001; and domain, al., GRAF2 et Shibata GRAF1, al., et 1996; 1999). Billuart al., with 2013; et al., domain, Hildebrand et GAP (Bai 1998; central GTPases Rho a (also and small and for membrane-binding specificity domains ARHGAP42) GRAF2 PH N-terminal and called have BAR ARHGAP26), (also (OPHN1)] called GRAF3 oligophrenin-1 ARHGAP10), (also called [GRAF1 label to 2006). family but al., 2006), al., et al., et (Ohsaki et populations Larigauderie Sztalryd LD 2009; 2010; distinct al., somewhat al., et et are Bulankina McIntosh growth (TIP47) 2009; LD 2006; PLIN3 promote al., to and et al., thought (Buers (ADRP) are et Both PLIN2 (Bickel expressed. LDs only ubiquitously bind these, share to Of capacity respectively) 2009). the and PLIN1–PLIN5, similarity renamed sequence recently LSDP5, cells. control non-adipocyte to and intrinsic likely adipocytes the the thus in in are homeostasis hence difference mechanisms LD low a Distinct and also LDs. but ratio, the of in core, curvature surface-to-volume results LD in only the in to not diameters accessibility difference This 2011). with major al., a et LDs, (Suzuki range several micrometre contain generally 02 uiooe l,20;SulyadHsan 02 Thiele 2012; the Hussain, and and Sturley Wolins, 2008; bilayer al., and (ER) et (Brasaemle Fujimoto reticulum proteins two 2012; LD-associated the endoplasmic between of lipids the recruitment neutral of of accumulation leaflets the with start is to brain conditions. progression the neurological and of onset in the variety in a LDs homeostasis of of role the LD determine system to controlling essential nervous mechanisms central understanding and Therefore, the other 2007). schizophrenia Hatcher, and in diseases, (Adibhatla epilepsy involved Niemann–Pick is be including metabolism lipid disorders to Deregulated al., thought 2007). et al., also (Eastman LDs et to Szymanski target 2009; diseases, neuronal with associated hc,udrrsigcniin,hv n ag Dwt a with LD 100 large to one up have conditions, of resting diameter under al., which, et (Binns 2008), Parton, peroxisomes and and (Martin 2006) ER al., 2006). the et with (Nan and mitochondria 2011), (Bostro al., LDs et other their Gong with facilitating on exchange by changing lipid and travel by and 2013), contact al., homeostasis LD then et 2003). lipid (Orlicky al., distribution could to cytoplasmic et contribute that Targett-Adams can 2004; monolayer mobility al., et lipid (Pol LDs a microtubules small to by lead would scission surrounded and Budding 2008). Spandl, and rtiso h Taergltrascae ihFK(GRAF) FAK with associated regulator GTPase the of Proteins and S3-12 TIP47, ADRP, (perilipin, family PAT the of Proteins lhuhdtisaecretylcig Dfraini thought is formation LD lacking, currently are details Although Dhmotsshspicplybe tde nadipocytes, in studied been principally has homeostasis LD nvivo in ntecs fGA1 eue expression reduced GRAF1, of case the In . m .I otat o-dpct cells non-adipocyte contrast, In m. me l,2005; al., et ¨m nvitro in

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Fig. contained segment (S/P, GRAF1a residues GRAF1b, 37-amino-acid proline 55-amino-acid to a and comparison the lacked serine In in to GRAF1c domain predicted enriched GRAF1b, GRAF1c). corresponded for stretch SH3 to no 8/20 cDNAs GRAF1b; comparison for terminal with abundant In clones (11/20 identified most region the isoforms shorter The a and The preceding 1A). GRAF1c. in (Fig. clones structure and differed 20 GRAF1b secondary encoded GRAF1a, sequenced in variants, proteins We enriched splice three is 1998). library. al., amplified GRAF1 et cDNA Taylor therefore that 1996; al., We et shown (Hildebrand brain have mammalian studies brain neonatal in Previous enriched protein LD-associated a is GRAF1a RESULTS homeostasis. specifically LD GRAF1a of regulator GRAF1b, a is to GRAF1c and whereas LDs distribution with that, associates similar here report a We far. vesiclesshows have GRAF1c so and and examined GRAF1a been of tubules roles not the but dynamic al., 2008), al., et with et (Barresi (Lundmark associates reported is GRAF1b GRAF1c, isoform, 2010). third anti-GRAF1 a and addition, databases, 2010). al., et In Jarius 2013; al., cerebellar 2010). et inflammatory (Jarius with ataxia al., patients in found et are autoantibodies (Barresi 2005), retardation al., mental syndrome alpha-thalassemia et X-linked Wilda lung with 2011; patients al., of in et primary and Qian cases 2000; al., from multiple et Borkhardt in 2006; al., tumours et 2007), (Bojesen leukemia brain myeloid al., syndrome/acute myelodysplastic et metastatic (Zohrabian in adenocarcinoma reported is controls. negative as used were Rab11 of and top Actin the 1). at (fraction PLIN3 gradient and the GRAF1a blotting, endogenous western of by enrichment analysed relative were showing fractions gradient odd and density cytoplasmic input by of The cells Separation centrifugation. glial (J) primary image. oleic-acid-fed each of of components right bottom the at 10 bars: enlarged Scale LDs. using visualize and to antibodies, (I), 493/503 anti-GRAF1 BODIPY GRAF1a or untagged anti-Myc or with (H) stained Myc–GRAF1a respectively, expressing cells microscopy HeLa illumination of structured (SIM) by acquired images of 15 projections bars: intensity corresponding Scale (see 1–3). c Movies or material live b supplementary of GRAF1a, images GFP-tagged plane expressing focal cells Single HeLa (see (G) immunoprecipitations arrows. S1A,B). the by Fig. from indicated material absent are supplementary band GRAF1 non-specific of a c indicates and * b F Bands a, and experiments. isoforms E independent to in two corresponding Results than antibody. was more anti-calnexin loading of an Equal representative with lane. are glial blot marker of M, same or brain. the cells rat on glial adult checked of in cultures and primary neurones, in and GRAF1 antibody.cells of anti-Rab8 analysis an blot with Western blot (F) same loading the Equal on proteins. verified the GRAF showing was the adult, E18 of an and expression from E16 regulated and developmentally from neonates, extracts P7 brain and mouse P1 in embryos, GRAF2 and blot Western GRAF1 (E) of cells. analysis HeLa in Myc-tagged overexpressed of GRAF1c, or analysis GRAF1b blot GRAF1a, western and (IP) Immunoprecipitation (D) w RF sfrs RFaadGA1,aelse in listed are GRAF1b, and GRAF1a isoforms, GRAF1 Two A ceai ersnaino RF sfrs /,serine- S/P, isoforms. GRAF1 of representation Schematic (A) GRAF1 eune rmabrain a from sequences m .Bxdaesi – are G–I in areas Boxed m. m .(,)Maximum (H,I) m. aeldtblsadsalvsce Fg G.A previously As 1G). (Fig. vesicles small that and suggesting tubules 1F). labelled neurones co-cultures, (Fig. of cells than glial devoid in enriched cultures GRAF1 is in GRAF1a glial 1F). more predominant amounts, (Fig. embryos also protein contained E18 of was equal brains GRAF1a the At from neonates. isolated cells GRAF1a in primary whereas brain, are abundant adult GRAF1c in GRAF1a is isoforms and as GRAF1 mass GRAF1b major to the molecular S1A). thus same Fig. switch the of material antibody with anti-GRAF1 a (supplementary Immunoprecipitation protein an a with 1E). was in extracts (Fig. brain resulted (P7) there adults 7 day in which postnatal and isoforms (E16) after longer 16 shorter the day postnatally, embryonic of before was Expression increased started expression brain. S1A,B). (GRAF1a) mouse GRAF1 isoform Fig. developing of in material pattern different two the supplementary as however, 1E; GRAF1b migrated (Fig. Interestingly, of adult GRAF1 masses GRAF1c in molecular extracts, the data, and to protein PCR corresponding the brain bands, major with rat agreement adult or In in mouse 1D). (Fig. abundant sizes least their corresponded the clones is it 1/20 that Only brain. suggesting 1A–C). GRAF1a, Fig. to (Hyd, residues ne etn odtos navreyo ellnsadprimary and lines cell of variety a 1J). LDs in (Fig. cells. with conditions, associates PLIN3 1 that GRAF1 resting neonates in a fraction of under brain is enriched in the GRAF1a in was that enriched enriched isoform 1 show also experiments fraction these was as been Collectively, GRAF1a gradient, has at were 1J). the what were (Fig. of LDs to cells 2006), top Similar Wolins, glial the and gradient. endogenous (Brasaemle primary sucrose reported of previously a of distribution on extracts In subcellular fractionated low. cytoplasmic the was number examine GRAF1a, staining reticular LD upon to the A seen when order was S1E,F). antibodies ER, primary anti-GRAF1 the Fig. and with of material S1D) typical staining, Fig. (supplementary background material cells (supplementary U- glioblastoma glial human cells 1I), MG (Fig. GRAF1a cells HeLa 87 untagged were in LDs organelles overexpressed on these found Similarly, was 1H), LDs. neutral (Fig. as the (supplementary 493/503 identified and BODIPY human and cells GRAF1a probe Myc-tagged BSC1 fibroblasts lipid Using PC12, S1C). epithelial 3T3 Fig. pheochromocytoma monkey material NIH green rat murine African GRAF1a–GFP SH-SY5Y, in when neuroblastoma not cytoplasmic labelled transfected (data were immobile spherical was structures were relatively Similar were surrounding they which shown). 1G), 2008), membrane mostly (Fig. GRAF1a inclusions al., contrast, the striking et In labelled shown). (Lundmark not (data GRAF1b dynamic for reported ie ht nieGA1 Fg A,GA1 i o associate not addition, additional did GRAF1b In 2A), short (Fig. interactions. GRAF1a unlike hydrophobic binding a that, mediates through given therefore has In linker membranes This GRAF1a S1G). to 1A). (Fig. Fig. GRAF1b, linker internal material to (supplementary sodium the comparison to resistant extraction GRAF1b, was unlike membranes carbonate with addition, GRAF1a same of In the association target not compartments. do are they intracellular GRAF1b but and proteins, membrane-binding GRAF1a domains both that PH showed and an BAR observation on its Microscopy both and relies linker LDs hydrophobic with internal GRAF1a of association Specific hnoeepesd F-agdGA1 n GRAF1c and GRAF1b GFP-tagged overexpressed, When by discriminated be could GRAF1c and GRAF1b GRAF1a, ora fCl cec 21)17 6241 doi:10.1242/jcs.147694 4602–4619 127, (2014) Science Cell of Journal

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Journal of Cell Science mgsaeo ofclsak.Bxsnmee niaeLscontaining LDs indicate 1 numbered 2 Boxes bar: Scale stacks. LD. confocal the of of are surface images the from intensity extending Maximum tubule of (A) GRAF1a bottom SIM. 15 the by bar: at visualized Scale enlarged were projection. is Cells area (A,C) in boxed image. GRAF1a A each of LDs. presence of the visualize sub-populations to different (B) anti- antibodies with anti-PLIN3 with analysis and immunofluorescence stained volume. PLIN2 for either LD processed were or in (A,C) GRAF1a–GFP increase Red with an LipidTOX transfected to cells leads HeLa GRAF1a (A–C) of Overexpression 3. Fig. ARTICLE RESEARCH Fg B.PI3wssihl oeaudn nGRAF1a- on vice-versa abundant but S2A), and Fig. more material PLIN3, (supplementary slightly PLIN2 not than reported, was LDs and positive previously PLIN3 PLIN2 As of 3B). devoid 3). contained (Fig. were labelled LDs few a boxes three some and anti- 3B, for the 2), (Fig. positive or labelled with only had GRAF1a boxes were detected some 3B, LDs S1D–F) 1), (Fig. were labelled some GRAF1a boxes Fig. antibodies, 3B, LDs (Fig. anti-PLIN3 proteins If material and 3A). (Fig. PLIN2 supplementary Red 493/ BODIPY 1H,I; most LipidTOX as (Fig. on such 2011; found probes, lipophilic al., was 503 with protein cells et visualized HeLa the as Storey when GRAF1a, LDs, 2006; conditions, with resting al., transfected Under states et were 2005). metabolic Ohsaki al., different et 2005; in Wolins reflect be al., of to can et terms proposed LDs (Martin in individual been that heterogeneous has fact this is the composition; population protein LD and the lipid volume cell, LD a in increase Within an induces overexpression GRAF1a (supplementary GRAF1a- localization LD S1J). exclusive Fig. more GRAF1a- material acid, a oleic with cells acquired incubating ER LDs by the When and enlarged S1I). IV were Fig. COX material with (supplementary also calreticulin but protein GM130, protein Golgi the GRAF1a- IV with S1H). COX Fig. protein material mitochondrial (supplementary the with colocalization extensive 15 bar: Scale GRAF1a. of only devoid containing LDs LDs 3, 2, numbered boxes numbered and boxes GRAF1a, PLIN3, and PLIN2 GRAF1a, 1 n8idpneteprmns eut r mean 189 are and Results CTRL experiments. 213 ** experiments, independent independent 8 11 PLIN3, in in experiments; G1b G1a independent 204 7 and in CTRL 5 G1b independent 152 111 in 13 and G1a in CTRL G1a 233 193 243 and and experiments, CTRL untransfected CTRL 181 156 and PLIN2, Red, transfected experiments; LipidTOX per independent LD (F) quantified. mean number was the LD cell (D), mean (CTRL) volume the LD and total (E) staining the anti- volume condition, after or each identified anti-PLIN2 For with were antibodies. analysis LDs PLIN3 immunofluorescence indicated, or As Red, h. LipidTOX GRAF1b–GFP 48 with or after (G1a) fixed GRAF1a–GFP were with (G1b) transfected cells HeLa (D–F) uat splmnaymtra i.SL.I addition, MG In U-87 transfected S1L). GRAF1a- of GRAF1a–GFP, that Fig. extracts showed deletion cells cytoplasmic GRAF1a material GFP of the the fractionation to (supplementary that corresponded and mutants proteins thus overexpressed examined the fusions of cleavage little a material had (supplementary it LDs that S1K). for suggesting Fig. affinity membranes, reduced other substantially to binding residual nyncsayrgo,btisBRadP oan r both are surface. domains LD the PH to GRAF1a and of BAR distribution the the is its restrict linker to but required hydrophobic region, Its necessary binding. only LD specific for a sufficient to S1M). present Fig. were material two (supplementary latter extent the lesser although fraction, LD-enriched GRAF1a- and linker-SH3–GFP P etr ltaayi fcl xrcscnimdta hr was there that confirmed extracts cell of analysis blot Western hs xeiet hwta osnl oano RFais GRAF1a of domain single no that show experiments These , 0.01. m .()Snl oa ln.A ro onsa a at points arrow An plane. focal Single (C) m. D D A–F eealfudi the in found all were BAR–GFP A,hwvr tl showed still however, BAR, D HGP GRAF1a- PH–GFP, D 6 A colocalized BAR ...* s.e.m. P , m 0.05; .I B, In m. D m PH m. oiiepnt eese Fg B oe) fe 03 min, 20–30 After boxed). with 4B, labelled (Fig. seen BODIPY-C were small were LDs puncta pre-existing positive addition, C BODIPY of Within 1). Movie min material supplementary Wang 5–15 4A,B; 2005; with al., (Fig. LDs et 2010) (Liao al., into acids et fatty incorporates unmodified that to kinetics analogue similar acid fatty fluorescent pftyais rteefcec ficroainit D,but LDs. LDs, larger into of incorporation number smaller of by take a efficiency to to altered cells the leads of or not the rapidly capacity acids, was the cells, fatty change LDs up of not resting does Overexpression to therefore S2K). to PLIN3 GRAF1a Fig. Similarly material of (supplementary 4F). binding GRAF1a (Fig. 4D; distinct of of (Fig. reduced number efficiency the to as GRAF1a was 4E) lead volume (Fig. not volume LD LDs overexpressing did LD total mean this in However, increase cells the an S2J). acid, Fig. in material oleic supplementary with increased incubation was 1-h was a decrease after this that S2I). Fig. suggesting material supplementary 4C; clearly, for (Fig. GRAF1a as positive by stimulated LDs all diminish into of arrowheads). almost density not transformed were the did cells, h, 4B, LDs untransfected 1 (Fig. in small after Interestingly, GRAF1a. which, of not LDs, number larger sometimes fewer large this and Thereafter, GRAF1a, with n nrnfce el splmnaymtra i.S2D–F), Fig. material (supplementary Myc–GRAF1a-transfected cells between untransfected LD of compared PLIN3-positive and or number were 493/503 the BODIPY properties increased Red, obtained mostly LipidTOX were results when it Similar 3F). volume whereas mean (Fig. the structures LipidTOX 3E), in PLIN3-positive of (Fig. increase case an LDs through the and so In of did 3D). it untransfected PLIN2, (Fig. and volume significant Red LD between a total in induced compared increase GRAF1a cells, and GRAF1a–GFP-expressing objects, quantified PLIN3-positive or PLIN2-positive were as by or with Red, formed the LipidTOX structure association of specific exploration. its further artefacts a require to or will were GRAF1a growth tubules leading tubules LD GRAF GRAF1a of endogenous these phases of PLIN3 during Whether overexpression or induced below). not S2C) were (see Fig. and not material 2A), did (Fig. (supplementary S2B), neutral Fig. 2A; PLIN2 with material (Fig. stained supplementary contain LDs not 3C; (Fig. were from probes however, extend lipid These tubules, to These 3C). seemed LDs. Fig. sometimes GRAF1a that or tubules presence all the to on heterogeneity GRAF1a. of LD found absence extend was thus observations protein neither eas RFbepeso i o hneL properties LD change S2H). se, not Fig. LD-targeting per material transfection did the supplementary by 3D–F; expression to caused (Fig. not GRAF1b specific was was and because GRAF1, increase of size LD isoform the to This S2G), access hinder Fig. surface. sterically material GRAF1a (supplementary as overexpressed LDs addition, did to nor In binding PLIN3 phenotype. of and efficiency this PLIN2 the on of change tag not the did overexpression of GRAF1a effect an out ruling OIY5858C 558/568 and BODIPY formation, acid LD oleic during with incubated GRAF1a were of cells behaviour GRAF1a–GFP-transfected the follow to cells order of In size loading LD acid in fatty increase upon an promotes overexpression GRAF1a hntesz n ubro D a uniidi ie cells fixed in quantified was LDs of number and size the When hntesz n ubro D,ietfe ysann with staining by identified LDs, of number and size the When small on found was GRAF1a LDs, to addition in cells, some In ora fCl cec 21)17 6241 doi:10.1242/jcs.147694 4602–4619 127, (2014) Science Cell of Journal 12 Fg B ros.A hstm,nwGRAF1a- new time, this At arrows). 4B, (Fig. 12 pstv D perd oeie co-stained sometimes appeared, LDs -positive 12 hratrcle OIYC BODIPY called (hereafter 12 4607 ,a ),

Journal of Cell Science EERHARTICLE RESEARCH 4608 cells. of loading acid fatty upon volume LD C mean BODIPY in and increase acid an oleic induces GRAF1a 4. Fig. nrnfce CR)cl eeqatfe ntreidpneteprmnsuig8 TLad16Gacls eut r mean are Results cells. G1a 106 and CTRL 85 using experiments independent three in quantified were cell (CTRL) untransfected BC tl mgso ein oe nAa h niae iepit hwn rnfce B n nrnfce C el.I ,arw on opre-ex to point arrows B, In cells. (C) untransfected and (B) C BODIPY transfected indicate showing arrowheads time-points and indicated LDs, the nascent at GRAF1a-positive around A placed in are boxed boxes regions LDs, of images Still (B,C) D eeietfe sBODIPY-C as identified were LDs 12 AC ielpemve flv el eeaqie nasnl oa ln.()Siliae t0ad6 i.Saebr:15 bars: Scale min. 60 and 0 at images Still (A) plane. focal single a on acquired were cells live of movies Time-lapse (A–C) . 12 pstv rpes h enL oue() oa Dvlm E n enL ubr()prtasetd(1)and (G1a) transfected per (F) number LD mean and (E) volume LD total (D), volume LD mean The droplets. -positive ora fCl cec 21)17 6241 doi:10.1242/jcs.147694 4602–4619 127, (2014) Science Cell of Journal eaclstasetdwt RFaGPwr nuae with incubated were GRAF1a–GFP with transfected cells HeLa 12 pstv acn D.(–)Clswr ie fe hour. 1 after fixed were Cells (D–F) LDs. nascent -positive 6 ...* s.e.m. P , 0.05. isting m m.

Journal of Cell Science EERHARTICLE RESEARCH u ti huh opoedtruhacmiaino oa lipid local of combination a through proceed known, to not thought is is growth it LD for but responsible mechanism motility precise LD The reduces GRAF1a of Overexpression 5. Fig. e etpg o legend. for page next See ehrisl noi,adaetu ihl ikdt Dmobility. LD to linked tightly thus and are target its and reach The to it, 2012). LD onto a al., of itself et ability the tether (Yang on depend fusion will LD two latter and transfer lipid synthesis, ora fCl cec 21)17 6241 doi:10.1242/jcs.147694 4602–4619 127, (2014) Science Cell of Journal 4609

Journal of Cell Science GRAF1 ihti,OlRdOsann fP ri etossoe nya only showed sections brain P7 of In staining O S3D–F). agreement Red In Oil Fig. low. this, were material brains with their in supplementary levels triglyceride 6B,C; addition, (Fig. adults or agdGA1 Ga,GA1 Gb rGA2(2 eeicbtdwith incubated C BODIPY were and (G2) GRAF2 acid oleic or (G1b) GRAF1b (G1a), the GRAF1a potentiates tagged loading. and acid LDs fatty down by slows induced GRAF1a clustering of Overexpression 5. Fig. ARTICLE RESEARCH 4610 GRAF1 between differences overall, lipid significant that no development. showed were mouse there analysis for (TLC) essential chromatography Fig. were not no Thin-layer material therefore revealed is (supplementary organs GRAF1 architecture pups of S3C). various tissue material of and in (supplementary analysis fertile abnormality Knockout animals Histological were GRAF1- S3B). wild-type and Fig. as 6A). littermates. normally, weights Fig. wild-type developed similar mice their S3A; knockout from Fig. indistinguishable growth LD in role material a plays vivo GRAF1a whether in examine to order In abnormal strong a show phenotype not do mice and GRAF1-knockout mobility LD limits clustering. overexpression their potentiates GRAF1a cluster. S2O). LDs GRAF1a- Fig. with material in treatment (supplementary tethering upon network by and microtubule explained disperse be the not Fig. not to control could did it material LDs that in suggesting stage, nocodazole, supplementary this both 5G; At (Fig. S2N). seen perinuclear cells the was in clustered overexpressing fatty were This with LDs incubated imaged, were and region. cells h when 24 result, for showing a acids clearly As without fusion. often movements of other, 5F), directed signs each (Fig. to of rearrangements bound cytoplasmic result remained large-scale and a of or as 5E) together (Fig. the came of analysis LDs not the could during images. sometimes objects they independent that as other discriminated LDs each be Indeed, to size. bound LD tightly mean so in were in increase striking the explained particularly S2M) partially Fig. was and material This (supplementary 5D). GRAF1a overexpressing (Fig. cells clusters tight 5B,C). as was LD (Fig. seen it GRAF2 total because by of GRAF1a, nor not to GRAF1b but specific by area, induced was overexpression LD neither This GRAF1a mean 5B,C). cells, in (Fig. S2L, increase area fixed Fig. an with induced material above in also supplementary As decrease 5A; 2). significant (Fig. Movie velocity a LD induced mean GRAF1a of Overexpression curdo igefclpae A niiulBODIPY-C Individual (A) plane. focal single a on acquired xeiet nattlo 3CR n 7Gacls – fe li acid oleic after h BODIPY-C 2–5 of cells, area surface G1a (C) 27 total and and (B) CTRL Mean (B,C) 53 addition. of total a independent on three experiments and in (CTRL) quantified untransfected was in cells velocity (G1a) GRAF1a-transfected LD mean The tracked. and identified mgsaepoetoso ofclsak.Saebr:15 bars: Scale stacks. confocal of lines. projections dashed are and by untransfected Images surrounded both are of latter region The perinuclear addition, cells. the acid GRAF1a–GFP-expressing oleic in after LDs h of clustering 24 imaging.showing fixed the cells of of course analysis Immunofluorescence the (G) during followed individual arrows LDs of E, individual because In to clustering (F). rearrangements point LD cytoplasmic showing large-scale of F or and (E) E movements in areas Boxed enlarged addition. acid are oleic D after two min in and 60 two starting four, cells, of in images cells Still (D–F) G2 mean 50 are CTRL, Results respectively. 45 experiments, and independent 34 G1b; CTRL, 27 54 CTRL, from 16 quantified G1a; cell transfected and (CTRL) untransfected per hnclswr nuae ihftyais D eeoften were LDs acids, fatty with incubated were cells When hs xeiet hsso htdrn hsso Dgrowth, LD of phases during that show thus experiments These ncotmuewsegnee (supplementary engineered was mouse knockout a , 2 / 2 ie hte okn ttsusfo 7neonates P7 from tissues at looking whether mice, 12 AF ielpemve flv el were cells live of movies Time-lapse (A–F) . eaclstasetdwt GFP- with transfected cells HeLa 12 6 m pstv D were LDs -positive ...** s.e.m. (D,G). m 12 pstv LDs -positive P , +/+ 0.01. and flre lses hte D eeietfe sBODIPY-C as identified LDs, were number smaller LDs a whether the into clusters, of LDs larger of expression of association of cells, tight the together surface untransfected to to LDs led whole GRAF1a comparison tethering In in the 8B,C). participates GFP (Fig. on GRAF1a whereas that with distributed contacts, suggesting inter-LD was at not clustered super- PLIN3 were by that but GRAF1a proteins showed cells (SIM) GRAF1a of untagged microscopy S4A) illumination Imaging with structured S4B). resolution seen Fig. Fig. material also (supplementary material was This GRAF1a– (supplementary overexpressing 8A). cells 24-h (Fig. in a occur GFP after to not Strikingly, did similar 2006). dispersion is is wash, al., lipolysis This et when (Marcinkiewicz 8A). fibroblasts stimulated (Fig. PLIN1-transfected in dispersed removed happens were LDs what acids medium, fatty the When from 5G). (Fig. region pericentriolar i.SH,btas eas twsasn rmbanetat of 1E). extracts (Fig. brain abundant from was absent GRAF1a was where it pups, because P7 material also (supplementary but LDs not S3H), target GRAF1a, Fig. not for no did GRAF2 was compensate because there to only unlikely but S3G). Fig. out, however, material ruled was, (supplementary be GRAF2 level not expression its expressed could in also 1E) change was (Fig. and brain organization adult domain in difference similar a gross has no which 6D). and (Fig. ventricles animals the wild-type and lining knockout cells between in LDs few hni GRAF1 in than splmnaymtra oi ) h rsneo RF thus GRAF1 of presence anchor The of 3). point Movie their around material movement (supplementary of freedom more had LDs oa Dvlm nGRAF1 in volume LD total mobility. 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(Fig. growth 7A) (Fig. glia an their primary was there in during acid LDs oleic of with incubation accumulation Upon (supplementary S3I). conditions glial Fig. resting under material primary LDs few in contained cells accumulation GRAF1 LD from compare isolated to cultures decided We GRAF1 Primary fe 4ho li cdlaig D accumulated LDs loading, acid oleic of h 24 After the acids after fatty dispersion of LD washout prevents GRAF1a of Overexpression Fg F upeetr aeilFg 4) ugsigan washout suggesting acid S4E), fatty Fig. the upon material seen attenuated volume supplementary overexpression LD the 8F; GRAF1a total (Fig. alter in objects addition, not decrease Fig. did In material PLIN3-positive (supplementary but LDs S4F). to or PLIN3 S4C,D), of efficiency Fig. binding 8D,E) (Fig. material (supplementary structures positive GRAF1 in GRAF1a gemn ihtehsooyaayi,GRAF1 analysis, histology the with agreement aiiae ne-Dcnat n otiue oL rwhin growth LD to contributes cells. and (supplementary glial mobility, primary contacts LD h regulates inter-LD 48 to GRAF1a not cells, similar facilitates HeLa did that, at in show but observed thus seen we experiments 7F), what These (Fig. clustering S3K). addition Fig. the material acid enhance oleic after further h 24 clumping ata opnaino RFbadGA1 yGRAF2, by GRAF1c and GRAF1b of compensation Partial hnpiaygi eeicbtdwt li cdfr4 ,the h, 48 for acid oleic with incubated were glia primary When ora fCl cec 21)17 6241 doi:10.1242/jcs.147694 4602–4619 127, (2014) Science Cell of Journal +/+ 2 / 2 la el hwipie Daccumulation LD impaired show cells glial el Fg CD.Taseto funtagged of Transfection 7C,D). 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Journal of Cell Science EERHARTICLE RESEARCH cd splmnaymtra i.SH.Hwvr D could GRAF1a- LDs a However, fatty when S4H). of washout Fig. spread the material after (supplementary clumping LD acids caused still were deleted were LDs 4). Movie material cells, immobile supplementary relatively and were untransfected and 8G overexpressing clusters (Fig. cells as in in remained they whereas that, GRAF1a mobile, highly showed and during cells independent of wash of imaging population Live mixed the cells. a untransfected GRAF1a- was effect and it the because transfected controls in of partly in small, magnitude than remaining relatively The was higher S4G). was levels Fig. material chase (supplementary the triglyceride after after cells cells by the stored overexpressing triglycerides uptake, of amount was the there the although in this, difference with no agreement In (Cohen lipolysis. myelin of of are inhibition component they major because a but GRAF1 acids, sulfatides, of fatty to with staining correspond co-migrate O probably C Red they in Oil S3D), ** Fig. by (D) indicated material 2010). spots (supplementary al., The organs et B). other Paglia in all (* 2005; from included and not brain was P7 mean corresponding from the level, ( background composition at their is in difference significant no showing littermates, GRAF1 6. Fig. hshtdltaoaie(E,popaiycoie(C,shnoyln(M n hlseo CO ewe needn ar fGRAF1 of pairs independent between (CHO) cholesterol and (SM) sphingomyelin (PC), phosphatidylcholine (PE), phosphatidylethanolamine sn nat-anxnatbd.Tersl hw srpeettv fmr hntrepiso nml.(,)Autban er,ln n pen(B) spleen and lung heart, brain, Adult (B,C) animals. of GRAF1 pairs from three than extracts more lipid of (C) representative is brain shown P7 result The antibody. anti-calnexin an using mg.Saebr:50 bars: Scale image. GRAF1 RFacntut nwihteP,GPo H domains SH3 or GAP PH, the which in constructs GRAF1a 2 / 2 ie hwn t niheti h ri,adiskoku nGRAF1 in knockout its and brain, the in enrichment its showing mice, 2 / 2 ied o hwasrn rs phenotype. gross strong a show not do mice m m. D A osrc a overexpressed was construct BAR +/+ n GRAF1 and 2 / 2 iewr nlsdb L.()Means (B) TLC. by analysed were mice +/+ n GRAF1 and n 5 A muorcptto fGA1i ri,hat ugadsle xrcso GRAF1 of extracts spleen and lung heart, brain, in GRAF1 of Immunoprecipitation (A) )(e upeetr aeilFg 3) stetilcrd ptitniyo ri samples brain of intensity spot triglyceride the As S3D). Fig. material supplementary (see 3) 2 / 2 ri etosfo 7pp.Bxdaesaemgiida h o eto each of left top the at magnified are areas Boxed pups. P7 from sections brain 2 soitswt D.Ti stu o agdaduntagged and tagged mostly for but GRAF1c, true tubules, and is dynamic GRAF1b This on to LDs. are found with contrast GRAF1c seldom associates and In is GRAF1b adults. GRAF1a whereas in neonates, enriched isoform GRAF1 in predominant the expressed as GRAF1a identified have We DISCUSSION ceeae niiulL rwhdrn at cdutk,and uptake, This acid washout. mobility. fatty upon their during lipolysis restricts growth retards and LD LDs individual of accelerates association the the causes from prevent the acids of presence fatty to the of on relies GRAF1a domain. washout and BAR specific, the thus of is during cells overloaded LDs capacity of The redistribution 8A,D,E). (Fig. / 2 uigL rwhaddrn ioyi,GA1 therefore GRAF1a lipolysis, during and growth LD During ie qa rti muti h nu a hce ywsenblotting western by checked was input the in amount protein Equal mice. ora fCl cec 21)17 6241 doi:10.1242/jcs.147694 4602–4619 127, (2014) Science Cell of Journal 6 ...o h ptitniyrtoo rgyeie(TG), triglyceride of ratio intensity spot the of s.e.m. 2 / 2 n GRAF1 and rautand adult or , absent +/+ 4611 tal., et and +/+

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Journal of Cell Science upie Student’s (unpaired eoiyprcl uniidi rmr la el sltdfo ih GRAF1 eight from GRAF1 isolated at eight LD cells and point Mean glial cells) and primary (B) (16 in positions imaging. quantified fixed the cell at during per on placed immobile velocity enlarged are essentially is Arrows remaining area intervals. LDs upon boxed h three starting The 1 plane, 493/503. at focal BODIPY right single and the a cells. acid on glial oleic cells primary of glial addition in primary growth live LD of to images contributes GRAF1a 7. Fig. ARTICLE RESEARCH ahdlns CEF mgscrepn opoetoso ofclstacks. confocal of projections 15 to bars: correspond Scale Images by surrounded (C,E,F) are lines. cells immunofluorescence GRAF1a- Transfected dashed for showing accumulation. processed and 493/503, and clumping BODIPY LD h and induced (E) antibody 48 anti-GRAF1 or an (F) with 24 for acid oleic with ensont neta ari ntehdohbclpdlayer lipid al., et hydrophobic Moessinger the 1993; al., in et hairpin Dupree 2003; a has Herman, it as and to examined, (Caldas insert shown was to it been when has shown and segment been localization, this LD for proteins, required these be of all For 2008). 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Stevanovic al., 2011; et al., et Spandl 2011; 9/0,1 fe li cdadto.Rslsaemean are Results addition. acid oleic after h 10 493/503, GRAF1 idtp n he ncotebys D h oa Dvlm e elwas cell per volume LD GRAF1 total 840 three The for with (D) from quantified embryos. stained isolated knockout and cells three fixed of and h, images wild-type 48 Representative for (C) acid 493/503. oleic BODIPY with incubated were cultures aelte n omlzdb h au bandfrwl-yeembryos. wild-type for obtained value mean the are by Results normalized and litter same itr.Frec mro h enttlL oueprcl a uniid It quantified. was cell per GRAF1 volume all for LD averaged total then mean was the embryo, each For litters. sltdfo GRAF1 9 from isolated ta. 03 pnle l,21) BD Zhe ta. 2009), (Jo al., et AUP1 (Zehmer 2013), UBXD8 2011), Napoli, al., (Turro and et ALDI Spandl Jiang and 2013; 2012; al., LRAT LPCAT1 and et al., Rdh10 Napoli, 2001), 2013), et al., and (Fujimoto et al., Pol (Jiang caveolins 2004; al., et 2011), et Ostermeyer al., (Wilfling 2001; et GPAT4 (Moessinger 2008), LPCAT2 al., et nerirsuis h A-Hdmi fGA1hsbeen has GRAF1 of domain BAR-PH the studies, earlier In hydrophobic GRAF1a-specific the is it that show results Our +/+ F la el eetasetdwt nagdGA1,incubated GRAF1a, untagged with transfected were cells glial (F) ´ m ta. 06,AMBadCBR Zhe tal., et (Zehmer CYB5R3 and AAM-B 2006), al., et m. c 6 ...** s.e.m. 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LDs characterize examine of to further preferences chain and to acyl cells fatty essential non-adipocyte to and in cholesterol be order LDs In will of 2006). composition it of al., GRAF1a LDs, recruitment of et binding specific to high-affinity 2013; (Ehehalt the allow determines al., proteins what would et understand of (Arisawa and pools LD be 2011), the different might al., of This et status 2002). metabolic Storey al., the et to Tauchi-Sato 2010; al., linked 2010; et al., a (Blouin chains et containing acyl phospholipids McIntosh fatty in saturated It and of 2013). proportion cholesterol al., high free et in Penno other enriched 2007; any al., is from et different phospholipid (Bartz is of membrane it ratio intracellular that relative shows of analysis terms detailed in species, ER the of LDs. reminiscent to is binding high-affinity unlikely the of is for domain it account PH localization, wholly the 2006). LD to of perturbs deletion phosphoinositides marginally al., that any only given contain GRAF1a and et 2013), to al., (Andersson known et not (Penno cells are in LDs 3T3 formation phosphatidylinositol-4,5- However, LD NIH for a important insulin-stimulated D1, is protein, phospholipase bisphosphate-binding that proposed soitdwt ahidpnetL ihnacutrmight Cav cluster of a the overexpression motors within However, from LD other. molecular independent each result because counteract each also with clustering could associated LD overexpression observed GRAF1a-induced mobility GRAF1a to LD in leading decreased upon The ultimately particular, aggregation. tethered, in LD remain extensive other GRAF1a, and, each of LD rearrangements, contact presence disrupted also the potentiates cytoplasmic can LDs of also is case, the because but that travel, in if mobility Indeed, prevented LD clustering. 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Rohit for of and laboratory Evergren analysis McMahon Emma histology the thank H&E to the wish particular for we in Howard, Finally, Tissue and Susan Research Trust, and Human Foundation Zhao expert the NHS Wanfeng for acknowledge Hospitals Pasche to University and Mathias wish Cambridge handling thank We Bank, the to SIM. of like the aspects would with We various assistance mice. with the help facility of for Mead, LMB facility, housekeeping genotyping Annie ARES the particular the of in of members facility, and and Biomed Robinson, LMB Karen the and Brown of Michael members thank to wish We Acknowledgements mean the provided, quantifications all For analysis data Statistical otiuin fcl oeet oL mobility. 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Journal of Cell Science oe .B,Mrh,D . rdr . utr . rsel,D and D. Brasaemle, S., Rueter, T., Grider, D., D. Murphy, B., N. Cole, H., A. Futerman, A., Fein, J., Bodennec, L., Ravid, P. W., M. Auerbach, T., Czech, Cohen, and A. Chawla, V., Puri, E., Boutet, L., J. Christianson, G., J. Wittmann, and K., J. Mutenda, D., N. Wenzel, Severs, A., Deggerich, Y., V., Nitschke, A. Bulankina, S., Lorkowski, H., Robenek, I., Buers, E. N. Wolins, and L. D. Brasaemle, E. N. Wolins, and L. D. Brasaemle, Bostro Weinha O., Ammerpohl, E., S. Bojesen, Ko A., Eberl, S., Lay, Le M., C. Blouin, dbal,R .adHthr .F. J. Hatcher, and M. R. Adibhatla, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.147694/-/DC1 online available material Supplementary material Supplementary release. PMC immediate in Deposited for PA00P3-124164]. PBGE1-121206, numbers [grant fellowships ARTICLE RESEARCH 4618 E. G. Herman, and H. Caldas, D., Bartelheimer, U., Fuchs, A., O. Haas, S., Bojesen, A., Borkhardt, C., Gilpin, Y., Zhao, S., V. Markin, J., Hill, Y., Chen, T., Januszewski, D., R., Binns, Zemni, C., M. Vinet, V., Portes, des N., Ronce, T., Bienvenu, P., Billuart, ikl .E,Tne,J .adWle .A. R., M. Welti, Welte, and R., T. M. J. Tansey, Roth, E., P. K., Bickel, J. Zehmer, B., Venables, H., W. Li, R., Bartz, G., Rappazzo, L., Castiglia, N., Musso, M., Fichera, A., Ragusa, V., Barresi, F., Li, M., Caban I., Kakoki, Barba, M., Rojas, A., A. Suen, E., K. Bird, C., K. Lenhart, X., Bai, Bostro L., Andersson, rsw,K,Ih,I,Yskw,Y,Sn,Y n uiaa Y. Fujiwara, and Y. Sone, Y., Yasukawa, I., Ichi, K., P.,Arisawa, Li, L., N. Schieber, K., Green, L., Wu, A., N. Hamilton, S., Murphy, N., Ariotti, usam .L. R. and Nussbaum, function, lysosomal development, mouse for dispensable secretion. is variant M. splice Horowitz, and L. A. protein. Joyner, Cidea the of domain targeting droplet Res. lipid the of Identification Ho droplets. and lipid N. of K. biogenesis Burger, G., M. 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