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Series Editors Leslie Wilson Department of Molecular, Cellular and Developmental Biology University of California Santa Barbara, California Paul Matsudaira Department of Biological Sciences National University of Singapore Singapore Phong Tran Department of Cell and Developmental Biology University of Pennsylvania Philadelphia, Pennsylvania AcademicPressisanimprintofElsevier 525BStreet,Suite1800,SanDiego,CA92101-4495,USA 225WymanStreet,Waltham,MA02451,USA TheBoulevard,LangfordLane,Kidlington,Oxford,OX51GB,UK 32JamestownRoad,LondonNW17BY,UK Radarweg29,POBox211,1000AEAmsterdam,TheNetherlands Firstedition2013 Copyright#2013ElsevierInc.Allrightsreserved Nopartofthispublicationmaybereproduced,storedinaretrievalsystemortransmittedin anyformorbyanymeanselectronic,mechanical,photocopying,recordingorotherwise withoutthepriorwrittenpermissionofthepublisher PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRights DepartmentinOxford,UK:phone(+44)(0)1865843830;fax(+44)(0)1865853333; email:permissions@elsevier.com.Alternativelyyoucansubmityourrequestonlineby visitingtheElsevierwebsiteathttp://elsevier.com/locate/permissions,andselecting ObtainingpermissiontouseElseviermaterial Notice Noresponsibilityisassumedbythepublisherforanyinjuryand/ordamagetopersonsor propertyasamatterofproductsliability,negligenceorotherwise,orfromanyuseor operationofanymethods,products,instructionsorideascontainedinthematerialherein. Becauseofrapidadvancesinthemedicalsciences,inparticular,independentverificationof diagnosesanddrugdosagesshouldbemade ISBN:978-0-12-408051-5 ISSN:0091-679X ForinformationonallAcademicPresspublicationsvisit ourwebsiteatstore.elsevier.com PrintedandboundinUSA 13 14 15 16 11 10 9 8 7 6 5 4 3 2 1 Contributors KimberlyK. Buhman Department ofNutrition Science,Purdue University, West Lafayette, Indiana,USA GregoryCamus Gladstone Instituteof Virology and Immunology, University ofCalifornia, San Francisco, Owens Street, San Francisco, CA, USA Ji-Xin Cheng Interdisciplinary Life Science Program, Department of Chemistry,and Weldon School of BiomedicalEngineering, PurdueUniversity, West Lafayette, Indiana,USA Jinglei Cheng DepartmentofAnatomyandMolecularCellBiology,NagoyaUniversityGraduate SchoolofMedicine, Nagoya, Japan Simon Cichello SchoolofLife Sciences, La Trobe University, Melbourne, Victoria, Australia Gu¨nther Daum Institute ofBiochemistry, Graz University ofTechnology, Graz, Austria Yunfeng Ding NationalLaboratory ofBiomacromolecules, Institute ofBiophysics, Chinese Academy ofSciences, Beijing, China Jilian Fan BiosciencesDepartment,BrookhavenNationalLaboratory,Upton,NewYork,USA ToyoshiFujimoto DepartmentofAnatomyandMolecularCellBiology,NagoyaUniversityGraduate SchoolofMedicine, Nagoya, Japan Jingyi Gong Tsinghua-Peking Center for Life Sciences, SchoolofLife Sciences, Tsinghua University, Beijing, China David A.Gross BiochemistryDepartment,AlbertEinsteinCollegeofMedicine,Bronx,NewYork, USA,andPrograminCardiovascular&MetabolicDisorders,Duke-NUSGraduate MedicalSchool Singapore,Singapore Yi Guo Department ofBiochemistry and MolecularBiology, and Division of Gastroenterologyand Hepatology, Mayo Clinic, Rochester, Minnesota, USA Lydia-Ann L. S. Harris CenterforHumanNutrition,WashingtonUniversitySchoolofMedicine,St.Louis, Missouri, USA ix x Contributors Bradlee L. Heckmann Department ofBiochemistry and Molecular Biology, and Metabolic HEALth Program, Mayo Clinicin Arizona, Scottsdale, Arizona, USA Ru-ching Hsia CoreImaging Facility, DentalSchool, University of Maryland,Baltimore, Maryland, USA Barbara Koch Institute ofBiochemistry, Graz University ofTechnology, Graz, Austria Andrew S.Kondratowicz Gladstone Institute ofVirology and Immunology, University ofCalifornia, San Francisco, Owens Street,SanFrancisco, CA, USA HanLee Graduate Programin Neurobiology of Disease, Mayo Clinic, Rochester, Minnesota,USA HyeonJeong Lee Interdisciplinary Life Science Program, and Department ofChemistry, Purdue University, West Lafayette, Indiana,USA Richard Lehner GrouponMolecularandCellBiologyofLipids,DepartmentsofPediatricsandCell Biology, University of Alberta, Edmonton,Alberta, Canada MingLei VisualizationSciencesGroup,AnFEICompany,Burlington,Massachusetts,USA PengLi Tsinghua-Peking Center for LifeSciences, Schoolof LifeSciences, Tsinghua University, Beijing, China Jun Liu Department ofBiochemistry and Molecular Biology, and Metabolic HEALth Program, Mayo Clinicin Arizona, Scottsdale, Arizona, USA Pingsheng Liu National Laboratoryof Biomacromolecules, InstituteofBiophysics, Chinese Academy of Sciences, Beijing, China Ho Yi Mak Stowers Institutefor MedicalResearch, Kansas City, Missouri, and Department of Molecularand Integrative Physiology, University ofKansas MedicalCenter, Kansas City, Kansas, USA Huimin Na National Laboratoryof Biomacromolecules, InstituteofBiophysics, Chinese AcademyofSciences,andUniversityofChineseAcademyofSciences,Beijing, China Contributors xi Yuki Ohsaki DepartmentofAnatomyandMolecularCellBiology,NagoyaUniversityGraduate SchoolofMedicine, Nagoya, Japan Melanie Ott Gladstone Instituteof Virology and Immunology, University ofCalifornia, San Francisco, Owens Street, San Francisco, CA, USA Ying Peng Department ofBiochemistry and MolecularBiology, Mayo Clinic,Rochester, Minnesota, USA BirgitPloier Institute ofBiochemistry, Graz University ofTechnology, Graz, Austria ArielD.Quiroga Instituto de Fisiologı´a Experimental, Consejo Nacionalde Investigaciones Cientı´ficas yTe´cnicas (CONICET), Facultad de CienciasBioquı´micasy Farmace´uticas, Universidad Nacional de Rosario, Rosario,Argentina Claudia Schmidt Institute ofBiochemistry, Graz University ofTechnology, Graz, Austria David L. Silver PrograminCardiovascular&MetabolicDisorders,Duke-NUSGraduateMedical SchoolSingapore,Singapore James R.Skinner CenterforHumanNutrition,WashingtonUniversitySchoolofMedicine,St.Louis, Missouri, USA Zhiqi Sun Tsinghua-Peking Center for Life Sciences, SchoolofLife Sciences, Tsinghua University, Beijing, China Michitaka Suzuki DepartmentofAnatomyandMolecularCellBiology,NagoyaUniversityGraduate SchoolofMedicine, Nagoya, Japan CaroleSztalryd Division ofEndocrinology, Department ofMedicine, School of Medicine, University ofMaryland,and Geriatric Research,Education, and Clinical Center, Baltimore Veterans Affairs Health Care Center,Baltimore, Maryland,USA Aki Uchida Department ofNutrition Science,and Interdisciplinary Life Science Program, PurdueUniversity, West Lafayette, Indiana,USA DorotheeA. Vogt Gladstone Instituteof Virology and Immunology, University ofCalifornia, San Francisco, Owens Street, San Francisco, CA, USA xii Contributors HongWang Division ofEndocrinology, Department ofMedicine, School ofMedicine, University ofMaryland, Baltimore, Maryland, USA Huajin Wang Department ofCell Biology, YaleUniversity SchoolofMedicine, NewHaven, Connecticut,USA YangWang National Laboratoryof Biomacromolecules, InstituteofBiophysics, Chinese AcademyofSciences,andUniversityofChineseAcademyofSciences,Beijing, China Nathan E.Wolins CenterforHumanNutrition,WashingtonUniversitySchoolofMedicine,St.Louis, Missouri, USA Lizhen Wu Tsinghua-Peking Center for LifeSciences, Schoolof LifeSciences, Tsinghua University, Beijing, China Zhensheng Xie National Laboratoryof Biomacromolecules, InstituteofBiophysics, Chinese Academy of Sciences, Beijing, China Changcheng Xu Biosciences Department,BrookhavenNational Laboratory, Upton,NewYork, USA Chengshi Yan Biosciences Department,BrookhavenNational Laboratory, Upton,NewYork, USA Fuquan Yang National Laboratoryof Biomacromolecules, InstituteofBiophysics, Chinese Academy of Sciences, Beijing, China Li Yang National Laboratoryof Biomacromolecules, InstituteofBiophysics, Chinese AcademyofSciences,andUniversityofChineseAcademyofSciences,Beijing, China HuinaZhang National Laboratoryof Biomacromolecules, InstituteofBiophysics, Chinese Academy of Sciences, Beijing, China PengZhang National Laboratoryof Biomacromolecules, InstituteofBiophysics, Chinese AcademyofSciences,andUniversityofChineseAcademyofSciences,Beijing, China Xiaodong Zhang Department ofBiochemistry and Molecular Biology, and Metabolic HEALth Program, Mayo Clinicin Arizona, Scottsdale, Arizona, USA Preface Lipiddroplets(LDs)havenowbeenwellrecognizedasimportantcellularorganelles thatcanbefoundfrombacteriatoman.LDscompriseahydrophobic,neutrallipid core which is enclosed by a monolayer of phospholipids with proteins embedded. LDs have important roles in cell biology as they not only serve as cellular energy reservoir, but also take part in membrane trafficking, protein degradation, histone storage,andeventhereplicationofpathogenicviruses.LDsarealsocloselyassoci- atedwithhumanmetabolicdisorderssuchasatherosclerosis,fattyliver,andobesity, whicharecharacterizedbyexcessivelipidstorageandLDgrowth.Inaddition,LDs arehighlyrelevanttoplantoilandbiodieselproduction.However,manyfundamental questionsregardingthecellbiologyofLDsremainunanswered.Forinstance,where andhow do LDsoriginate? HowisthegrowthofLDs regulated? Howdo LDsin- teract with other cellular organelles? Clearly, more intense investigations into the cell biology ofLDs are required inthe near future. Inthisspecialvolume,expertshavebeeninvitedtoprovidein-depthdescription ofcommontechniquesusedinthestudyoftheLDs.AsLDsareconservedfrombac- teriatoman,thefirstsectionwilldiscussthetechniquesusedtostudyLDsinmodel organisms, including bacteria (Liu), yeast (Daum), Caenorhabditiselegans (Mak), Drosophila(Guo),andmicroalgae(Xu).ThesecondsectionfocusesonLDsindif- ferent mammaliancells/tissuesasLDsdemonstrate differentdynamicsindifferent tissues.Theseincludeadiposetissue/adipocytes(Liu),hepatocytes(Lehner),muscle (Sztalryd),intestine(Buhman),andcellsunderviralinfection(Ott).Inthefinalsec- tion,specifictechniquesarediscussedonneutrallipid–proteininteractions(Silver), imagingLDs(Wolins),measuringLDfusionandgrowth(Li),andimagingLDsby electronmicroscopy (Fujimoto). Althoughthisvolumedoesnotcomprehensivelycoverallaspectsanddevelop- mentsofthestudyofLDs,itishopedthatthevolumewillserveasageneralrefer- enceandasteppingstoneformoreexitingdiscoveriesofLDbiology,aswellasfor the development of more advanced technologies for the studyof LDs. Finally,wewouldliketothankthestaffofElsevier/AcademicPress,especially ZoeKruzeandPaulMatsudaira,fortheirenthusiasticsupportofourMethodsinCell Biologyvolumeon Lipid Droplets. Hongyuan Yang and Peng Li xiii CHAPTER 1 Proteomic Studies of Isolated Lipid Droplets from Bacteria, C. elegans, and Mammals Huimin Na*,{, Peng Zhang*,{,Yunfeng Ding*,Li Yang*,{, Yang Wang*,{,Huina { Zhang*,ZhenshengXie*,Fuquan Yang*, Simon Cichello ,and Pingsheng Liu* *NationalLaboratoryofBiomacromolecules,InstituteofBiophysics,ChineseAcademyofSciences, Beijing,China {UniversityofChineseAcademyofSciences,Beijing,China {SchoolofLifeSciences,LaTrobeUniversity,Melbourne,Victoria,Australia CHAPTER OUTLINE Introduction................................................................................................................2 1.1 Equipment...........................................................................................................3 1.2 Reagents.............................................................................................................3 1.3 Methods..............................................................................................................4 1.4 Discussion.........................................................................................................10 Acknowledgment.......................................................................................................12 References...............................................................................................................12 Abstract Lipiddroplets(LDs)areanintracellularorganelle,consistingofaneutrallipidcore covered by a monolayer of phospholipids and proteins. It primarily mediates lipid storage, metabolism, and transportation. Recently, research of LDs has emerged as a rapidly developing field due tothe strong linkage between ectopic lipid accu- mulationandmetabolicsyndromes.Recently,morethan30proteomicstudiesofiso- lated LDs have identified many important LD proteins that have highlighted and have also predicted the potential biological roles of the organelle, motivating the fieldtodevelopquiterapidly.Thischaptersummarizesmethodsusedinproteomic studiesforthreerepresentativespeciesreportedanddiscussestheiradvantagesand disadvantages.Webelievethatthischapterprovidesusefulinformationandmethods for future LDproteomic studies especially for LDs inother species. 1 MethodsinCellBiology,Volume116 ISSN0091-679X Copyright©2013ElsevierInc.Allrightsreserved. http://dx.doi.org/10.1016/B978-0-12-408051-5.00001-2 2 CHAPTER 1 Lipid Droplet Proteomics INTRODUCTION Lipiddroplets(LDs)storeandmetabolizealmostallneutrallipidsinthecell,across all species, and its formation and dynamics are directly linked to metabolic syn- dromesinhumansandtofoodproductionandbiofueldevelopmentinotherorgan- isms(Farese&Walther,2009;Martin&Parton,2006;Murphy,2011;Zehmeretal., 2009).Althoughitisanessentialcellularorganelle,comparedtoothersimilarcel- lularorganellessuchasthemitochondriaandendoplasmicreticulum(ER),ourun- derstanding of LDs still remains elusive, especially its biogenesis, dynamics, and functions.Therefore,knowingitstwoonlycomponents,namelylipidsandproteins, isofcriticalimportance.StudiesofLDlipidandproteincompositioncanbetraced back to 1970s. In the early stage, LDs were isolated and their lipids and proteins analyzed from mammal tissues and cells, plants, and yeast (Comai, Farber, & Paulsrud, 1975; DiAugustine, Schaefer, & Fouts, 1973; Hood & Patton, 1973; Jacks, Yatsu, & Altschul, 1967; Lang & Insull, 1970; Nissen & Bojesen, 1969; Yatsu,Jacks,&Hensarling,1971).Informationofbothlipidandproteincomposition providedbythosestudiesisverylimited,aslipidomicsandproteomicswerenotyet employed.InlastdecadetheHumanGenomeProjectandnewproteomictechnology allowedproteomicstudypossible.OurgroupandotherlabshaveisolatedLDsand performed proteomic analyses on them, and thereafter gained dramatic and useful knowledgethatproposesseveralputativerolesforLDsaccordingtotheirproteomes, further evolving the classic definition of LDs, as a lipid inclusion to a functional organelle (Athenstaedt, Zweytick, Jandrositz, Kohlwein, & Daum, 1999; Bartz, Zehmer, et al., 2007; Beller et al., 2006; Brasaemle, Dolios, Shapiro, & Wang, 2004; Cermelli, Guo, Gross, & Welte, 2006; Fujimoto et al., 2004; Jolivet et al., 2004;Liuet al., 2004; Sato et al., 2006; Yang et al., 2012). Someoftheseproteomicsfindingshavebeensupportedbyfurtherfunctionalstud- ies.Forexample,LD-associatedlipidsyntheticenzymesidentifiedfromyeastindicate thatLDfunctionsasoneofthecellularlipidsyntheticsites(Athenstaedtetal.,1999), andfurtherverifiedbyinvitrolipidsynthesisusingisolatedLDs.Moreover,thedis- coveryofRabproteinsandSNAREsinChinesehamsterovaryCHOK2cellssuggests thatLDsareinvolvedinmembranetrafficking(Liuetal.,2004),whichhasthereafter beenconfirmedbyRab-mediatedinteractionsbetweenLDsandER(Martin,Driessen, Nixon,Zerial,&Parton,2005;Ozekietal.,2005),aswellasLDsandendosome(Liu etal.,2007).Furthermore,thediscoveryofproteinsmovingonandoffLDssuggests thatLDsareverydynamicorganelles(Bartz,Zehmer,etal.,2007;Brasaemleetal., 2004).TheidentificationofprostaglandinsynthesispathwayshowsthatLDsproduce signalmolecules(Acciolyetal.,2008).Theobservationofubiquitination-relatedpro- teinssuggeststhatLDsareinvolvedinproteindegradation(Ploegh,2007),whichhas beenconfirmed bythe identificationofLD-mediatedHMG-CoA reductase recently (Hartman et al., 2010). Lastly, the discovery of histone proteins in Drosophila LDs suggeststhatLDsbehaveasaprotein-storagedepot(Cermellietal.,2006). TheimprovementofLDisolationmethods(Dingetal.,2013)andtheincreas- ing development of proteomic technologies play an essential role in the recent 1.2 Reagents 3 emergingLDproteomics.Theseproteomicstudieshavespannedmultiplespecies from bacteria to humans and demonstrated that LDs have quite different protein compositions, suggesting that the function of LDs varies between organisms, but alsotheregulation.However,moreresearchneedstobeconductedasonlylimited information is currently available, including further LD isolation and proteomic studiessoastofullyunderstandtheorganelle.WehavesuccessfullyisolatedLDs and carried out proteomic analyses from a bacteriumRhodococcus RHA1 (Ding etal.,2012),anematodeCaenorhabditiselegans(Zhangetal.,2012),amamma- lian cell line CHO K2 (Bartz, Zehmer, et al., 2007; Liu et al., 2004), and mam- malian tissues, that is, mouse liver (Bartz, Li, et al., 2007) and skeletal muscle (Zhang et al., 2011). Herein we provide a detailed experimental procedure that summarizes and standardizes LD proteomic studies from these three representa- tive species. We believe that the established method sufficiently allows the iso- lation of relative purer LDs (Ding et al., 2013) and proteomic analysis theoretically from all species mentioned. 1.1 EQUIPMENT 1. Frenchpresscell(JNBIO,cat.no.JN-3000,high-pressurehomogenizer)isused for homogenization ofbacteria such as Rhodococcus sp. RHA 1. 2. Polytron homogenizer (Cole-Parmer, LabGEN700 homogenizer) isused to disrupt the cellsofC. elegans. 3. Dounce homogenizer (Wheaton, cat. no. 357542) isused todissect the cells of mouse skeletal muscle and liver. 4. Nitrogen bomb (PARR Instrument, 4639celldisruptionbomb)isused to homogenizetissueculturecells,C.elegans,andmouseskeletalmuscleandliver. 5. Ultracentrifuge (Beckman, Optima™L-100 XPultracentrifuge) andSW40 Ti rotorare used toseparate LDs from othercellular fractions. 6. Ultracentrifuge(BeckmanCoulter,OptimaMAXultracentrifuge)andTLA100.3 rotorare used toobtaincytosol (Cyto) fraction. 7. Particleanalyzer(BeckmanCoulter,Delsa™NanoCparticleanalyzer)isused toanalyze LDsize. 8. Confocalmicroscope(Olympus,FV1000)andtransmissionelectronmicroscope (FEI, Tecnai 20) are used for morphological study of LDs. 9. Nano-LC-ESI-LTQmassspectrometerandnano-LC-ESI-LTQOrbitrapXLmass spectrometerare used for the proteomic analysisof different LD samples. 1.2 REAGENTS 1. Phosphate buffered saline (PBS) isused tocleansamples. 2. Buffer A (20mM tricine and 250mM sucrose, pH7.8) isused to protect intracellular organelles inthe sample homogenization.
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