Table Of ContentSeries 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
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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.
