Endo-Lysosomal Dysfunction in Human Proximal Tubular Epithelial Cells Deficient for Lysosomal

18 Pages·2015·2.87 MB·English

RESEARCHARTICLE
Endo-Lysosomal Dysfunction in Human
Proximal Tubular Epithelial Cells Deficient for
Lysosomal Cystine Transporter Cystinosin
EkaterinaA.Ivanova1☯,MariaGiovannaDeLeo2☯,LambertusVanDenHeuvel1,3,
AnnaPastore4,HenryDijkman5,MariaAntoniettaDeMatteis2,ElenaN.Levtchenko1*
1 DepartmentofPediatricNephrology&GrowthandRegeneration,UniversityHospitalsLeuven&
KatholiekeUniversiteit,Leuven,Belgium,2 TelethonInstituteofGeneticsandMedicine(TIGEM),Naples,
Italy,3 DepartmentofPediatricNephrology,RadboudUniversityMedicalCenterNijmegen,Nijmegen,The
a11111 Netherlands,4 LaboratoryofProteomicsandMetabolomics,Children‘sHospitalandResearchInstitute
"BambinoGesù"IRCCS,Rome,Italy,5 DepartmentofPathology,RadboudUniversityNijmegenMedical
Centre,Nijmegen,TheNetherlands
☯Theseauthorscontributedequallytothiswork.
* [email protected]
Abstract
OPENACCESS
Citation:IvanovaEA,DeLeoMG,VanDenHeuvel
L,PastoreA,DijkmanH,DeMatteisMA,etal.(2015) NephropathiccystinosisisalysosomalstoragedisordercausedbymutationsintheCTNS
Endo-LysosomalDysfunctioninHumanProximal geneencodingcystinetransportercystinosinthatresultsinaccumulationofaminoacidcys-
TubularEpithelialCellsDeficientforLysosomal
tineinthelysosomesthroughoutthebodyandespeciallyaffectskidneys.Earlymanifesta-
CystineTransporterCystinosin.PLoSONE10(3):
tionsofthediseaseincluderenalFanconisyndrome,ageneralizedproximaltubular
e0120998.doi:10.1371/journal.pone.0120998
dysfunction.Currenttherapyofcystinosisisbasedoncystine-loweringdrugcysteamine
AcademicEditor:BenedettaBussolati,Centerfor
thatpostponesthediseaseprogressionbutoffersnocurefortheFanconisyndrome.We
MolecularBiotechnology,ITALY
studiedthemechanismsofimpairedreabsorptioninhumanproximaltubularepithelialcells
Received:September5,2014
(PTEC)deficientforcystinosinandinvestigatedtheendo-lysosomalcompartmentsofcysti-
Accepted:February9,2015
nosin-deficientPTECbymeansoflightandelectronmicroscopy.Wedemonstratethat
Published:March26,2015 cystinosin-deficientcellshadabnormalshapeanddistributionoftheendo-lysosomalcom-
Copyright:©2015Ivanovaetal.Thisisanopen partmentsandimpairedendocytosis,withdecreasedsurfaceexpressionofmultiligandre-
accessarticledistributedunderthetermsofthe ceptorsanddelayedlysosomalcargoprocessing.Treatmentwithcysteamineimproved
CreativeCommonsAttributionLicense,whichpermits
surfaceexpressionandlysosomalcargoprocessingbutdidnotleadtoacompleterestora-
unrestricteduse,distribution,andreproductioninany
tionandhadnoeffectontheabnormalmorphologyofendo-lysosomalcompartments.The
medium,providedtheoriginalauthorandsourceare
credited. obtainedresultsimproveourunderstandingofthemechanismofproximaltubulardysfunc-
tionincystinosisandindicatethatimpairedproteinreabsorptioncan,atleastpartially,be
DataAvailabilityStatement:Allrelevantdataare
withinthepaper. explainedbyabnormaltraffickingofendosomalvesicles.
Funding:ThisworkwassponsoredbyCystinosis
ResearchFoundation(www.cystinosisresearch.org).
ElenaLevtchenkoissupportedbythefundfor
ScientificResearch,Flanders(F.W.O.Vlaanderen)
grant1801110N(www.fwo.be).MariaAntoniettaDe
Introduction
MatteiswassupportedbyTelethonFoundation(www.
telethon.it). Nephropathiccystinosis(MIM219800)isanautosomalrecessivedisordercausedbymutations
intheCTNSgeneencodingcystinosin,alysosomalcystinetransporter[1,2].Cystinosinisa
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist. 367-aminoacidlysosomalmembraneproteinwithpredicted7transmembranedomainsand
PLOSONE|DOI:10.1371/journal.pone.0120998 March26,2015 1/18
Endo-LysosomalDysfunctioninCystinosis
twolysosomaltargetingmotifslocatedintheC-terminusandinthe5thcytosolicloop[3].Asec-
ondisoformofcystinosinhasbeendescribed,inwhichtheC-terminaltargetingmotifissubsti-
tutedbyalongersequence.Thisisoformhasadifferentlocalizationwithinthecell,beingfound
ontheplasmamembrane,inthelysosomesandonothercytosolicstructuressuchasendoplas-
micreticulumandGolgiapparatus[4,5].Cystinosinwasdemonstratedtoactasaproton-
dependenttransporter[6,7],thus,effectivecystinetransportisstrictlydependentontheacidic
pHinsidethelysosomallumen.Cystinosindeficiencyresultsinlysosomalcystineaccumulation
inallbodyorgansandtissues.Treatmentwithcystine-loweringdrugcysteamineformsthebasis
ofcurrenttherapyofcystinosis[2,8].Cysteamineentersthelysosomeandsplitscystinemole-
culeintocysteineandcysteine-cysteaminemixeddisulphide.Bothproductscanthenbereleased
fromthelysosomethroughcysteineandPQLC2transportersrespectively[9].
Themostsevereinfantilenephropathicclinicalformofcystinosisistypicallyassociated
withmutationsresultinginacompletelossoffunctionofcystinosin[10].AmongtheCauca-
siansoriginatingfromtheNorthernEurope,themostprevalentmutationisa57-kbdeletion,
whichaffectsthefirst10exonsoftheCTNSgene[1].Cellsderivingfrompatientsbearingthis
deletionexpressnocystinosinandaccumulatecystineinthelysosomesinnormalculturing
conditions[11].
TheinitialclinicalmanifestationofcystinosisdevelopingduringinfancyisrenalFanconi
syndrome,ageneralizedrenalproximaltubulardysfunction,characterizedbypolyuriaandab-
normalurinarylossofaminoacids,glucose,low-molecular-weight(LMW)andintermediate
weightproteinsandothersolutes[2,11,12].Inhumans,generalizedaminoaciduriaappearsas
thefirstbiochemicalsign,alreadyinaneonatalperiodfollowedbyglucosuria,phosphaturia
andurinarylossofbicarbonateandproteins,graduallydevelopingintoafull-blownFanconi
syndromeby~6monthsofage[13].Laterinlife,untreatedpatientsdevelopprogressiverenal
damageleadingtotheend-stagerenaldisease(ESRD)andmultipleextra-renalcomplications
affectingeyes,endocrineorgans,liver,musclesandcentralnervoussystem[2,14,15].Treat-
mentwithcysteaminepreventslysosomalaccumulationofcystine,improvesgrowthandpost-
ponestheprogressionofrenaldiseaseandthedevelopmentextra-renalcomplications,
however,itoffersnocureforrenalFanconisyndrome,althoughsomeimprovementhasbeen
reportedinpatientstreatedstartingfromtheearlyage[14–18].
ThemechanismofrenalFanconisyndromeincystinosisisstillnotcompletelyunderstood
[11].Impairedapicaltransporterfunctionwasdemonstratedinculturedproximaltubularcells
fromcystinosispatients[19].Reabsorptionofproteinsfromtheprimaryurineisperformedby
endocytosisandisdependentontheconcertedfunctioningofmultiligandscavengerreceptors
presentontheapicalsurfaceofproximaltubules.Recentstudiesincystinosismousemodel
(Ctns-/-mice)haveshownadecreasedexpressionofthemulti-ligandreceptorsmegalinand
cubilinattheproximaltubuleapicalsurfacesassociatedwithcelldedifferentiation[20,21].
Therefore,theimpairedreabsorptionintheaffectedproximaltubulescanresulteitherfrom
thedecreasedexpressionofthemultiligandreceptorsorfromtheimpaireddeliveryofthere-
ceptorsand,possibly,othertransporterstotheapicalsurfaceofproximaltubularcellsdueto
deficientvesiculartraffickingandrecycling.Acombinationofbothpathologicalchangesis
alsopossible.However,anaccuratestudyoftheendocytosisandvesiculartraffickinginhuman
proximaltubularcellsdeficientforcystinosinhasnotbeenperformed.
Themajorchallengeinstudyingthepathogenesisofrenaldysfunctionincystinosisduring
manyyearswastheabsenceofasuitablemodel.ThefirstmousemodeldevelopedonaFVB/N
geneticbackgroundshowednosignsofkidneydiseasedespitepronouncedcystineaccumula-
tioninthekidney[22].Subsequently,anovelmurinemodelofcystinosisgeneratedonapure
C57BL/6backgroundshowedsignsofproximaltubulopathyandkidneyfailureandtherefore
offeredanopportunityforstudyingpathogenesis[23,20,21].However,itremainsunclearto
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Endo-LysosomalDysfunctioninCystinosis
whichextentthepathogenicmechanismsdescribedinmicecanbeextrapolatedtohuman
proximaltubularcells,especiallytakingintoaccountthatrenalphenotypeinmicedonotfully
replicatehumandisease[23].Furthermore,itisnotalwaysconceivabletodifferentiatebetween
pathologicandcompensativemechanismsinvivo[21].
Inourstudyweexaminedthedirectconsequencesofcystinosindeficiencyonproximaltu-
bularreabsorptionmachineryinhumanproximaltubularcellsafterdown-regulationofthe
CTNSgenewithspecificsiRNAandconfirmedtheresultsinproximaltubularcellsderived
frompatientswithcystinosis.Wefoundthattheabsenceofcystinosinresultedinanacutedis-
organisationofendo-lysosomalcompartments,withclusteringofendocytoticvesiclesinthe
perinuclearregion,decreasedexpressionofmultiligandreceptorsonthecellsurfaceandde-
layedprocessingoftheligands.Thesedata,togetherwithourfindingofabnormalco-localiza-
tionoflateendosomalandlysosomalmarkerLAMP-1withthemotorproteinkinesin-1in
cystinosin-deficientcells,suggestthatcystinosinfunctionsasanimportantregulatorofendo-
lysosomaldynamics.Thus,theabsenceofcystinosinmightleadtoadefectivemovementofen-
dosomestothecellsurface.Importantly,pre-treatmentwithcysteamineresultedinpartialim-
provementofthesurfaceexpressionofreceptorsandrescuedligandprocessing,butcouldnot
restoretheabnormalmorphologyoftheendo-lysosomalcompartments.Ourworkmakesa
contributiontotheunderstandingofpathologicalprocessesleadingtotherenalFanconisyn-
dromeincystinosisandprovidesfurtherdirectionsforstudyingpossibletreatmentsofthedis-
easeaimedtorestorethedefectivetraffickingmechanisms.
MaterialsandMethods
Antibodiesandreagents
HumanrecombinantGST-RAPwasproducedandisolatedaspreviouslydescribed[24].
ChemicalreagentsofanalyticalgradeorhigherwerefromSigma-Aldrich.Theantibodiesused
inthisstudyincludeAlexa-488-conjugatedanti-LAMP1(SantaCruz),anti-LAMP1rabbit
monoclonalantibody(CellSignaling),anti-GST(16B12)(Covance),anti-EEA1(CellSignal-
ing),anti-KIF5b(Millipore),anti-HAmousemonoclonalandrabbitpolyclonalantibodies
(Covance).TheAlexa-Fluor-488,Alexa-Fluor-568andAlexa-Fluor-633secondaryantibodies
werefromLifeTechnologies.
Cellcultureandtransfection
HumankidneyproximaltubularepithelialHK-2cellsoriginatefromtheAmericanTissue
TypeCollection(ATTC,USA)andwereusedtostudytheendocytosispreviously[25].Human
conditionallyimmortalizedPTEC(ciPTEC)wereobtainedfromlivingkidneycellsexfoliated
intourineasdescribedpreviously[26].ProximaltubularcellsweregrowninDMEM-HAM’s
F12culturemedium(Lonza)supplementedwith10%foetalbovineserum(Gibco),50IU/mL
penicillinand50mg/mLstreptomycin(Lonza).ForciPTEC,themediumwasadditionallysup-
plementedwith5μg/mlofinsulin,5μg/mloftransferrin,5ng/mlofselenium,40pg/mloftri-
iodothyronine,36ng/mlofhydrocortisoneand10ng/mlofEGF(allfromSigma)[26,27].
HK-2andciPTECcellsweretransfectedwithplasmidvectorsusingeitherTransIT-LT1
(MirusBioLLC)orjetPRIME(PolyplusTransfection),accordingtothemanufacturer’sin-
structions,andincubatedfor16–20hbeforefixation.
Down-regulationoftheCTNSgene
ThesiRNAoligonucleotidestargetingCTNSgenewerepurchasedfromDharmacon(ON--
TARGETplusSMARTpoolJ-019914-05–08).HK-2cellsweretransfectedwithsiRNAusing
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Endo-LysosomalDysfunctioninCystinosis
Dharmafect4(Dharmacon)transfectionreagentaccordingtothemanufacturer’sinstruction
andincubatedfor2to3daysbeforeperformingfurtherexperiments.Down-regulationwas
confirmedbyreal-timeRT-PCRusingprimers,specificforCTNS.Actinwasusedasahouse-
keepinggenecontrol.
Immunocytochemistryandconfocalmicroscopy
Forimmunocytochemistry,cellsgrownonglasscoverslipswerefixedwith4%paraformalde-
hydefor10minatroomtemperatureandwashedonceinPBS.Blockingreagentwasaddedto
thecellsfor20min,followedbya1hincubationwithappropriateconcentrationsoftheprima-
ryantibodiesinblockingreagent(0.5%BSA,0.05%saponine,50mMNH4Cl,0.02%NaN3in
PBS).ThecellswerethenwashedwithPBSandincubatedwithsecondaryantibodies(1:400)
andDAPI(1:1000)fordetectionofnuclei,dilutedinblockingsolution.Thecellswerewashed
twiceinPBSandonceinion-freewatertoremovesalts.Thecoverslipswerethenmountedon
glassmicroscopeslides(CarloErba,Italy)withMowiol(Calbiochem).Thesampleswereexam-
inedundereitherZeissLSM710orZeissAxiovert100Mconfocallaser-scanningmicroscopes,
inbothcasesequippedwitha63X(NA1.4)oil-immersionobjective.5–10randomlychosen
fieldswereanalyzedforeachcondition.ForRAPuptakeexperiments,atleast150individual
cellswereanalyzedforeachtimepoint.Imageprocessingandanalysiswereperformedusing
ImageJsoftware.Fluorescenceintensitywasmeasuredingreenandredchannelsaftersubtract-
ingbackgroundinsideaselectedareacorrespondingtoanindividualcell.Microscopicimages
werepreparedforpublicationusingImageJ(http://rsb.info.hin.gov/ij/)andAdobePhotoshop
CS4softwareforcropping.Themeasurementofthelysosomalsizewasperformedasdescribed
previously[28].LysosomediameterwasmeasuredusingImageJsoftware,withlysosomesap-
proximatedascircleswiththesizefrom0.1to5.0μm2.Thresholdswerekeptthesamefor
allimages.
Electronmicroscopy
Forelectronmicroscopy,smallpiecesofcystinotickidneybiopsydescribedpreviously[29]and
controlkidneybiopsyavailableatthePathologyDepartmentofRadboudUniversityMedical
Centre(Nijmegen,TheNetherlands)obtainedfromasubjectwithoutcystinosisorcystinosis
patienthavingFanconisyndromewerefixedin2.5%glutaraldehydedissolvedin0.1Msodium
cacodylatebuffer,pH7.4overnightat4°Candwashedinthesamebuffer.Thetissuefragments
werepostfixedinpalade-buffered2%OsO for1h,dehydrated,andembeddedinEpon812
4
(Merck,Darmstadt,Germany).Ultrathinsectionswerecutonanultratome(Leica,Reichert
Ultracuts,Wien,Austria),andcontrastedwith4%uranylacetatefor45minandsubsequently
withleadcitratefor4minatroomtemperature.SectionswereexaminedinaJeol1200EX2
electronmicroscope(JEOL,Tokyo,Japan).
Endocytosisassay
Cellswereserum-starvedfor1handwashedtwiceincoldPBScontaining1%BSA.Thecells
wereincubatedfor30minonicewith2.5μg/mlrecombinantGST-RAPinserum-freemedi-
um.Tomeasuresurfacebindingoftheligand,cellswerewashedwithcoldcompletemedium
andfixedin4%paraformaldehyde.Toassesstheendocytosis,thecellswerewashedwithcom-
pletemediumandincubatedat37°Ctoallowinternalizationoftheligandattheindicated
times.Attheendofthisincubation,thecellswerebrieflyacidwashed(150mMNaCl,10mM
aceticacid,pH3.5)andfixed.
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Endo-LysosomalDysfunctioninCystinosis
Megalinuptakeandrecyclingassay
FortheHA-Meg4internalisationassaycontrolandCTNSKDHK-2cellsweretransiently
transfectedwithHA-Meg4andpcDNA3-RAP,serumstarvedfor2hat37°Cinserum-free
DMEM,washedtwiceincoldPBSwith1%BSA,chilledto4°C,andincubatedwiththeanti-
HAmonoclonalantibodyforindicatedtimesinanice/waterbath,tolabelthecellsurface.The
unboundantibodywasrinsedoffwithcoldHEPES-bufferedDMEM,andthecellswereincu-
batedinpreheatedcompletemediumforindicatedtimesat37°C.Afterinternalisation,thean-
tibodiesremainingonthecellsurfacewereremovedbya30sacidwash(150mMNaCl,
10mMaceticacid,pH3.5),withabriefwashinPBS,andthenthecellswerefixed.
BSAdegradationassay
Lysosomaldegradationwasmeasuredasdescribedpreviously[30].Briefly,controlandcysti-
nosisciPTECgrowninglass-bottomcellculturechambers(LabTek)wereincubatedwith
5μg/mlDQ-BSAand5μg/mlAlexa-Fluor-555-BSA(LifeTechnologies)at37°Cfor2h,
washedandanalyzedunderaconfocalmicroscope.Cellstreatedwith100nMofbafilomycin
A1wereusedasacontrol.
MeasurementoflysosomalpH
ControlandcystinoticciPTECandcontrolandCTNSKDHK-2cellswereloadedwith10μM
ofacridineorangefor15minat37°C,washedinHEPES-bufferedcompletemediumandana-
lyzedunderaconfocalmicroscope.Acridineorangewasexcitedat450–490nmandthesignal
recordedat520–560nmor>620nm.Typically,5–10imageswerecollectedfrom3separate
experimentsandthered/greenratiowascalculated.
Results
Morphologyofendosomalcompartmentsisalteredincystinosin-
deficientcells
Westudiedthechangesintheendo-lysosomalcompartmentsincystinosisusingtwoworking
models.First,weusedhumanproximaltubularHK-2celllinetreatedwithsiRNAspecificto
theCTNSgene(CTNSKD).Theefficacyofdown-regulationwasevaluatedbyreal-timePCR
priortoeachexperimentandwas80%orhigherafter2days.Thecystineaccumulationwas
notdetectableafter2daysofdown-regulationwhenthemorphologyandendocytosisassess-
mentswereperformed.Afteraprolonged4-dayexposure,HK-2CTNSKDcellsdemonstrated
a~7-fold(p<0.005)increaseofcystinecontentincomparisontothecontrol,confirmingthe
disruptedfunctionofthetransporter.However,theformationofthedensecobblestone-like
monolayerofHK-2cellsobstructedtheimagingexperimentsatthistimepoint.Next,wecon-
firmedtheresultsinciPTECthatwereestablishedasdescribedpreviouslyfromlivingexfoliat-
edcellsextractedfromurineofacystinosispatientbearingthe57-kbdeletionandofahealthy
volunteer[19].
Tostudytheendosomalcompartments,controlandcystinosisciPTECcellsandCTNSKD
HK-2cellswerestainedwithantibodiesspecifictomarkersofearlyendosomesandlateendo-
somes/lysosomes(EEA1andLAMP1respectively),theER(VAP-A),Golgi(GM130)andTGN
(TGN46).Tomeasurethediameterofindividuallysosomes,weusedthemethod,described
previously[28].Clusteredlysosomeswereexcludedfromtheanalysisbythesizefiltering.In
cystinosisciPTEC,averagesizeoftheindividualLAMP1-positivevesicleswasenlargedby35%
(p<0.01),andthevesiclesdemonstratedadifferentdistributionpatternthanintheciPTEC
fromahealthydonor(Fig.1a).InHK-2cells,accuratemeasurementofthelysosomalsizewas
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Endo-LysosomalDysfunctioninCystinosis
notfeasibleduetohighlevelofclusterizationofendosomalvesiclesinthemajorityofCTNS
KDcells:EEA1-positiveandLAMP1-positivevesicleswereabnormallyclusteredinperinuclear
area(Fig.1b).Toestimatequantitativelytheeffect,twoindependentobserversanalyzed2dis-
tinctgroupsofcells,characterizedbya‘perinuclear’localizationofthevesicles,inwhichnola-
belledcompartmentswerevisibleoutsidethecompactperinuclearcluster,orbya‘perinuclear
+peripheral’localization,inwhichendosomesweredistributedthroughoutthecentralandpe-
ripheralareas.Theformerphenotypewasdefinedascystinotic,andthelatter—asnormal,
sinceitwasobservedinmostofthecontrolHK-2cells.InCTNSKDHK-2cells,thenumberof
cellswithperinucleardistributionofendosomesincreasedbymorethan2foldsincomparison
withthecontrol(Fig.1c).
AscystinoticciPTECarecharacterizedbyasignificantcystineaccumulationundernormal
cultureconditions,wetestedwhethercystine-loweringtreatmentcouldrescuetheabnormal
phenotypeobserved.ControlandcystinosisciPTECcellsweretreatedwith100μMofcyste-
aminefor24h.Suchtreatmentwaspreviouslyshowntodecreasethecystineaccumulationby
~91%withoutcausingtoxiceffectsonculturedcells.Longertreatmentdidnotresultinasig-
nificantfurtherdecreaseofcystinecontent(S1Fig.).Wefoundthatthecysteaminetreatment
hadnosignificanteffectonlysosomalsizeanddistribution(Fig.1a).
Toconfirmourinvitrofindings,weperformedelectronmicroscopystudyofabiopsymate-
rialobtainedfromacystinosispatienttreatedwithcysteaminethatwaspreviouslydescribed
[29].Wefoundanincreasednumberofelectron-denselysosomesclusteredintheperinuclear
regioninproximaltubularcellscomparedtocontroltissuesamples(Fig.1d).
Toidentifydifferentendosomalcompartments,wetransfectedcontrolandCTNSKDHK-2
cellswithGFP-taggedRabGTPases,commonlyusedforthispurpose[31–33].Theco-
localizationofRab5andRab7,correspondingtoearlyandlateendosomes,withEEA1and
LAMP1markersrespectively,wasunchanged,andnomixingofthedifferentendocyticcom-
partmentswasdetectedinCTNSKDcells.ThesubcellularlocalizationofRab6andRab9,cor-
respondingtotrans-Golginetworkandlateendosome-to-Golgitransportdomains,was
alsounaffected.
Distributionofkinesin-1isalteredincystinosin-deficientcells
Theperinuclearclusteringoflysosomessuggestedapossibleinvolvementofthemicrotubule-
basedmotorproteinkinesin-1responsibleforthetransportofendosomalvesiclestowardsmi-
crotubulesplus-end.Visualizationofkinesin-1withanti-kinesin-1heavychainB(KIF5b)anti-
bodiesshowedanalteredsubcellularlocalizationoftheproteininCTNSKDHK-2cellsas
comparedtocontrolcells.Moreover,a~8-foldincreaseofco-localizationofkinesin-1with
LAMP1wasobserved(Fig.2a,b,p<0.001).Togethertheseobservationssuggestthattheal-
teredendosomalandlysosomaltraffickinganddistributionincystinosin-deficientcellsmight
beexplainedbyincorrectdynamicsoflysosome-associatedmotorproteins,asdiscussedlater.
Multiligandreceptor-mediatedendocytosisisimpairedincystinosin-
deficientcells
Toestimatethesurfaceexpressionofmegalin,weusedarecombinantGST-taggedRAPpro-
tein,whichbindsmegalinwithhighaffinityandhasbeenusedtostudyendocytosisinproxi-
maltubularcellspreviously[25].HK-2orciPTECcellswereincubatedwithexogenous
GST-RAPat4°Cfor30minutes,fixedandstainedwithantibodiesagainstGSTtovisualisesur-
face-boundGST-RAP.WefoundthatsurfacebindingofGST-RAPwasreducedby~30%
(p<0.05)inCTNSKDHK-2cells(Fig.3a,b)andby~60%(p<0.01)inciPTECderivedfroma
cystinosispatient(Fig.3c,d)ascomparedtocontrol.Theseobservationsindicateareduced
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Fig1.Alteredendosomal/lysosomalcompartmentsincystinosin-deficientcells.(a)AlteredmorphologyofearlyandlateendosomesinciPTEC
derivingfromthecystinosispatient:lateendosomesappearedenlargedandabnormallyclusteredaroundthenuclei(*),andEEA1-positiveendosomes
tendedtoclusterinperinucleararearatherthantospreadthroughoutthecellasincontrolciPTEC(arrows).Cysteaminetreatmenthadnoinfluenceonthe
abnormalphenotypeincystinoticcells.Scalebar=10μm.(b)Immunostainingwithantibodiestoearlyendosomal(EEA1)andlateendosomal/lysosomal
(LAMP1)markersrevealedalteredmorphologyinCTNSKDHK-2cells.Scalebar=10μm.(c)Numberofcellswith“cystinotic”phenotype(endosomes
clusteredinperinuclearareaanddisappearanceofperipheralvesicles)wasquantifiedinCTNSKDcells;representationof3independentdown-regulation
experiments(**p<0.01).(d)Transmittedelectronmicroscopyofkidneybiopsymaterialobtainedfromacystinosispatienttreatedwithcysteamineanda
controlsubjectand.Incystinosistissue,perinuclearlocalizationofelectron-denselysosomes(asterisk)wasdemonstratedinPTEC.Scalebar=3μm.
doi:10.1371/journal.pone.0120998.g001
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Fig2.Abnormaldistributionofkinesin-1motorproteinincystinosin-deficientcells.(a)ControlandCTNSKDHK-2cellswerefixedandstainedwith
antibodiesspecifictoKIF5b,acomponentofkinesin-1complex,andLAMP1.CTNSKDcellshadanabnormaldistributionofkinesin-1toLAMP1-positive
enlargedstructures.Scalebar=10μm.(b)Co-localizationanalysiswasperformedusingImageJJACoPplug-inon50controlandCTNSKDcells.CTNSKD
cellshada8-foldincreaseofco-localizationbetweenKIF5bandLAMP1(***p<0.001).
doi:10.1371/journal.pone.0120998.g002
surfaceexpressionofmegalinincellsdeficientforcystinosin.TreatmentofcystinoticciPTEC
with100μMofcystine-loweringdrugcysteaminefor24hresultedinapartialimprovementof
GST-RAPsurfacebinding(Fig.3c,d).
Tostudyendocytosis,cellspre-incubatedwithGST-RAPligandweretransferredto37°Cto
allowinternalizationoftheligandboundtothecellsurface.Atindicatedtimepoints,cellswere
fixedandstainedforGST-RAP.Atearliertimepoints,thestainingwasvisibleinvesicular
structurescorrespondingtoendosomesandthengraduallydisappeared.InCTNSKDHK-2
cells,GST-RAPremainedvisibleforsignificantlylongertimeafterinternalizationincompari-
sontocontrols(Fig.4a).
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Fig3.Decreasedsurfaceexpressionofmegalinincystinosin-deficientcells.(a,b)ControlandCTNSKDHK-2cellswereincubatedwithGST-RAPfor
30minonice.Asevaluatedbyfluorescenceintensityofindividualcells,surfaceexpressionofmultiligandreceptorswasdecreasedincystinosin-deficient
cells(*p<0.05).(c,d)ciPTECcellsfromhealthydonorandcystinosispatientwereincubatedwithGST-RAP.Cystinoticcellsdemonstratedadecreased
surfaceexpressionofmultiligandreceptors(**p<0.01).TreatmentofcystinoticandcontrolciPTECwith100μMcysteaminefor24hpartiallyrestored
GST-RAPsurfacebinding(*p<0.05).Scalebar=10μm.
doi:10.1371/journal.pone.0120998.g003
TheobservationwasalsoconfirmedincystinoticciPTEC(Fig.4b).Importantly,pre-treat-
mentofcystinoticciPTECwith100μMcysteaminefor24hpriortotheexperimentpartially
rescuedthedelayedcargoprocessing(Fig.4b).
Decreasedsurfaceexpressionofmegalinisassociatedwithimpaired
trafficking
Toexcludethepossibilitythatreducedmegalinsurfaceexpressionwascausedbydecreasedex-
pressionofendogenousmegalinincystinosindeficientcellsandtostudymegalinlocalization
andtrafficking,weusedHA-Meg4mini-receptorconstructtransientlyexpressedinHK-2cells.
PLOSONE|DOI:10.1371/journal.pone.0120998 March26,2015 9/18
Endo-LysosomalDysfunctioninCystinosis
Fig4.EndocyticuptakeandprocessingofGST-RAPligand.(a)ControlandCTNSKDHK-2cellswereincubatedwithGST-RAPfor30minoniceand
thenwereallowedtointernalizetheligandat37°Cfor15and45min.GST-RAPprocessingwasevaluatedbytheintensityofanti-GSTstainingwithspecific
antibodiesatindicatedtimepoints.DegradationoftheligandwasdelayedinCTNSKDcellsincomparisonwiththecontrol,asexpressedafternormalization
ofthesignaltotheinitialvalue(surfacebindingat0min).(b)TheobservationwasconfirmedincontrolandcystinoticciPTECcells(**p<0.01after45min).
TreatmentwithcysteamineresultedinpartialrescueoftheGST-RAPprocessing(*p<0.05after45min).Scalebar=10μm.
doi:10.1371/journal.pone.0120998.g004
PLOSONE|DOI:10.1371/journal.pone.0120998 March26,2015 10/18