hadbeenchosenasacontrolItseemsser - PDF document

1 hadbeenchosenasacontrol.Itseemsser- endi
hadbeenchosenasacontrol.Itseemsser- endipitousthattheyneverthelessdecidedto storethesamplesintherefrigeratorand reassaythematalatertime.Theresults obtained
vedayslatercametosteerthe researchersontoanewpaththatledthem totheirdiscovery,
rstofthelysosomeand latertheperoxisome. DeDuveandhisgroupfoundthat,with theexceptionoftheactivityinthe
nalsu- pernatant,acidphosphataseactivitieshad risenproportionatelyinallofthefractions, aswellasintheunprocessedhomogenate, whoseactivitynowcorrespondedtothesum oftheactivitiesinallofthefractions.They soonshowedthattheeffectof “ ageing ” the fractionsintherefrigeratorcouldberecre- atedbytreatmentsthatdisruptmembranes, suchasblenderhomogenizationorrepeated cyclesoffreeze-thawing.Onthisbasis,de Duveinsightfullyconcludedthatthe “ latent enzyme ” wassequesteredwithin “ mem- branesacs ” thatmadeitinaccessibleto thesubstrates. Thestudiesonacidphosphataseprompted deDuve ’ sgrouptodevelopaprocedurethat separatedfromthefractionrichinmitochon- driaa “ lightmitochondrialfraction ” orL fraction,whichcontainedmostoftheacid phosphatasebutverylittlecytochromeoxi- daseactivity.WhatdeDuve ’ slaboratory,in fact,accomplishedwasthepuri
cationofa neworganellesolelyonthebasisofanalytical biochemicalprocedures,guidedbymeas- urementsofspeci
cenzymaticactivities, whicharenowregardedas “ marker enzymes. ” The
ndingthatfourotheracid hydrolases —
-glucuronidase,cathepsinD, ribonuclease,andDNAse — displayedlatency andwerealsoenrichedintheLfractionled deDuvetoformulatethe “ lysosome ” con- cept:thatis,amembrane-boundedorganelle thatcontainsacidhydrolaseswithvarious speci
citiesandwhosemainfunctionisthe intracellulardigestionofmacromolecules. Later,asprogresswasbeingmadeineluci- datingthebroadfunctionoflysosomes,De Duvealsocoinedtheterms “ endocytosis, ” “ phagocytosis, ” and “ autophagy ” todesig- natepathwaysthatbringsubstratesfordi- gestioninlysosomesand,today,areactive
eldsofresearchincellbiology. Remarkably,deDuvearrivedatthelyso- someconceptwithoutresortingtoany microscopicexaminationofhissamples. Infact,therewasnomicroscopeinhis laboratoryandheentitledhisNobellecture “ ExploringCellswithaCentrifuge. ” The lysosomeobtainedamorphologicalidentity in1955asaresultofabriefcollaboration withAlexNovikoff,avisitingscientistfrom theAlbertEinsteinCollegeofMedicinein NewYork,whohadexpertiseinelectron microscopy.Novikoff ’ smicrographsshowed thatthe “ lightmitochondrial ” fractioncon- tainedmembranebounded “ densebodies ” similartothosepresentintheperi-canalicular regionofhepatocytes. Thediscoveryofthelysosomeinaugurated aneweraincellularphysiologyandpath- ophysiology,whichwasfollowedbythe identi
cation,
rstinLouvainandthen throughouttheworld,ofmorethan40 lysosomalstoragediseasesresultingfrom mutationsingenesforspeci
chydrolases. The
rstinklingthat,inadditionto lysosomes,thelightmitochondrialfraction alsoharboredanasyetunknownorganelle, wasthe
ndingthaturateoxidase — anen- zymethatisnotanacidhydrolaseanddoes notshowlatency — hadasimilardistribution insubcellularfractionsasacidphosphatase. By1960,deDuvehadfoundthatthiswas alsotrueforcatalaseandfor D -aminoacid oxidase,thenthoughttobemitochondrial enzymes.Helaterextendedthese
ndings toseveralotherperoxide-producingoxidases withasedimentationbehaviorsimilartocat- alase,anenzymethatbreaksdowntheir product.DeDuvehadtheinsightthatafunc- tionallinkagebetweentheseenzymesexisted, whichwasmadepossiblebytheirinclusion inthesameparticle.Thus,theconceptof aperoxisomewasbeingborn,butitwas nottobepresentedpubliclyuntilseveral yearslater,afterdeDuvehadbeguntosplit histimebetweenLouvainandNewYork. In1962deDuveacceptedanattractive offertocreateanddirectalaboratoryatThe RockefellerInstituteinNewYork,while maintaininghislaboratoryinLouvain.He wasabletotransfertohisnewlaboratorythe varioustechnologiesdevelopedinLouvain byarrangingforregularvisitsofhismajor BelgianassociatestoNewYork.Inbothlab- oratories,deDuvecontinuedthecharacter- izationofthenewlydiscoveredoxidase- containingparticles
rstidenti
edinrat liver.Threeyearslater,onlyafterparticles withasimilarsedimentationbehaviorand biochemicalpropertieswerefoundinrat kidneyandintheciliatedprotozoan Tetrahy- menapyriformis ,didheannounce,atameet- ingoftheAmericanSocietyofCellBiology, thathehaddiscoveredaneworganelle,for whichheproposedthename “ peroxisome. ” Againinthiscase,electronmicroscopy showedthat,morphologically,thenewor- ganellecorrespondedtomembrane-bounded particlesofunknownfunctionthathad beenrecognizedbymicroscopiststobe presentinalmostalltissuesandhadbeen designated “ microbodies. ” Subsequentstudiesfrommanylaborato- ries,includingthosefromdeDuve ’ sandhis formerassociatesandstudents,showedthat peroxisomes —
rstdiscoveredinmammalian tissues,wheretheyplayimportantmetabolic roles,includingthe
-oxidationofverylong- chainfattyacidsbyapathwaydifferentfrom thatinmitochondria — aremembersofalarge familyofevolutionarilyrelatedorganelles presentinmanydifferenteukaryoticcell typesandorganisms,includingplants,and protozoa,wheretheycarryoutdistinctfunc- tionsandhavebeengivenspeci
cnames, suchasglyoxysomesandglycosomes.Thus, withhisdiscoveryofperoxisomes,deDuve oncemorelaidthefoundationforthe growthofanewchapterintheburgeoning
eldofCellBiology. In1974,soonafterreceivingtheNobel Prize,deDuve,inspiredbyhisexperienceat TheRockefellerInstitute,championedthe creationinBrusselsofanewmultidisci- plinary “ InternationalInstituteofCellular andMolecularPathology, ” withatransla- tionalmission,whichheoriginallydirected andathis80thbirthdaywasrenamedthe “ deDuveInstitute. ” DeDuveleftamajorimprintinthebi- ologicalsciencesthroughtheworkhecarried outonbothsidesoftheAtlanticandthrough themanyscientistswhotrainedwithhim.He wasahighlyc

2 ulturedpersonwhospokefour languages 
ulturedpersonwhospokefour languages  uentlyandwroteelegantprosein atleasttwoofthem.DeDuve ’ sinterestsex- tendedwellbeyondtheareasofhisscienti
c contributions,intotherealmsofphilosophy, thetheoryofknowledge,theoriginoflife, andtheevolutionoftheeukaryoticcell.He publishedextensivelyhisthoughtsonques- tionsfromalmostallthese
elds,inlucid articlesaswellasinbooks.DeDuvealso wrotemanyengaginghistoricalaccountsof themajorscienti
cdiscoveriesmadeinhis laboratoriesandinallofthemhetookgreat caretogivecredittohisyoungerassociates andtopointouttheirspeci
ccontributions. ChristiandeDuvewasawarmcolleague andafascinatingconversationalist.Thoseof uswhohadthegoodfortuneofknowinghim personallywillsorelymisshim. SabatiniandAdesnik PNAS | August13,2013 | vol.110 | no.33 | 13235 RETROSPECTIVE RETROSPECTIVE ChristiandeDuve:Explorerofthecellwho discoveredneworganellesbyusingacentrifuge DavidD.Sabatini 1 andMiltonAdesnik DepartmentofCellBiology,NewYorkUniversitySchoolofMedicine,NewYork,NY10016 ChristiandeDuve,whoselaboratoryin Louvaindiscoveredlysosomesin1955and de
nedperoxisomesin1965,diedathis homeinNethen,Belgiumattheageof95,on May4,2013.DeDuvewasthelastofagroup ofeminentphysiologicalchemistswho,by the1940sand1950s,begantoexplorethe subcellularorganizatio nofbiochemicalpath- waysandthusforgedtheemergenceof ModernCellBiology.ChristianDeDuve, AlbertClaude,andGeorgePaladereceived theNobelPrizein1974 “ fortheirdiscoveries concerningthestructuralandfunctionalor- ganizationofthecell. ” DeDuvewasbornonOctober2,1917in ThamesDitton,UnitedKingdom,atownnot farfromLondonwherehisfamilyhadsought refugeduringWorldWarI.Afteraclassic educationinaJesuitschoolinAntwerp,De DuveenteredtheMedicalSchoolofthe CatholicUniversityofLouvainin1934,with nointentionofbecomingascientist.He creditedastudentapprenticeshipwithJoseph Bouckaert,whoheadedthephysiologylabo- ratory,forsparkinghisinterestinbasic research.AmajorconcernofBouckaert ’ sre- searchwasthemechanismofactionofin- sulin.DeDuveparticipatedinexperimentsin whichrathercrudepreparationofthehor- monewereadministeredtohepatectomized animals,whichledhimtoadopttheideathat insulinactedprimarilyontheliver,andfor manyyearsheinvestigatedwithintensitythe validityofthisnotion. DeDuvewasinhislastyearofmedical schoolwhentheGermansinvadedBelgium in1940.Hisinvolvementinthewarwas minor,ashewasdraftedasamedic,and soonwasabletoreturntoLouvainto
nish medicalschool.However,bythattimede Duve ’ scommitmenttoresearchwastoo strongforhimtopursueacareerinmedi- cine.AftercompletingaMaster ’ sthesisin chemistryatLouvainin1946,deDuvespent overayearasapostdoctoralfellowinStock- holmwithHugoTheorell,apioneerinthe studyofoxidizingenzymeswhoreceivedthe NobelPrizein1955.Theorell ’ slaboratory providedanidealplacefordeDuvetolearn themostadvancedtoolsofenzymology, whichwerecentraltohislaterwork.His Swedishsojournwasfollowedbyavisitto thelaboratoryofCarlandGertyCoriinSt. Louis,theMeccaofcarbohydrateresearchat thetime,whereheworkedforafewmonths withEarlSutherland,withwhomheidenti-
edglucagonasacontaminantofinsulin preparationswidelyusedinthosedays.Glu- cagonwasoftenreferredtoasthe “ hypergly- cemicglycogenolyticfactor ” anddeDuve laterproudlyreferredtothisworkashis “ re-discoveryofglucagon. ” Sutherland ’ sfur- therworkonthehormonalcontrolofglyco- genolysisledhimtothediscoveryofcAMP, forwhichhereceivedtheNobelPrize in1971. In1948,deDuvereturnedtoLouvain, whereheintendedtopursuehisinterestin carbohydratemetabolismandtheactionof insulin.Withanewlyassembledgroup ofyoungcollaborators,deDuvedecided tocharacterizethehexosephosphatase, which — followingtheactionofphosphorylase onglycogen — wasresponsiblefortheunique propertyofthelivertoreleaseglucoseintothe blood.Theresearchersidenti
edaliverphos- phatasespeci
cforglucose-6-phosphateand correctlyconcludedthatitwasresponsiblefor thateffect.Theirsubsequentattemptstopu- rifythatenzymesetthemonthetracktothe discoveryoflysosomes. DeDuveandhisgroupobservedthatan acidicpHcausedanirr eversibleprecipitation oftheglucose-6-phosphatase,whichledde Duvetoinferthattheenzymecouldbeas- sociatedwithagglutinatedcytoplasmicmem- branes.Hence,thegroupdecidedtofollow thedistributionoftheenzymeinthevarious cellfractionsthatcouldbeobtainedfrom liverhomogenatesbyaproceduredeveloped byClaude,whichusedmildhomogenization conditionsandwasdesignedtopreservethe integrityofsubcellularorganelles. Itwasmostfortunatethatinthecourseof theseexperiments,inadditiontofollowing thedistributionofglucose-6-phosphatase — whichwasfoundtobeprimarilyinthe smallgranulefractioncalled “ microsomes ” byClaude — deDuve ’ sgroupalsofollowed, asacontrol,thedistributionandactivityin thesubcellularfractionsofacidphospha- tase,anenzymewithanoptimumpHof 5andaverybroadsubstratespeci
city, whichisfoundinalmostalltissues.Be- causethisenzymewassolublewhenhomo- genateswerepreparedinaWaringblender, theresearchersexpectedto
nditinthe
nalsupernatantobtainedbyClaude ’ spro- cedure.However,theactivitywasfoundto bepresenttovariousextentsinallofthe fractionsand,inparticular,inthelarge granulefractionknowntocontainthemi- tochondria.This
ndingwaspuzzling,as werealsothefactsthatthesumoftheactiv- itiesinallofthefractionswasmuchgreater thantheactivityinthewholehomogenate, whoseactivitywasmuchlowerthanwhen theWaringblenderwasusedfortheho- mogenization.Theseintriguingobservations wereobtainedinDecember1949justbe- foreaweekend,andcouldhavediscour- ageddeDuve ’ sgroupfromfurtherstudies onacidphosphatase,anenzymethat,after all,wasnotofmajorinteresttothemand ChristiandeDuve. Authorcontributions:D.D.S.andM.A.wrotethepaper. 1 Towhomcorrespondenceshouldbeaddressed.E-mail:david. sabatini@nyumc.org. 13234 – 13235 | PNAS | August13,2013 | vol.110 | no.33www.pnas.org/cgi/doi/10.1073/pnas.131208411