MitochondrialandMitochondrialDNAInheritance CheckpointsintheBuddingYeast, Saccharomyces cerevisiae DavidGarryCrider Submittedinpartialfulfillmentoftherequirementsforthe degreeofDoctorofPhilosophyintheGraduateSchoolof ArtsandSciences COLUMBIAUNIVERSITY 2012 ©2011 DavidGarryCrider Allrightsreserved ABSTRACT MitochondrialandMitochondrialDNAInheritance CheckpointsintheBuddingYeast, Saccharomycescerevisiae DavidGarryCrider This dissertation analyzes the importance of mitochondria and mitochondrial DNA in Saccharomyces cerevisiae during cell division . Movement and positional control of mitochondria and other organelles are coordinated with cell cycle progression in the buddingyeast,Saccharomycescerevisiae.Recentstudieshaverevealedacheckpoint thatinhibitscytokinesiswhenthereareseveredefectsinmitochondrialinheritance.An establishedcheckpointsignalingpathway,themitoticexitnetwork(MEN),participatesin this process. Here, we describe mitochondrial motility during inheritance in budding yeast, emerging evidence for mitochondrial quality control during inheritance, and organelleinheritancecheckpointsformitochondriaandotherorganelles. TABLE OF CONTENTS CHAPTER1................................................................................................1 INTRODUCTION........................................................................................1 MITOCHONDRIA....................................................................................................................2 CELLCYCLE..........................................................................................................................2 CHECKPOINTS......................................................................................................................5 MITOCHONDRIALINHERITANCE.......................................................................................10 QUALITYCONTROLDURINGMITOCHONDRIALINHERITANCE......................................11 MITOTICEXITNETWORKFUNCTIONINTHEMITOCHONDRIALINHERITANCE CHECKPOINT......................................................................................................................16 CHAPTER2 ........................................................................................................................20 MITOCHONDRIALINHERITANCEISREQUIREDFORMEN- REGULATEDCYTOKINESISINBUDDINGYEAST ..........................................20 SUMMARY...........................................................................................................................21 RESULTSANDDISCUSSION..............................................................................................22 Mutationsthatinhibitmitochondrialinheritanceproducemultibuddedcellsinbuddingyeast...........22 mdm10 ∆cellsexhibitdefectsincontractileringclosure....................................................................26 RolefortheMENinregulationofcellcycleprogressioninmdm10 ∆cells........................................32 ExperimentalProcedures...................................................................................................................38 Yeaststrains,plasmids,andgrowthconditions:................................................................................42 Yeaststrainsusedforthisstudy........................................................................................................43 CHAPTER3..............................................................................................46 MtDNAINHERITANCECHECKPOINT.....................................................46 BACKGROUND....................................................................................................................47 OTHERPROTEINSIMPLICATEDINmtDNAINHERITANCE..............................................48 mtDNAMUTATIONS:rho 0ANDrho -CELLS........................................................................49 Rad53ANDTHEDNADAMAGECHECKPOINT..................................................................51 LINKSBETWEENRad53ANDmtDNA................................................................................54 RESULTS.............................................................................................................................56 LOSSOFmtDNAINDUCESAG1ARRESTINCELLCYCLEPROGRESSION............................56 i THEG1TOSPROGRESSIONDEFECTOBSERVEDINCELLSLACKINGmtDNAISNOTDUE TOLOSSOFMITOCHONDRIALRESPIRATORYACTIVITYORENERGYPRODUCTION..........58 THEDEFECTINCELLCYCLEPROGRESSIONOBSERVEDINrho 0CELLSISDUETOLOSSOF DNAINMITOCHONDRIAANDNOTGENESENCODEDBYTHATDNA.......................................61 ROLEFORAKNOWNCHECKPOINTPROTEIN(Rad53)INREGULATIONOFCELLCYCLE PROGRESSIONINCELLSLACKINGmtDNA..................................................................................62 DISCUSSION.......................................................................................................................65 EXPERIMENTALPROCEDURES........................................................................................68 Yeaststrains,plasmids,andgrowthconditions:................................................................................68 CHAPTER4..............................................................................................72 DISCUSSION............................................................................................72 DISCUSSION.......................................................................................................................73 SUMMARYOFPROJECT1:................................................................................................74 FUTUREEXPERIMENTS:....................................................................................................75 SUMMARYOFPROJECT2:................................................................................................77 ROLEFORDNAPol γASASENSORFORTHEmtDNAINHERITANCECHECKPOINT:...79 HOWISTHESIGNALTHATmtDNALOSSTRANSMITTEDFROMMITOCHONDRIATO THENUCLEUS?..................................................................................................................83 POSSIBLEROLEFORPROHIBITINSINTHEmtDNAINHERITANCECHECKPOINT.......85 HOWMYSTUDIESMAYCONTRIBUTETOOURUNDERSTANDINGOF MITOCHONDRIALDISEASES.............................................................................................87 BIBLIOGRAPHY.......................................................................................90 ii ACKNOWLEDGEMENTS IwouldliketothankLizaPonforhermentorshipoverthepast3years.Thelab environmentshecreatedgiveseverygraduatestudenttheopportunityto succeed.Shesettheperfectbalancebetweenscientificcuriosityandreality;for whentofocusandfinishupthestory.Icannotthankherenoughforher dedicationandguidance. ThePonlabwouldnotfunctionasefficiently,oraswellasitdoes,withoutIstvan Boldogh.Heisthebackboneofthelab.Hehassuchawealthofknowledge,and suchaselflesspersonalitythathewilldropeverythingandhelpyouwheneverhe can.Iamsogratefulforallourconversationsandthetimewespenttogether.He trulymadethelabafunplace. IwouldliketothankTomLipkinfortakingthetimetograbacupofcoffeeand encouragemetojointhePonlab.TheresaSwaynemightnotrealizethatshewas thefirstpersonthatItalkedtowhenIcametoColumbia(duringmyorientation) andI’mhonoredtobeabletothankheronmylastdayatColumbia.ToJose “Ricky”RicardoMcFalineFigueroa(pickanameman!)whoislikeanolder,I meanyounger,brotherthatIneverhad,thankyou.Wediscussedverylittle scienceandforthatIcreditmycurrentsanity.ToLuisGarcia,whoIhavenever met,butwouldnothavehadsuchanexcitingprojectifitwasn’tforhisdiscovery andpreviouswork. iii Iwouldliketothankmyfirstscientificmentor,KennethBoheler,whoencouraged out-of-the-boxthinkingandmoldedmeintoascientist.Tothisdaymymost excitingexperimentwaswatchingmystemcellderivedcardiomyocytesbeaton thedish.WhowouldhavethoughtthatsingleexperimentwouldbethereasonI metmywife.I’dliketothankthoselittlecardiomyocytesforgrabbingmywife’s attentionandgivingmethechancetomeether,fallinlove,andgetmarried…my nextmostexcitingexperimentsareyettocome. Myfamilyhasprovidedunconditionalsupportformethroughoutthelastdecade asIwouldfinishonedegreeandstartthenext.Theyneverquestionedmy dedicationandalwaysunderstoodandacceptedthesacrificethatittooktoreach mygoal.Icannevergetthoselostholidaysandtimespentawayfromthemback butIhopethesacrificeisjustifiedintheend. Tomypap,thebestself-taughtscientistthatIknow. Everyonementionedhadahugeroleintogettingmetothisdaybutnoone deservesmorecreditthanmywifeCheryl.SheisthereasonIcamebackto graduateschoolmorefocusedanddeterminedthanever.Icannotthankher enoughforherpatienceandunderstandingwhenIhadtoworkeveryweekend andstayinlablatebutalsoforthosedayswheresheputherfootdownand physicallydraggedmeoutoflab.SheknewwhenIwasburningmyselfoutand whentotellmetogetmyassoffthecouchandfinish.Ican’tdescribehowmuch youmeantomeandIcouldn’thavecompletedthisjourneywithoutyouandI cannotwaittostartthenextjourneywithyou. iv 1 CHAPTER1 INTRODUCTION 2 MITOCHONDRIA Mitochondria are essential organelles that perform fundamental cellular functions includingaerobicmetabolicfattyacidoxidation,aminoacidmetabolism,apoptosisand biosynthesis of many cellular metabolites. Mitochondria contain their own DNA (mtDNA), which encodes respiratory chain components and tRNAs necessary for synthesis of mtDNA-encoded respiratory chain components, and cannot be made de novo. Saccharomyces cerevisiae has become an ideal model system for studying mitochondrialfunctionandinheritance,processesthatarerequiredforcellsurvival.The vital process of replicating and transferring ‘fit’ mitochondria