Mitosis

Mitosis is a dynamic and stochastic process that has evolved to accurately segregate the geneticmaterialintotwodaughtercells,therebypreventinggenomicinstabilityandthe developmentofdisease. Mitosisisaphenomenallycomplicatedprocessthatinvolvesh- dreds,ifnotthousands,ofproteincomponentsandregulatorystepsthatoperateinboth spaceandtimetodrivecelldivision. Modernmethodsmakeitpossibletoaskformechan- ticprinciplesunderlyingthisbewilderingcomplexity. Themainpurposeofthisvolumeis toprovideanup-to-datecollectionofmethodsandapproachesthatareusedtoinvestigate themechanismofmitosisatthemolecularlevel. Whilemanyofthesemethodsarefocused onmammaliancells,wehave,whereappropriate,includedchaptersusingmodelorg- isms. We hope to capture both current approaches and the future direction of method development,withcontributionsfrombothestablishedresearchersandemergingyoung scientists. Thisbookisdesignedwithtwogeneralgroupsofreadersinmind:First,graduates- dentsandpostdoctoralresearcherswhoarebeginningworkforthe?rsttimeinamitosis laboratory. Second,researcherswhoarealreadyworkinginthemitosis?eldwhorequire a resource for both established and newly developed methodologies. To achieve this, the organization of this book developed into three general areas: First, we cover me- ods that can be used to inactivate your gene of interest, or deplete proteins of interest (chapters1–3). Second,welearnaboutspeci?cbiochemicalandmicroscope-basedme- ods (chapters 4–9). Third, we discover approaches to monitor and measure key mitotic processes(chapters9–20). Giventhecomplexitiesofmitosis,itseemshighlyprobablethat suchacombinationofimaging,biochemicalandgeneticmethodologieswillbecrucialto ourfutureunderstandingofmitoticregulation. I would like to thank all of the authors for their enthusiasm and effort in putting togetherthissetofmethods,aswellasallthemembersofmylaboratoryforproofreading and correcting the chapters and especially Sarah McClelland who read all the chapters atleasttwiceatvariousstages. Finally,IwishtothankLyndyRasmusenforherhelpin administeringtheprojectandkeepingmeorganized. Andrew D. McAinsh v Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix 1 Knock-inandKnock-out:TheUseofReverseGeneticsinSomatic CellstoDissectMitoticPathways . . . . . . . . . . . . . . . . . . . . . . . 1 AlexisR. Barr, DeborahZyssandFanniGergely 2 FunctionalDissectionofMitoticRegulatorsThroughGene TargetinginHumanSomaticCells. . . . . . . . . . . . . . . . . . . . . . . 21 EliBerdougo, Marie-EmilieTerretandPrasadV. Jallepalli 3 RNAiinDrosophilaS2CellsasaToolforStudyingCell CycleProgression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Monica ´ Bettencourt-DiasandGohtaGoshima 4 ProductionofMitoticRegulatorsUsinganAutoselectionSystem forProteinExpressioninBuddingYeast. . . . . . . . . . . . . . . . . . . . 63 MarcoGeymonat, AdonisSpanosandStevenSedgwick 5 HydrodynamicAnalysisofHumanKinetochoreComplexes DuringMitosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 SarahE. McClellandandAndrewD. McAinsh 6 IsolationofProteinComplexesInvolvedinMitosisandCytokinesis fromDrosophilaCulturedCells. . . . . . . . . . . . . . . . . . . . . . . . . 99 PierPaoloD’Avino,VincentArchambault,MarcinR. Przewloka, WeiZhang,ErnestD. LaueandDavidM. Glover 7 AutomatedLiveMicroscopytoStudyMitoticGeneFunctionin FluorescentReporterCellLines . . . . . . . . . . . . . . . . . . . . . . . . 113 MichaelH. A. SchmitzandDanielW. Gerlich 8 ElectronTomographyofMicrotubuleEnd-Morphologies inC. elegansEmbryos. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 EileenO’TooleandThomasMuller-Reicher ¨ t 9 DissectingMitosiswithLaserMicrosurgeryandRNAi inDrosophilaCells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 AntonioJ. Pereira,IrinaMatos,MarianaLince-Faria ´ andHelderMaiato 10 FluorescenceImagingoftheCentrosomeCycleinMammalianCells . . . . 165 SuzannaL. ProsserandAndrewM. Fry vii viii Contents 11 VisualizationofFluorescence-TaggedProteinsinFissionYeast:The AnalysisofMitoticSpindleDynamicsUsingGFP-Tubulin UndertheNativePromoter. . . . . . . . . . . . . . . . . . . . . . . . . . . 185 MasamitsuSato, MikaToyaandTakashiToda 12 AnalysingKinetochoreFunctioninHumanCells:Spindle CheckpointandChromosomeCongression. . . . . . . . . . . . . . . . . . 205 ChristianeKlebig,AlbertoToso,SatyarebalaBorusu andPatrickMeraldi 13 ProbingKinetochoreStructureandFunctionUsingXenopuslaevis FrogEggExtracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 MichaelJ. EmanueleandP.