Mitosis

in putting togetherthissetofmethodsaswellasallthemembersofmylaboratoryforproofreading and correcting the chapters and especially Sarah McClelland who read all the chapters atleasttwiceatvariousstages. FinallyIwishtothankLyndyRasmusenforherhelpin 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’AvinoVincentArchambaultMarcinR. Przewloka WeiZhangErnestD. 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. PereiraIrinaMatosMarianaLince-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 ChristianeKlebigAlbertoTosoSatyarebalaBorusu andPatrickMeraldi 13 ProbingKinetochoreStructureandFunctionUsingXenopuslaevis FrogEggExtracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 MichaelJ. EmanueleandP.