Mitosis: Methods and Protocols (2009)

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. Emanue