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.
