Sinopsis
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...2 1 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.
De la contraportada
With the advent of modern methodology, the phenomenal complexity of the protein components and regulatory steps involved in mitosis has become approachable. In Mitosis: Methods and Protocols, experts in the field provide an up-to-date collection of methods and approaches that are used to investigate the mechanism of mitosis at the molecular level. Ideal for both experienced and novice scientists, the volume includes step-by-step methods covering inactivation of genes of interest, depletion of proteins of interest, biochemical and microscope-based techniques, and procedures to monitor and measure key mitotic processes. Written in the highly successful Methods in Molecular Biology™ series format, chapters include brief introductions to the respective topics, lists of the necessary materials and reagents, readily reproducible laboratory protocols, and notes on troubleshooting and avoiding known pitfalls.
Comprehensive and authoritative, Mitosis: Methods and Protocols combines imaging, biochemical, and genetic methodologies in order to best guide researchers in their attempts to better understand the crucial processes of mitotic regulation.
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