Mitosis: Methods And Protocols

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Humana Press Mitosis: Methods And Protocols by Andrew D. Mcainsh

Mitosis Is A Dynamic And Stochastic Process That Has Evolved To Accurately Segregate The GeneticmaterialintotwodaughtercellsTherebypreventinggenomicinstabilityandthe Developmentofdisease. Mitosisisaphenomenallycomplicatedprocessthatinvolvesh- DredsIfnotthousandsOfproteincomponentsandregulatorystepsthatoperateinboth Spaceandtimetodrivecelldivision. Modernmethodsmakeitpossibletoaskformechan- Ticprinciplesunderlyingthisbewilderingcomplexity. Themainpurposeofthisvolumeis Toprovideanup-To-Datecollectionofmethodsandapproachesthatareusedtoinvestigate Themechanismofmitosisatthemolecularlevel. Whilemanyofthesemethodsarefocused OnmammaliancellsWehaveWhereappropriateIncludedchaptersusingmodelorg- Isms. We Hope To Capture Both Current Approaches And The Future Direction Of Method DevelopmentWithcontributionsfrombothestablishedresearchersandemergingyoung Scientists. Thisbookisdesignedwithtwogeneralgroupsofreadersinmind:FirstGraduates- Dentsandpostdoctoralresearcherswhoarebeginningworkforthe?Rsttimeinamitosis Laboratory. SecondResearcherswhoarealreadyworkinginthemitosis?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). SecondWelearnaboutspeci?Cbiochemicalandmicroscope-Basedme- Ods (Chapters 4-9). Third We Discover Approaches To Monitor And Measure Key Mitotic Processes(Chapters9-20). GiventhecomplexitiesofmitosisItseemshighlyprobablethat SuchacombinationofimagingBiochemicalandgeneticmethodologieswillbecrucialto Ourfutureunderstandingofmitoticregulation. I Would Like To Thank All Of The Authors For Their Enthusiasm And Effort 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 PierpaolodAvinoVincentarchambaultMarcinr. Przewloka WeizhangErnestd. Laueanddavidm. Glover 7 Automatedlivemicroscopytostudymitoticgenefunctionin Fluorescentreportercelllines .113 Michaelh. A. Schmitzanddanielw. Gerlich 8 Electrontomographyofmicrotubuleend-Morphologies Inc. Elegansembryos.135 EileenoTooleandthomasmuller-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.Show More