Overview

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.

Full Product Details

Author:   Andrew D. McAinsh
Publisher:   Humana Press Inc.
Imprint:   Humana Press Inc.
Volume:   545
Weight:   0.759kg
ISBN:  

9781627038508

ISBN 10:   1627038507
Pages:   345
Publication Date:   24 November 2014
Audience:   Professional and scholarly ,  Professional & Vocational
Format:   Paperback
Publisher's Status:   Active
Availability:   Manufactured on demand  
We will order this item for you from a manufactured on demand supplier.

Table of Contents

Knock-in and Knock-out: The Use of Reverse Genetics in Somatic Cells to Dissect Mitotic Pathways.- Functional Dissection of Mitotic Regulators Through Gene Targeting in Human Somatic Cells.- RNAi in Drosophila S2 Cells as a Tool for Studying Cell Cycle Progression.- Production of Mitotic Regulators Using an Autoselection System for Protein Expression in Budding Yeast.- Hydrodynamic Analysis of Human Kinetochore Complexes During Mitosis.- Isolation of Protein Complexes Involved in Mitosis and Cytokinesis from Drosophila Cultured Cells.- Automated Live Microscopy to Study Mitotic Gene Function in Fluorescent Reporter Cell Lines.- Electron Tomography of Microtubule End-Morphologies in C. elegans Embryos.- Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells.- Fluorescence Imaging of the Centrosome Cycle in Mammalian Cells.- Visualization of Fluorescence-Tagged Proteins in Fission Yeast: The Analysis of Mitotic Spindle Dynamics Using GFP-Tubulin Under the Native Promoter.- Analysing Kinetochore Function in Human Cells: Spindle Checkpoint and Chromosome Congression.- Probing Kinetochore Structure and Function Using Xenopus laevis Frog Egg Extracts.- Live Cell Imaging of Kinetochore Capture by Microtubules in Budding Yeast.- The Spindle Checkpoint: Assays for the Analysis of Spindle Checkpoint Arrest and Recovery.- Measuring Proteolysis in Mitosis.- An In Vitro Assay for Cdc20-Dependent Mitotic Anaphase-Promoting Complex Activity from Budding Yeast.- In Vitro Assays for the Anaphase-Promoting Complex/Cyclosome (APC/C) in Xenopus Egg Extracts.- Preparation of Synchronized Human Cell Extracts to Study Ubiquitination and Degradation.- Biochemical Analysis of the Anaphase Promoting Complex: Activities of E2 Enzymes and Substrate Competitive (Pseudosubstrate) Inhibitors.

Reviews

From the reviews: McAinsh ... introduces mitosis as a phenomenally complicated process of the splitting of genetic material into two daughter cells that prevents genomic instability. Twenty chapters comprise this collection of laboratory protocols of up-to-date approaches and methods ... used to investigate the mechanism of mitosis at the molecular level. ... The volume is intended for graduate students and researchers in the field. (SciTech Book News, March, 2010)

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