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Main description:
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.
Contents:
1. Knock-In and Knock-Out: The Use of Reverse Genetics in Somatic Cells to Dissect Mitotic Pathways
Alexis R. Barr, Deborah Zyss, and Fanni Gergely
2. Functional Dissection of Mitotic Regulators through Gene Targeting in Human Somatic Cells
Eli Berdougo, Marie-Emilie Terret, and Prasad V. Jallepalli
3. RNAi in Drosophila S2 Cells As A Tool For Studying Cell Cycle Progression
Monica Bettencourt-Dias and Gohta Goshima
4. Production of Mitotic Regulators Using an Autoselection System for Protein Expression in Budding Yeast
Marco Geymonat, Adonis Spanos, and Steven Sedgwick
5. Hydrodynamic Analysis of Human Kinetochore Complexes during Mitosis
Sarah E. McClelland and Andrew D. McAinsh
6. Isolation of Protein Complexes Involved in Mitosis and Cytokinesis from Drosophila Cultured Cells
Pier Paolo D'Avino, Vincent Archambault, Marcin R. Przewloka, Wei Zhang, Ernest D. Laue, and David M. Glover
7. Automated Live Microscopy to Study Mitotic Gene Function in Fluorescent Reporter Cell Lines
Michael H. A. Schmitz and Daniel W. Gerlich
8. Electron Tomography of Microtubule End-Morphologies in C. elegans Embryos
Eileen O'Toole and Thomas Muller-Reichert
9. Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells
Antonio J. Pereira, Irina Matos, Mariana Lince-Faria, and Helder Maiato
10. Fluorescence Imaging of the Centrosome Cycle in Mammalian Cells
Suzanna L. Prosser and Andrew M. Fry
11. Visualization of Fluorescence-Tagged Proteins in Fission Yeast: The Analysis of Mitotic Spindle Dynamics Using GFP-Tubulin under the Native Promoter
Masamitsu Sato, Mika Toya, and Takashi Toda
12. Analysing Kinetochore Function in Human Cells: Spindle Checkpoint and Chromosome Congression
Christiane Klebig, Alberto Toso,Satyarebala Borusu, and Patrick Meraldi
13. Probing Kinetochore Structure and Function Using Xenopus laevis Frog Egg Extracts
Michael J. Emanuele and P. Todd Stukenberg
14. Live Cell Imaging of Kinetochore Capture by Microtubules in Budding Yeast
Kozo Tanaka and Tomoyuki U. Tanaka
15. The Spindle Checkpoint: Assays for the Analysis of Spindle Checkpoint Arrest and Recovery
Josefin Fernius and Kevin G. Hardwick
16. Measuring Proteolysis in Mitosis
Catherine Lindon and Barbara Di Fiore
17. An In Vitro Assay for Cdc20-dependent Mitotic Anaphase-Promoting Complex Activity from Budding Yeast
Scott C. Schuyler and Andrew W. Murray
18. In vitro Assays for the Anaphase-Promoting Complex/Cyclosome (APC/C) in Xenopus Egg Extracts
Hiroyuki Yamano, Michelle Trickey, Margaret Grimaldi, and Yuu Kimata
19. Preparation of Synchronized Human Cell Extracts to Study Ubiquitination and Degradation
Adam Williamson, Lingyan Jin, and Michael Rape
20. Biochemical Analysis of the Anaphase Promoting Complex: Activities of E2 Enzymes and Substrate Competitive ('Pseudosubstrate') Inhibitors
Matthew K. Summers and Peter K. Jackson
PRODUCT DETAILS
Publisher: Springer (Humana Press Inc.)
Publication date: November, 2014
Pages: 345
Weight: 759g
Availability: Available
Subcategories: Genetics
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