Mitosis: Methods and Protocols

ISBN-13: 9781603279925 / Angielski / Twarda / 2009 / 345 str.

Andrew D. McAinsh

Opis

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. Emanue

Recenzje

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)

Spis treści

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 Mónica 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 Müller-Reichert 9. Dissecting Mitosis with Laser Microsurgery and RNAi in Drosophila Cells António 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

Blurb

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.