Presents a collection of methods and approaches that are used to investigate the mechanism of mitosis at the molecular level. This book covers 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.
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 covers 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.
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