This detailed book collects the main methodologies used for the analysis of the activity, localization, and regulation of the comp...
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This detailed book collects the main methodologies used for the analysis of the activity, localization, and regulation of the components of the Mitotic Exit Network (MEN) pathway during mitotic exit in Saccharomyces cerevisiae, as well as for the evaluation of the roles of these proteins in other cellular processes, such as the condensation of the rDNA, the functionality of the mitotic checkpoints, and cytokinesis. Budding yeast serves as an ideal model system for dissecting the mechanisms that regulate cell cycle progression and providing new insights into the molecular basis of cell cycle control and, thus, into the origin of diseases that arise as a consequence of problems during cell division. Therefore, although this volume concentrates on Saccharomyces cerevisiae as a model, it also details the implications that the research about the MEN have on our understanding of the mitotic exit process in higher eukaryotes. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, The Mitotic Exit Network: Methods and Protocols will be a valuable reference for cellular and molecular biologists and biochemists as well as for all scientists interested in the study of the regulation of mitotic exit using budding yeast as a model organism.
Includes cutting-edge methods and protocols for studying the Mitotic Exit Network Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts Inhalt Part I: Analysis of Cell Cycle Progression and Mitotic Exit 1. Regulation of Mitotic Exit in Saccharomyces cerevisiae Bàrbara Baro, Ethel Queralt, and Fernando Monje-Casas 2. Methods of Synchronization of Yeast Cells for the Analysis of Cell Cycle Progression M. Angeles Juanes 3. Analysis of Cell Cycle Progression in Saccharomyces cerevisiae Using the Budding Index and Tubulin Staining Marta Muñoz-Barrera and Fernando Monje-Casas 4. Determination of Cell Cycle Stage and Mitotic Exit Through the Quantification of the Protein Levels of Known Mitotic Regulators Cristina Cepeda-García 5. Cdc14 Localization as a Marker for Mitotic Exit: In Vivo Quantitative Analysis of Cdc14 Release Gabriel Neurohr and Manuel Mendoza Part II: Biochemistry of the MEN 6. In Vitro Analysis of Tem1 GTPase Activity and Regulation by the Bfa1/Bub2 GAP Marco Geymonat, Adonis Spanos, and Katrin Rittinger 7. Analysis of Protein-Protein Interactions Between MEN Components Rami Rahal 8. A New Methodology for the Quantification of In Vivo Cdc14 Phosphatase Activity Ethel Queralt and José-Antonio Rodriguez-Rodriguez 9. Analysis of SUMOylation in the RENT Complex by Fusion to a SUMO-Specific Protease Domain Neus Colomina, Clàudia Guasch, and Jordi Torres-Rosell 10. A Substrate Trapping Method for Identification of Direct Cdc14 Phosphatase Targets Brendan L. Powers, Hana Hall, Harry Charbonneau, and Mark C. Hall Part III: Analysis of the Localization of MEN Components 11. Localizing MEN Components by Indirect Immunofluorescence Analysis of Budding Yeast Francesca Senic-Matuglia and Rosella Visintin 12. Analysis of the Localization of MEN Components by Live Cell Imaging Microscopy Zhiang Guo and Marisa Segal 13. Evaluation of the Dynamicity of Mitotic Exit Network and Spindle Position Checkpoint Components on Spindle Pole Bodies by Fluorescence Recovery After Photobleaching (FRAP) Ayse Koca Caydasi and Gislene Pereira 14. Asymmetric Localization of Components and Regulators of the Mitotic Exit Network at Spindle Pole Bodies Ilaria Scarfone and Simonetta Piatti 15. Evaluation of the Nucleolar Localization of the RENT Complex to Ribosomal DNA by Chromatin Immunoprecipitation Assays Julie Huang, Nahid Iglesias, and Danesh Moazed Part IV: Control of Cellular Processes by the MEN 16. Analysis of the Functionality of the Mitotic Checkpoints Roberta Fraschini 17. Cdc14 and Chromosome Condensation: Evaluation of the Recruitment of Condensin to Genomic Regions Facundo Ramos, Joanne Leonard, Andrés Clemente-Blanco, and Luis Aragón 18. Studying the Role of the Mitotic Exit Network in Cytokinesis Magdalena Foltman and Alberto Sanchez-Diaz Part V: Final Remarks: The MEN as a Model for Higher Eukaryotes 19. Hippo Signaling in Mitosis: An Updated View in Light of the MEN Pathway Alexander Hergovich