The Mitotic Exit Network

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 inMolecular 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

Regulation of Mitotic Exit in Saccharomyces cerevisiae .- Methods of Synchronization of Yeast Cells for the Analysis of Cell Cycle Progression.- Analysis of Cell Cycle Progression in Saccharomyces cerevisiae Using the Budding Index and Tubulin Staining.- Determination of Cell Cycle Stage and Mitotic Exit Through the Quantification of the Protein Levels of Known Mitotic Regulators.- Cdc14 Localization as a Marker for Mitotic Exit: In Vivo Quantitative Analysis of Cdc14 Release.- In Vitro Analysis of Tem1 GTPase Activity and Regulation by the Bfa1/Bub2 GAP.- Analysis of Protein-Protein Interactions Between MEN Components.- A New Methodology for the Quantification of In Vivo Cdc14 Phosphatase Activity.- Analysis of SUMOylation in the RENT Complex by Fusion to a SUMO-Specific Protease Domain.- A Substrate Trapping Method for Identification of Direct Cdc14 Phosphatase Targets.- Localizing MEN Components by Indirect Immunofluorescence Analysis of Budding Yeast.- Analysis of the Localization of MEN Components by Live Cell Imaging Microscopy.- Evaluation of the Dynamicity of Mitotic Exit Network and Spindle Position Checkpoint Components on Spindle Pole Bodies by Fluorescence Recovery After Photobleaching (FRAP).- Asymmetric Localization of Components and Regulators of the Mitotic Exit Network at Spindle Pole Bodies.- Evaluation of the Nucleolar Localization of the RENT Complex to Ribosomal DNA by Chromatin Immunoprecipitation Assays.- Analysis of the Functionality of the Mitotic Checkpoints.- Cdc14 and Chromosome Condensation: Evaluation of the Recruitment of Condensin to Genomic Regions.- Studying the Role of the Mitotic Exit Network in Cytokinesis.- Hippo Signaling in Mitosis: An Updated View in Light of the MEN Pathway.