The Mitotic Exit Network: Methods and Protocols

ISBN-13: 9781493965007 / Angielski / Twarda / 2016 / 281 str.

Fernando Monje-Casas; Ethel Queralt

Opis

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.

Spis treści

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