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Centrosome and Centriole
1st Edition, Volume 129 - July 10, 2015
Editors: Renata Basto, Karen Oegema
Language: English
Hardback ISBN:9780128024492
9 7 8 - 0 - 1 2 - 8 0 2 4 4 9 - 2
eBook ISBN:9780128026380
9 7 8 - 0 - 1 2 - 8 0 2 6 3 8 - 0
This new volume of Methods in Cell Biology looks at methods for analyzing centrosomes and centrioles. Chapters cover such topics as methods to analyze centrosomes, centriole biogen…Read more
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This new volume of Methods in Cell Biology looks at methods for analyzing centrosomes and centrioles. Chapters cover such topics as methods to analyze centrosomes, centriole biogenesis and function in multi-ciliated cells, laser manipulation of centrosomes or CLEM, analysis of centrosomes in human cancers and tissues, proximity interaction techniques to study centrosomes, and genome engineering for creating conditional alleles in human cells.
Covers sections on model systems and functional studies, imaging-based approaches and emerging studies
Chapters are written by experts in the field
Cutting-edge material
Researchers and students in cell, molecular and developmental biology
Series Editors
Preface
Chapter 1. Correlative light and electron microscopy analysis of the centrosome: A step-by-step protocol
Introduction
1. Experimental Procedure
Chapter 2. Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering
Introduction
1. CRISPR/Cas System
2. Analog-Sensitive Kinases
3. Methods
4. Functional Analysis
Conclusion
Chapter 3. Studying centrosome function using three-dimensional cell cultures
Introduction
1. Methods
Conclusion
Chapter 4. Analysis of centrosomes in human cancer
Introduction
1. Methods
Conclusions
Chapter 5. In vivo examination of the cortical cytoskeleton in multiciliated cells using electron tomography
Introduction
1. Rationale
2. Methods
3. Materials
Conclusion
List of Abbreviations
Chapter 6. Tackling centrosome biology through gene targeting in chicken B cells
Introduction
1. Methods
2. Analysis of DT40 Cells
Conclusion
Chapter 7. Centriole biogenesis and function in multiciliated cells
Introduction
1. Visualization of Centrioles and Related Structures in Xenopus MCCs
2. Driving Specific Expression in Xenopus MCCs
3. Generating Mosaic Xenopus Embryos to Circumvent Embryo Lethality
4. Ciliated Epithelial Spheroid Culture
5. Manipulating MCC Formation
6. Fully-Automatic, Program-Assisted Counting of Centrioles in Confocal Images Using ImageJ
Conclusion
Chapter 8. Subdiffraction resolution microscopy methods for analyzing centrosomes organization
Introduction
1. Methods
Summary and Outlook
Chapter 9. Probing mammalian centrosome structure using BioID proximity-dependent biotinylation
Introduction
1. Materials
2. Methods
3. Large-Scale BioID Pulldown of BirA*-Fusion Proteins
4. Data Analysis
Conclusion
Chapter 10. Purification of centrosomes from mammalian cell lines
Introduction
1. Methods
Conclusions
Chapter 11. Isolation, cryotomography, and three-dimensional reconstruction of centrioles
Introduction
1. Methods
Conclusions
Chapter 12. New insights into centrosome imaging in Drosophila and mouse neuroepithelial tissues
Introduction
1. Imaging Centrosomes in Drosophila Neuroepithelial Cells
2. High Spatial Resolution Imaging of Centrosomes in Neural Stem Cells of the Mouse Neocortex
Conclusion
Chapter 13. Microinjection techniques for studying centrosome function in Drosophila melanogaster syncytial embryos
Introduction
1. Protocol
2. Collection and Preparation of Drosophila Embryos
3. Preparing Samples for Microinjection
4. Mounting Embryos for Spinning Disc Confocal Microscopy
5. Microinjection of Embryos
6. Perturbation via Cold Treatment
7. Imaging and Data Analysis
Chapter 14. A yeast two-hybrid approach for probing protein–protein interactions at the centrosome
Introduction
1. Identifying Direct Protein Interactions Have Had a Profound Impact on Understanding Centrosome Function
2. Challenges to Understanding Protein–protein Interactions in the Centrosome
3. Dissection of Complex, Multicomponent Protein Machines Using Y2H
4. Performing an Array Based Screen for Centrosome Protein Interactions
5. Generating Specific, Separation of Function Mutations by Reverse Y2H
Summary
Chapter 15. Genome-wide RNAi screens in S2 cells to identify centrosome components
Introduction
1. Methods
2. Genome-Wide Screen
3. Image Acquisition
4. Scoring Phenotypes and Image Analysis
5. Validation of Potential Hits
Discussion
Chapter 16. Time-lapse recording of centrosomes and other organelles in Drosophila neuroblasts
Introduction
1. Primary Cultures
2. Ex vivo Whole Mount
3. Fluorescent Reporters
4. Remarks and Troubleshooting
Chapter 17. Centrosomes and spindles in ascidian embryos and eggs
Introduction
1. Methods
Chapter 18. Ultrastructural analysis of Caenorhabditis elegans cilia
Introduction
1. Methods
Discussion
Chapter 19. Method: In vitro analysis of pericentriolar material assembly
Introduction
1. Methods
Conclusion
Chapter 20. Monitoring SPB biogenesis in fission yeast with high resolution and quantitative fluorescent microscopy
Introduction
1. Monitoring SPB Duplication in Fixed SPB-Labeled Strains
2. Quantitative Analysis of SPB Biogenesis in Live Cells
Conclusion
Volumes in Series
Index
No. of pages: 434
Language: English
Edition: 1
Volume: 129
Published: July 10, 2015
Imprint: Academic Press
Hardback ISBN: 9780128024492
eBook ISBN: 9780128026380
RB
Renata Basto
Affiliations and expertise
Cell Biology Department, CNRS, Institut Curie, France
KO
Karen Oegema
Affiliations and expertise
Ludwig Institute for Cancer Research, University of California, San Diego, USA