The Mitotic Spindle: Methods and Protocols

ISBN-13: 9781493935406 / Angielski / Twarda / 2016 / 424 str.

Paul Chang; Ryoma Ohi

Opis

This volume includes a series of protocols focused on mitotic spindle assembly and function. The methods covered in this book feature a broad range of techniques from basic microscopy to the study of spindle physiologies relevant to cancer. These methods can be applied to diverse model systems that range from the cell-free Xenopus egg extract system to the moss Physcomitrella patens, in an effort to demonstrate the key contributions made by researchers using multiple model organisms. Chapters in The Mitotic Spindle: Methods and Protocols integrate cutting-edge technologies that have only become available due to the cross-disciplinary efforts, such as ATP analogue sensitive inhibition of mitotic kinases. Written in the highly successful Methods in Molecular Biology series format, 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.

Thorough and informative, The Mitotic Spindle: Methods and Protocols, is a valuable resource for researchers who are new to mitosis or are already experts in the field.

Spis treści

Part I Methods Focused on Cytology 

1: Using Fluorescence Microscopy to Study Mitosis|Sai K. Balchand, Barbara J. Mann, and Patricia Wadsworth

2: Using Photoactivatable GFP to Study Microtubule Dynamics and Chromosome Segregation
Bin He and Daniela Cimini

Part II Methods Focused on Microtubules and the Mitotic Spindle

3: Purification and Fluorescent Labeling of Tubulin from Xenopus Laevis Egg Extracts
Aaron C. Groen and Timothy J. Mitchison

4: Measuring the Effects of Microtubule-Associated Proteins on Microtubule Dynamics In Vitro
Marija Zanic

5: Imaging and Quantifying the Dynamics of γ-Tubulin at Microtubule Minus Ends in Mitotic Spindles
Nicolas Lecland and Jens Lüders

6: Visualizing and Analyzing Branching Microtubule Nucleation Using Meiotic Xenopus Egg Extracts and TIRF Microscopy
Matthew King and Sabine Petry

7: Encapsulation of Xenopus Egg and Embryo Extract Spindle Assembly Reactions in Synthetic Cell-Like Compartments with Tunable Size
Matthew C. Good

Part III Methods Focused on Kinetochores and the Kinetochore-Microtubule Interface

8: In Vitro Kinetochore Assembly
Matthew D. D. Miell and Aaron F. Straight

9: Biochemical and Structural Analysis of Kinetochore Histone-Fold Complexes
Tatsuya Nishino and Tatsuo Fukagawa

10: Measuring Kinetochore-Microtubule Attachment Stability in Cultured Cells
Keith F. DeLuca, Jacob A. Herman, and Jennifer G. DeLuca

11: Studying Kinetochores In Vivo Using FLIM-FRET
Tae Yeon Yoo and Daniel J. Needleman

Section IV Methods Focused on the Spindle Pole

12: Purification of Fluorescently Labeled Saccharomyces Cerevisiae Spindle Pole Bodies
Kimberly K. Fong, Beth Graczyk, and Trisha N. Davis

13: A Cell-Free System for Real-Time Analyses of Centriole Disengagement and Centriole to Centrosome Conversion
Rajesh Kumar Soni and Meng-Fu Bryan Tsou

14: Assays to Study Mitotic Centrosome and Spindle Pole Assembly and Regulation
Vladimir Joukov, Johannes C. Walter, and Arcangela De Nicolo

Part V Methods Focused on the Cellular Functions of Microtubule Motor Proteins

15: Analyzing Spindle Positioning Dynamics in Cultured Cells
Tomomi Kiyomitsu

16: Quantification of Mitotic Chromosome Alignment
Cindy Fonseca and Jason Stumpff

17: Imaging Mitosis in the Moss Physcomitrella Patens
Moé Yamada, Tomohiro Miki, and Gohta Goshima

18: Small Molecule Approach to Study the Function of Mitotic Kinesins
Naowras Al-Obaidi, Johanna Kastl, and Thomas U. Mayer

Part VI Novel Approaches to Study Spindle Function and Regulation

19: Identification and Characterization of Mitotic Spindle-Localized Transcripts
Amy B. Emerman, Ashwini Jambhekar, and Michael D. Blower

20: Probing Mitosis by Manipulating the Interactions of Mitotic Regulator Proteins Using Rapamycin-Inducible Dimerization
Edward R. Ballister and Michael A. Lampson

21: Studying Kinetochore Kinases
Adrian T. Saurin and Geert J.P.L. Kops

22: Engineering and Functional Analysis of Mitotic Kinases through Chemical Genetics
Mathew J.K. Jones and Prasad V. Jallepalli

Part VII The Mitotic Spindle and Cancer

23: Using Cell Culture Models of Centrosome Amplification to Study Centrosome Clustering in Cancer
Mijung Kwon

24: Generation and Purification of Tetraploid Cells
Elizabeth M. Shenk and Neil J. Ganem

25: Anti-Microtubule Drugs
Stefan Florian and Timothy J. Mitchison