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Methods and Protocols
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1136, Methods in Molecular Biology

Part I: Imaging and Measurement of Mitotic Spindle Dynamics In Vivo

1. Analysis of Mitotic Protein Dynamics and Function in Drosophila Embryos by Live Cell Imaging and Quantitative Modeling

Ingrid Brust-Mascher, Gul Civelekoglu-Scholey, and Jonathan M. Scholey

2. Rapid Measurement of Mitotic Spindle Orientation in Cultured Mammalian Cells

Justin Decarreau, Jonathan Driver, Charles Asbury and Linda Wordeman

3. Automated Segmentation of the First Mitotic Spindle in Differential Interference Contrast Microcopy Images of C. elegans Embryos

Daniel Needleman

4. Imaging the Mitotic Spindle by Spinning Disk Microscopy in Tobacco Suspension Cultured Cells

Takashi Murata and Tobias I. Baskin

5. Image-Based Computational Tracking and Analysis of Spindle Protein Dynamics

Ge Yang

Part II: Analysis and Manipulation of the Microtubule Cytoskeleton in Cells and In Vitro

6. The Use of Cultured Drosophila Cells for Studying the Microtubule Cytoskeleton

Jonathan Nye, Daniel W. Buster, and Gregory C. Rogers

7. Measuring Microtubule Growth and Gliding in C. elegans Embryos

Justus Tegha-Dunghu, Eva M. Gusnowski, and Martin Srayko

8. Xenopus Egg Extracts as a Simplified Model System for Structure Function Studies of Dynein Regulators

Eliza ylkiewicz and P. Todd Stukenberg

Part III: Specialized Biophysical and Microscopy Approaches for Analyzing Microtubules and Microtubule Associated Proteins

9. Covalent Immobilization of Microtubules on Glass Surfaces for Molecular Motor Force Measurements and Other Single-Molecule Assays

Matthew P. Nicholas, Lu Rao, and Arne Gennerich

10. An Improved Optical Tweezers Assay for Measuring the Force Generation of Single Kinesin Molecules

Matthew P. Nicholas, Lu Rao, and Arne Gennerich

11. Seeded Microtubule Growth for Cryo-electron Microscopy of End Binding Proteins

Sebastian P. Maurer, Franck J. Fourniol, Andreas Hoenger, and Thomas Surrey

12. The Segmentation of Microtubules in Electron Tomograms using Amira

Stefanie Redemann, Britta Weber, Marit Möller, Jean-Marc Verbavatz, Anthony A. Hyman, Daniel Baum, Steffen Prohaska, and Thomas Müller-Reichert

Part IV: Extras

13. Manipulating Cell Shape by Placing Cells into Micro-fabricated Chambers

Fred Chang, Erdinc Atilgan, David Burgess, and Nicolas Minc

14. Four- color FISH for the Detection of Low Levels Aneuploidy in Interphase Cells

Francesca Faggioli, Jan Vijg, and Cristina Montagna
Mitosis: Methods and Protocols provides state-of-the-art overviews on the most important approaches currently used in mitosis research spanning from the analysis of single molecules in isolation to their utilization within the complex environment of the cell. The volume is divided into four parts, each focused on methods pertaining to distinct aspects of mitosis research. Part I presents approaches for visualizing and analyzing the dynamic behaviors of the spindle apparatus, the microtubule based machine that drives chromosome segregation. Part II focuses more generally on methods for studying and manipulating the microtubule cytoskeleton in cells and complex cell free extracts. Part III provides state of the art biophysical and high resolution microscopy approaches for assessing complex interactions between microtubules and microtubule-associated proteins in isolation as well as microtubule structure in cells. Part IV provides methods for studying the effects of cell shape on cell division and methods for quantifying aneuploidy (aberrant chromosome number) which frequently results from mitotic defects and has been linked to human maladies ranging from birth defects to cancer. 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 key tips on troubleshooting and avoiding known pitfalls.Authoritative and practical, Mitosis: Methods and Protocols seeks to provide diverse methods and new techniques to address new or old questions related to the mechanisms of mitosis.