Abstract
Many proteins act in multiple pathways which complicates phenotypic analysis. Xenopus egg extracts reconstitute complex reactions in vitro, and this can be used to develop assays that isolate a single function of a multifunctional protein. We have applied this system to study regulators of cytoplasmic dynein (dynein), which has numerous roles in the cell including trafficking, nuclear migration, and mitotic spindle formation. Here we describe a functional assay to specifically study the regulation of spindle pole self-organization by dynein and summarize an experimental approach that was used to perform a structure–function analysis of its regulator Ndel1. The approaches presented here can be generalized to isolate a single function of other multifunctional proteins.
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Abbreviations
- Σ:
-
Extinction coefficient
- MW:
-
Molecular weight
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Acknowledgements
We want to acknowledge Dr. Zygmunt S. Derewenda (University of Virginia) for insightful discussions on the structure of Ndel1 and generously sharing reagents. We also thank Drs. Won-Chan Choi and Ankoor Roy (University of Virginia) for generating Ndel1 constructs and help with CD spectroscopy experiments. This work was made possible by NIH grants R01-NS036267 and R01-GM063045.
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Żyłkiewicz, E., Stukenberg, P.T. (2014). Xenopus Egg Extracts as a Simplified Model System for Structure–Function Studies of Dynein Regulators. In: Sharp, D. (eds) Mitosis. Methods in Molecular Biology, vol 1136. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0329-0_8
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DOI: https://doi.org/10.1007/978-1-4939-0329-0_8
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