Summary
Since directed movement toward an extracellular chemoattractant requires rapid and continuous reorganization of the actin cytoskeleton to form complex structures such as a protruding lamellipodium, it is of great interest to analyze and understand the individual contribution of proteins specifically involved in this process. Over the last decade, enormous progress has been made toward understanding the versatile molecular mechanisms underlying actin-based cell motility and the regulation of site-specific F-actin assembly and disassembly. In spite of this wealth of knowledge and due to the constant discovery of novel regulatory factors, many questions remain to be answered. In this chapter, we describe a powerful method that allows to study the effects of actin-binding proteins on the assembly of single filaments by in vitro total internal reflection fluorescence (TIRF) microscopy using purified proteins and fluorescently labeled actin.
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Acknowledgments
We thank Drs. David Kovar, Emmanuèle Helfer, Christophe Le Clainche, and Marie-France Carlier for helpful advice to perform in vitro TIRF microscopy of actin assembly. This work was supported by a grant to J.F. (FA 330/4–1) from the Deutsche Forschungsgemeinschaft.
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Breitsprecher, D., Kiesewetter, A.K., Linkner, J., Faix, J. (2009). Analysis of Actin Assembly by In Vitro TIRF Microscopy. In: Jin, T., Hereld, D. (eds) Chemotaxis. Methods in Molecular Biology™, vol 571. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-198-1_27
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DOI: https://doi.org/10.1007/978-1-60761-198-1_27
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