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Cytoplasmic Actin: Purification and Single Molecule Assembly Assays

Part of the Methods in Molecular Biology book series (MIMB,volume 1046)

Abstract

The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of actin as well as the physical properties of these filaments. Development of an in vitro model of actin polymerization from purified components has allowed for great advances in determining the effects of these proteins on the actin cytoskeleton. Here we describe how to use the pyrene actin assembly assay to determine the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleation or capping factors. Secondly, we show how fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how proteins regulate actin filament structure. Finally, we describe a method for visualizing dynamic assembly and disassembly of single actin filaments and fluorescently labeled actin binding proteins using total internal reflection fluorescence (TIRF) microscopy.

Key words

  • Actin polymerization assay
  • TIRF
  • Single molecule imaging
  • Acanthamoeba
  • Fluorimetry
  • Pyrene
  • Cytoskeleton
  • Phalloidin

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Acknowledgments

The authors would like to thank their colleagues Orkun Akin, Margot Quinlan, and Peter Bieling for support and help in developing the techniques described. S.D.H was supported by a National Science Foundation predoctoral Fellowship. J.B.Z. was supported by an American Heart Association predoctoral fellowship, and is currently a Howard Hughes Medical Institute Fellow of the Life Sciences Research Foundation.

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Hansen, S.D., Zuchero, J.B., Mullins, R.D. (2013). Cytoplasmic Actin: Purification and Single Molecule Assembly Assays. In: Coutts, A. (eds) Adhesion Protein Protocols. Methods in Molecular Biology, vol 1046. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-538-5_9

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  • DOI: https://doi.org/10.1007/978-1-62703-538-5_9

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  • Publisher Name: Humana Press, Totowa, NJ

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