Cytoplasmic actin: Purification and single molecule assembly assays

Research output: Chapter in Book/Report/Conference proceedingChapter

Authors

External Institution(s)

  • University of California at San Francisco

Details

Original languageEnglish (US)
Title of host publicationAdhesion Protein Protocols
StatusPublished - Jan 1 2013

Publication series

NameMethods in Molecular Biology
Volume1046
ISSN (Print)1064-3745

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.

    Research areas

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

Citation formats

APA

Hansen, S. D., Zuchero, J. B., & Mullins, R. D. (2013). Cytoplasmic actin: Purification and single molecule assembly assays. In Adhesion Protein Protocols (pp. 145-170). (Methods in Molecular Biology; Vol. 1046). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-538-5_9

Harvard

Hansen, SD, Zuchero, JB & Mullins, RD 2013, Cytoplasmic actin: Purification and single molecule assembly assays. in Adhesion Protein Protocols. Methods in Molecular Biology, vol. 1046, Humana Press Inc., pp. 145-170. https://doi.org/10.1007/978-1-62703-538-5_9