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 and endocytosis. Actin polymerization is tightly regulated by various 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. The pyrene actin assembly assay is a powerful tool for determining the effect of a protein on the kinetics of actin assembly, either directly or as mediated by proteins such as nucleators or capping factors. In addition, fluorescently labeled phalloidin can be used to visualize the filaments that are created in vitro to give insight into how these proteins influence actin filament superstructure.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Pollard, T. D. and Borisy, G. G. (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112, 453–465.
Oosawa, F. and Kasai, M. (1962) A theory of linear and helical aggregations of macromolecules. J. Mol. Biol. 4, 10–21.
Frieden, C. (1983) Polymerization of actin: mechanism of the Mg2+-induced process at pH 8 and 20 degrees C. Proc. Natl. Acad. Sci. USA 80, 6513–6517.
Welch, M. D. and Mullins, R. D. (2002) Cellular control of actin nucleation. Annu. Rev. Cell Dev. Biol. 18, 247–288.
Baum, B. and Kunda, P. (2005) Actin nucleation: spire—actin nucleator in a class of its own. Curr. Biol. 15, R305–R308.
Quinlan, M. E., Heuser, J. E., Kerkhoff, E., and Mullins, R. D. (2005) Drosophila spire is an actin nucleation factor. Nature 433, 382–388.
Machesky, L. M., Mullins, R. D., Higgs, H. N., et al. (1999) Scar, a WASp-related protein, activates nucleation of actin filaments by the Arp2/3 complex. Proc. Natl. Acad. Sci. USA 96, 3739–3744.
Cooper, J. A., Walker, S. B., and Pollard, T. D. (1983) Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J. Muscle Res. Cell Motil. 4, 253–262.
Pollard, T. D. (1983) Measurement of rate constants for actin filament elongation in solution. Anal. Biochem. 134, 406–412
Mullins, R. D. and Machesky, L. M. (2000) Actin assembly mediated by Arp2/3 complex and WASP family proteins. Methods Enzymol. 325, 214–237.
Gordon, D. J., Eisenberg, E., and Korn, E. D. (1976) Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method. J. Biol. Chem. 251, 4778–4786.
Blanchoin, L. and Pollard, T. D. (1999) Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments. J. Biol. Chem. 274, 15,538–15,546.
Kelleher, J. F., Mullins, R. D., and Pollard, T. D. (1998) Purification and assay of the Arp2/3 complex from Acanthamoeba castellanii. Methods Enzymol. 298, 42–51.
Dayel, M. J., Holleran, E. A., and Mullins, R. D. (2001) Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments. Proc. Natl. Acad. Sci. USA 98, 14,871–14,876.
Mullins, R. D., Heuser, J. A., and Pollard, T. D. (1998) The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments. Proc. Natl. Acad. Sci. USA 95, 6181–6186.
Neff, R. J. (1958) Mechanisms of purifying amoebae by migration on agar surfaces. J. Protozool. 5, 226–231.
Schuster, F. L. (2002) Cultivation of pathogenic and opportunistic free-living amebas. Clin. Microbiol. Rev. 15, 342–354.
Higgs, H. N., Blanchoin, L., and Pollard, T. D. (1999) Influence of the C terminus of Wiskott-Aldrich syndrome protein (WASp) and the Arp2/3 complex on actin polymerization. Biochemistry 38, 15,212–15,222.
Zigmond, S. H. (2000) How WASP regulates actin polymerization. J. Cell Biol. 150, F117–F120.
Selden, L. A., Kinosian, H. J., Estes, J. E., and Gershman, L. C. (2000) Cross-linked dimers with nucleating activity in actin prepared from muscle acetone powder. Biochemistry 39, 64–74.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Zuchero, J.B. (2007). In Vitro Actin Assembly Assays and Purification From Acanthamoeba . In: Coutts, A.S. (eds) Adhesion Protein Protocols. Methods in Molecular Biology™, vol 370. Humana Press. https://doi.org/10.1007/978-1-59745-353-0_15
Download citation
DOI: https://doi.org/10.1007/978-1-59745-353-0_15
Publisher Name: Humana Press
Print ISBN: 978-1-58829-533-0
Online ISBN: 978-1-59745-353-0
eBook Packages: Springer Protocols