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Cytoplasmic Actin: Purification and Single Molecule Assembly Assays

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Adhesion Protein Protocols

Part of the book series: Methods in Molecular Biology ((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.

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References

  1. Pollard TD, Borisy GG (2003) Cellular motility driven by assembly and disassembly of actin filaments. Cell 112:453–465

    Article  PubMed  CAS  Google Scholar 

  2. Fletcher DA, Mullins RD (2010) Cell mechanics and the cytoskeleton. Nature 463:485–492

    Article  PubMed  CAS  Google Scholar 

  3. Oosawa F, Kasai M (1962) A theory of linear and helical aggregations of macromolecules. J Mol Biol 4:10–21

    Article  PubMed  CAS  Google Scholar 

  4. 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

    Article  PubMed  CAS  Google Scholar 

  5. Welch MD, Mullins RD (2002) Cellular control of actin nucleation. Annu Rev Cell Dev Biol 18:247–288

    Article  PubMed  CAS  Google Scholar 

  6. Quinlan ME, Heuser JE, Kerkhoff E, Mullins RD (2005) Drosophila spire is an actin nucleation factor. Nature 433:382–388

    Article  PubMed  CAS  Google Scholar 

  7. Campellone KG, Welch MD (2010) A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol 11:237–251

    Article  PubMed  CAS  Google Scholar 

  8. Cooper JA, Walker SB, Pollard TD (1983) Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J Muscle Res Cell Motil 4:253–262

    Article  PubMed  CAS  Google Scholar 

  9. Pollard TD (1983) Measurement of rate constants for actin filament elongation in solution. Anal Biochem 134:406–412

    Article  PubMed  CAS  Google Scholar 

  10. Mullins RD, Machesky LM (2000) Actin assembly mediated by Arp2/3 complex and WASP family proteins. Methods Enzymol 325:214–237

    Article  PubMed  CAS  Google Scholar 

  11. Gordon DJ, Eisenberg E, Korn ED (1976) Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method. J Biol Chem 251:4778–4786

    PubMed  CAS  Google Scholar 

  12. Blanchoin L, Pollard TD (1999) Mechanism of interaction of Acanthamoeba actophorin (ADF/Cofilin) with actin filaments. J Biol Chem 274:15538–15546

    Article  PubMed  CAS  Google Scholar 

  13. Kelleher JF, Mullins RD, Pollard TD (1998) Purification and assay of the Arp2/3 complex from Acanthamoeba castellanii. Methods Enzymol 298:42–51

    Article  PubMed  CAS  Google Scholar 

  14. Dayel MJ, Holleran EA, Mullins RD (2001) Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments. Proc Natl Acad Sci USA 98:14871–14876

    Article  PubMed  CAS  Google Scholar 

  15. Mullins RD, Heuser JA, Pollard TD (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

    Article  PubMed  CAS  Google Scholar 

  16. Kuhn JR, Pollard TD (2005) Real-time measurements of actin filament polymerization by total internal reflection fluorescence microscopy. Biophys J 88:1387–1402

    Article  PubMed  CAS  Google Scholar 

  17. Hermanson GT (1996) Bioconjugate techniques. Academic, San Diego, CA, pp 266–272 (Product # 20002T)

    Google Scholar 

  18. Bieling P, Telley IA, Hentrich C, Piehler J, Surrey T (2010) Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor, and + TIP dynamics. Methods Cell Biol 95:555–580

    Article  PubMed  CAS  Google Scholar 

  19. Kovar DR, Harris ES, Mahaffy R, Higgs HN, Pollard TD (2006) Control of the assembly of ATP- and ADP-actin by formins and profilin. Cell 124:423–435

    Article  PubMed  CAS  Google Scholar 

  20. Kuhn JR, Pollard TD (2007) Single molecule kinetic analysis of actin filament capping. J Biol Chem 282:28014–28024

    Article  PubMed  CAS  Google Scholar 

  21. Hansen SD, Mullins RD (2010) VASP is a processive actin polymerase that requires monomeric actin for barbed end association. J Cell Biol 191:571–584

    Article  PubMed  CAS  Google Scholar 

  22. Huang NP, Michel R, Voros J, Textor M, Hofer R, Rossi A, Elbert DL, Hubbell JA, Spencer ND (2000) Poly(l-lysine)-g-poly(ethylene glycol) layers on metal oxide surfaces: surface analytical characterization and resistance to serum and fibrinogen adsorption. Langmuir 17:489–498

    Article  Google Scholar 

  23. Stuurman N, Amodaj N, Vale RD (2007) μManager: open source software for light microscope imaging. Microsc Today 15:42–43

    Google Scholar 

  24. Neff RJ (1958) Mechanisms of purifying amoebae by migration on agar surfaces. J Protozool 5:226–231

    Google Scholar 

  25. Schuster FL (2002) Cultivation of pathogenic and opportunistic free-living amebas. Clin Microbiol Rev 15:342–354

    Article  PubMed  Google Scholar 

  26. Huxley HE, Brown W (1967) The low-angle x-ray diagram of vertebrate striated muscle and its behaviour during contraction and rigor. J Mol Biol 30:383–434

    Article  PubMed  CAS  Google Scholar 

  27. Higgs HN, Pollard TD (2000) Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex. J Cell Biol 150:1311–1320

    Article  PubMed  CAS  Google Scholar 

  28. Padrick SB, Cheng HC, Ismail AM, Panchal SC, Doolittle LK, Kim S, Skehan BM, Umetani J, Brautigam CA, Leong JM, Rosen MK (2008) Hierarchical regulation of WASP/WAVE proteins. Mol Cell 32:426–438

    Article  PubMed  CAS  Google Scholar 

  29. Selden LA, Kinosian HJ, Estes JE, Gershman LC (2000) Cross-linked dimers with nucleating activity in actin prepared from muscle acetone powder. Biochemistry 39:64–74

    Article  PubMed  CAS  Google Scholar 

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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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Authors and Affiliations

  1. Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA

    Scott D. Hansen & R. Dyche Mullins

  2. Department of Neurobiology, Stanford University, Stanford, CA, USA

    J. Bradley Zuchero

Authors
  1. Scott D. Hansen
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  2. J. Bradley Zuchero
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  3. R. Dyche Mullins
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Editor information

Editors and Affiliations

  1. , Department of Oncology, University of Oxford, Roosevelt Drive, Oxford, OX3 7DQ, United Kingdom

    Amanda S. Coutts

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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

  • Print ISBN: 978-1-62703-537-8

  • Online ISBN: 978-1-62703-538-5

  • eBook Packages: Springer Protocols

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