Abstract
We describe here a permeablization method that retains coupling between N-formylmethionyl- leucyl-phenylalanine (fMLP) receptor stimulation and barbed-end actin nucleation in neutrophils. Using fluorescently-tagged actin monomers, we are able to quantify and localize actin-free barbed ends generated downstream of chemoattractant receptors. Partial permeabilization of the neutrophils with the mild detergent n-octyl-β-glucopyranoside maintains signaling from membrane receptor to the actin cytoskeleton while allowing for the introduction of inhibitors and activators of signal transduction pathways implicated in regulating actin cytoskeleton dynamics. This is a useful assay for studying signal transduction to the actin cytoskeleton in neutrophils.
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References
Niggli, V. (2003) Signaling to migration in neutrophils: importance of localized pathways. Int. J. Biochem. Cell Biol. 35, 1619–1638.
Weiner, O. D., Servant, G., Welch, M. D., Mitchison, T. J., Sedat, J. W., and Bourne, H. R. (1999) Spatial control of actin polymerization during neutrophil chemotaxis. Nat. Cell Biol. 1, 75–81.
Fenteany, G. and Glogauer, M. (2004) Cytoskeletal remodeling in leukocyte function. Curr. Opin. Hematol. 11, 15–24.
Cicchetti, G., Allen, P. G., and Glogauer, M. (2002) Chemotactic signaling pathways in neutrophils: from receptor to actin assembly. Crit. Rev. Oral Biol. Med. 13, 220–228.
Sun, C. X., Downey, G. P., Zhu, F., Koh, A. L., Thang, H., and Glogauer, M. (2004) Rac1 is the small GTPase responsible for regulating the neutrophil chemotaxis compass. Blood 104, 3758–3765.
Hartwig, J. H., Bokoch, G. M., Carpenter, C. L., et al. (1995) Thrombin receptor ligation and activated Rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets. Cell 82, 643–653.
Glogauer, M., Hartwig, J., and Stossel, T. (2000) Two pathways through Cdc42 couple the N-formyl receptor to actin nucleation in permeabilized human neutrophils. J. Cell Biol. 150, 785–796.
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© 2007 Humana Press Inc., Totowa, NJ
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Glogauer, M. (2007). Quantifying and Localizing Actin-Free Barbed Ends in Neutrophils. In: Quinn, M.T., DeLeo, F.R., Bokoch, G.M. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology™, vol 412. Humana Press. https://doi.org/10.1007/978-1-59745-467-4_15
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DOI: https://doi.org/10.1007/978-1-59745-467-4_15
Publisher Name: Humana Press
Print ISBN: 978-1-58829-788-4
Online ISBN: 978-1-59745-467-4
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