Abstract
The development of both microscopic and flow cytometry techniques has allowed investigators to examine the individual behavior of migratory cells under many different conditions, such as inflammation, invasion and metastasis, normal recirculation of blood cells, and so forth. Cells belonging to the immune system provide a typical example of cells in which migration is a critical feature for them to exert their physiological function (1–3). Leukocyte migration requires the reorganization of the cytoskeleton of the cell to allow the transition from a medium of high flow stress, such as the blood, into the target tissues across narrow gaps provided by endothelial cell-cell junctions (4). Thus, cytoskeletal plasticity is a key requirement for leukocyte migration and its associated morphological changes (5). Such a property is provided by the actomyosin system, a flexible compartment of the cell cytoskeleton that is dynamically regulated by a number of signaling intermediates and adaptor molecules that ensure its proper polymerization/depolymerization and contractility (6–9). These molecules are regulated by extracellular cues, which function as molecular on/off devices, providing the cell with the appropriate signal that triggers changes in the cytoskeleton by the recruitment and/or activation of the adequate intracellular components. Chemoattractants are among the most widely studied of these signals. They can be simple molecules, such as fMLP, or complex polypeptides, such as chemokines.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Butcher, E. C. and Picker, L. J. (1996) Lymphocyte homing and homeostasis. Science 272, 60–66.
Ansel, K. M. and Cyster, J. G. (2001) Chemokines in lymphopoiesis and lymphoid organ development. Curr. Opin. Immunol. 13, 172–179.
Von Andrian, U. H. and Mackay, C. R. (2000) T-Cell function and migration. Two sides of the same coin. N. Engl. J. Med. 343, 1020–1034.
Worthylake, R. A. and Burridge, K. (2001) Leukocyte transendothelial migration: orchestrating the underlying molecular machinery. Curr. Opin. Cell Biol. 13, 69–577.
Brown, M. J., Hallam, J. A., Colucci-Guyon, E., and Shaw, S. (2001) Rigidity of circulating lymphocytes is primarily conferred by vimentin intermediate filaments. J. Immunol. 166, 6640–6646.
Bear, J. E., Krause, M., and Gertler, F. B. (2001) Regulating cellular actin assembly. Curr. Opin. Cell Biol. 13, 158–166.
Cooper, J. and Schafer, D. (2000) Control of actin assembly and disassembly at filament ends. Curr. Opin. Cell Biol. 12, 97–103.
Small, J. V., Rottner, K., and Kaverina, I. (1999) Functional design of the actin cytoskeleton. Curr. Opin. Cell Biol. 11, 54–60.
Volkmann, N. and Hanein, D. (2000) Actomyosin: law and order in motility. Curr. Opin. Cell Biol. 12, 26–34.
Moser, B. and Loetscher, P. (2001) Lymphocyte traffic control by chemokines. Nat. Immunol. 2, 123–128.
Horuk, R. (2001) Chemokine receptors. Cytokine Growth Factor Rev. 12, 313–335.
Thelen, M. (2001) Dancing to the tune of chemokines. Nat. Immunol. 2, 129–134.
Mellado, M., Rodríguez-Frade, J. M., Mañes, S., and Martínez-A, C. (2001) Chemokine signaling and functional responses: the role of receptor dimerization and TK pathway activation. Annu. Rev. Immunol. 19, 397–421.
Dancker, P., Low, I., Hasselbach, W., and Wieland, T. (1975) Interaction of actin with phalloidin: polymerization and stabilization of F-actin. Biochim. Biophys. Acta 400, 407–414.
Vandekerckhove, J., Deboben, A., Nassal, M., and Wieland, T. (1985) The phalloidin binding site of F-actin. EMBO J. 4, 2815–2818.
Bleul, C. C., Fuhlbrigge, R. C., Casasnovas, J. M., Aiuti, A., and Springer, T. A. (1996) A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J. Exp. Med. 184, 1101–1109.
Vicente-Manzanares, M., Cabrero, J. R., Rey, M., Pérez-Martínez, M., Itoh, K., and Sánchez-Madrid, F. (2002) Activation of the Rho-Rho-kinase pathway by the chemokine SDF-1α: a role for p160ROCK in the maintenance of lymphocyte morphology and migration. J. Immunol. 168, 400–410.
Roberts, A. W., Kim, C., Zhen, L., et al. (1999) Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity 10, 183–196.
Ridley, A. J., Paterson, H. F., Johnston, D., Diekmann, D., and Hall, A. (1992) The small GTP-binding protein Rac regulates growth factor-induced membrane ruffling. Cell 70, 401–410.
Nobes, C. and Hall, A. (1995) Rho, Rac and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamel-lipodia and filopodia. Cell 81, 52–62.
D’Souza-Schorey, C., Boettner, B., and Van Aelst, L. (1998) Rac regulates integrin-mediated spreading and increased adhesion of T lymphocytes. Mol. Cell Biol. 18, 3936–3946.
Haddad, E., Zugaza, J. L., Louache, F., et al. (2001) The interaction between Cdc42 and WASP is required for SDF-1-induced T-lymphocyte chemotaxis. Blood 91, 33–38.
del Pozo, M. A., Vicente-Manzanares, M., Tejedor, R., Serrador, J. M., and Sanchez-Madrid, F. (1999) Rho GTPases control migration and polarization of adhesion molecules and cytoskeletal ERM components in T lymphocytes. Eur. J. Immunol. 29, 3609–3620.
Vicente-Manzanares, M., Montoya, M. C., Mellado, M., et al. (1998) The chemokine SDF-1alpha triggers a chemotactic response and induces cell polarization in human B lymphocytes. Eur. J. Immunol. 28, 2197–2207.
Vicente-Manzanares, M., Rey, M., Jones, D., et al. (1999) Involvement of phosphatidylinositol 3-kinase in stromal-cell derived factor-1alpha-induced lymphocyte polarization and chemotaxis. J. Immunol. 163, 4001–4012.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Vicente-Manzanares, M., VitÓn, M., Sánchez-Madrid, F. (2004). Measurement of the Levels of Polymerized Actin (F-Actin) in Chemokine-Stimulated Lymphocytes and GFP-Coupled cDNA Transfected Lymphoid Cells by Flow Cytometry. In: D’Ambrosio, D., Sinigaglia, F. (eds) Cell Migration in Inflammation and Immunity. Methods in Molecular Biology™, vol 239. Humana Press. https://doi.org/10.1385/1-59259-435-2:53
Download citation
DOI: https://doi.org/10.1385/1-59259-435-2:53
Publisher Name: Humana Press
Print ISBN: 978-1-58829-102-8
Online ISBN: 978-1-59259-435-1
eBook Packages: Springer Protocols