Abstract
Invasive cell migration is critical for leukocyte trafficking into tissues. Podosomes are matrix-degrading adhesive structures that are formed by macrophages and are necessary for macrophage migration and invasion. Here, we describe methods for imaging and quantifying podosomes in primary human macrophages and in THP-1 cells, a monocyte cell line that can be differentiated to a macrophage-like state. Moreover, we outline detailed methods for live imaging of podosomes, which are highly dynamic, and for the quantification of rates of podosome turnover. Finally, we discuss methods for the quantitative analysis of matrix degradation on fluorescent-gelatin-coated cover slips.
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References
Tarone, G., Cirillo, D., Giancotti, F., Comoglio, P., and Marchisio, P. (1985) Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes., Exp Cell Res 159, 141–157.
Hai, C., Hahne, P., Harrington, E., and Gimona, M. (2002) Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells., Exp Cell Res 280, 64–74.
Lener, T., Burgstaller, G., Crimaldi, L., Lach, S., and Gimona, M. (2006) Matrix-degrading podosomes in smooth muscle cells., Eur J Cell Biol 85, 183–189.
Linder, S. (2007) The matrix corroded: podosomes and invadopodia in extracellular matrix degradation., Trends Cell Biol 17, 107–117.
Evans, J., Correia, I., Krasavina, O., Watson, N., and Matsudaira, P. (2003) Macrophage podosomes assemble at the leading lamella by growth and fragmentation., J Cell Biol 161, 697–705.
Linder, S., Nelson, D., Weiss, M., and Aepfelbacher, M. (1999) Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages., Proc Natl Acad Sci USA 96, 9648–9653.
Calle, Y., Antón, I., Thrasher, A., and Jones, G. (2008) WASP and WIP regulate podosomes in migrating leukocytes., J Microsc 231, 494–505.
Linder, S., and Aepfelbacher, M. (2003) Podosomes: adhesion hot-spots of invasive cells., Trends Cell Biol 13, 376–385.
Buccione, R., Orth, J., and McNiven, M. (2004) Foot and mouth: podosomes, invadopodia and circular dorsal ruffles., Nat Rev Mol Cell Biol 5, 647–657.
Ley, K., Laudanna, C., Cybulsky, M., and Nourshargh, S. (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated., Nat Rev Immunol 7, 678–689.
Carman, C., Sage, P., Sciuto, T., de la Fuente, M., Geha, R., Ochs, H., Dvorak, H., Dvorak, A., and Springer, T. (2007) Transcellular diapedesis is initiated by invasive podosomes., Immunity 26, 784–797.
Cougoule, C., Le Cabec, V., Poincloux, R., Al Saati, T., Mège, J., Tabouret, G., Lowell, C., Laviolette-Malirat, N., and Maridonneau-Parini, I. (2010) Three-dimensional migration of macrophages requires Hck for podosome organization and extracellular matrix proteolysis., Blood 115, 1444–1452.
Jones, G., Zicha, D., Dunn, G., Blundell, M., and Thrasher, A. (2002) Restoration of podosomes and chemotaxis in Wiskott-Aldrich syndrome macrophages following induced expression of WASp., Int J Biochem Cell Biol 34, 806–815.
Dovas, A., Gevrey, J., Grossi, A., Park, H., Abou-Kheir, W., and Cox, D. (2009) Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages., J Cell Sci 122, 3873–3882.
Tsuboi, S. (2006) A complex of Wiskott-Aldrich syndrome protein with mammalian verprolins plays an important role in monocyte chemotaxis., J Immunol 176, 6576–6585.
Svensson, H., West, M., Mollahan, P., Prescott, A., Zaru, R., and Watts, C. (2008) A role for ARF6 in dendritic cell podosome formation and migration., Eur J Immunol 38, 818–828.
Ochs, H., and Thrasher, A. (2006) The Wiskott-Aldrich syndrome., J Allergy Clin Immunol 117, 725–738; quiz 739.
Cortesio, C., Cooper, K., Wernimont, S., Kastner, D., and Huttenlocher, A. (2010) Impaired podosome formation and invasive migration of macrophages from patients with a PSTPIP1 mutation and PAPA syndrome., Arthritis Rheum.
Ruoslahti, E., Hayman, E., Pierschbacher, M., and Engvall, E. (1982) Fibronectin: purification, immunochemical properties, and biological activities., Methods Enzymol 82 Pt A, 803–831.
Tsuboi, S., Takada, H., Hara, T., Mochizuki, N., Funyu, T., Saitoh, H., Terayama, Y., Yamaya, K., Ohyama, C., Nonoyama, S., and Ochs, H. (2009) FBP17 Mediates a Common Molecular Step in the Formation of Podosomes and Phagocytic Cups in Macrophages., J Biol Chem 284, 8548–8556.
Dostert, C., Pétrilli, V., Van Bruggen, R., Steele, C., Mossman, B., and Tschopp, J. (2008) Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica., Science 320, 674–677.
Carrithers, M., Chatterjee, G., Carrithers, L., Offoha, R., Iheagwara, U., Rahner, C., Graham, M., and Waxman, S. (2009) Regulation of podosome formation in macrophages by a splice variant of the sodium channel SCN8A., J Biol Chem 284, 8114–8126.
Reiner, N. (2009) Methods in molecular biology. Macrophages and dendritic cells. Methods and protocols. Preface., Methods Mol Biol 531, v–vi.
Mosier, D. E. (2004) Introduction for “Safety Considerations for Retroviral Vectors: A Short Review”, pp 68–75, Applied Biological Safety Association, Applied Biosafety.
Zhang, X., Edwards, J., and Mosser, D. (2009) The expression of exogenous genes in macrophages: obstacles and opportunities., Methods Mol Biol 531, 123–143.
Riedl, J., Crevenna, A., Kessenbrock, K., Yu, J., Neukirchen, D., Bista, M., Bradke, F., Jenne, D., Holak, T., Werb, Z., Sixt, M., and Wedlich-Soldner, R. (2008) Lifeact: a versatile marker to visualize F-actin., Nat Methods 5, 605–607.
Schnoor, M., Buers, I., Sietmann, A., Brodde, M., Hofnagel, O., Robenek, H., and Lorkowski, S. (2009) Efficient non-viral transfection of THP-1 cells., J Immunol Methods 344, 109–115.
Calle, Y., Carragher, N., Thrasher, A., and Jones, G. (2006) Inhibition of calpain stabilises podosomes and impairs dendritic cell motility., J Cell Sci 119, 2375–2385.
Webb, D., Donais, K., Whitmore, L., Thomas, S., Turner, C., Parsons, J., and Horwitz, A. (2004) FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly., Nat Cell Biol 6, 154–161.
Chan, K., Cortesio, C., and Huttenlocher, A. (2007) Integrins in cell migration., Methods Enzymol 426, 47–67.
Zamir, E., Katz, B., Aota, S., Yamada, K., Geiger, B., and Kam, Z. (1999) Molecular diversity of cell-matrix adhesions., J Cell Sci 112 (Pt 11), 1655–1669.
Yamaguchi, H., Pixley, F., and Condeelis, J. (2006) Invadopodia and podosomes in tumor invasion., Eur J Cell Biol 85, 213–218.
Tu, C., Ortega-Cava, C., Chen, G., Fernandes, N., Cavallo-Medved, D., Sloane, B., Band, V., and Band, H. (2008) Lysosomal cathepsin B participates in the podosome-mediated extracellular matrix degradation and invasion via secreted lysosomes in v-Src fibroblasts., Cancer Res 68, 9147–9156.
Mulari, M., Zhao, H., Lakkakorpi, P., and Väänänen, H. (2003) Osteoclast ruffled border has distinct subdomains for secretion and degraded matrix uptake., Traffic 4, 113–125.
Cougoule, C., Carréno, S., Castandet, J., Labrousse, A., Astarie-Dequeker, C., Poincloux, R., Le Cabec, V., and Maridonneau-Parini, I. (2005) Activation of the lysosome-associated p61Hck isoform triggers the biogenesis of podosomes., Traffic 6, 682–694.
Artym, V., Zhang, Y., Seillier-Moiseiwitsch, F., Yamada, K., and Mueller, S. (2006) Dynamic interactions of cortactin and membrane type 1 matrix metalloproteinase at invadopodia: defining the stages of invadopodia formation and function., Cancer Res 66, 3034–3043.
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Starnes, T.W., Cortesio, C.L., Huttenlocher, A. (2011). Imaging Podosome Dynamics and Matrix Degradation. In: Wells, C., Parsons, M. (eds) Cell Migration. Methods in Molecular Biology, vol 769. Humana Press. https://doi.org/10.1007/978-1-61779-207-6_9
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DOI: https://doi.org/10.1007/978-1-61779-207-6_9
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