Abstract
Fractalkine (CX3CL1) is a membrane-anchored chemokine whose N-terminus contains a unique CX3C motif that is cleaved and released. The membrane-bound form functions as an adhesion molecule and the secreted form as a chemotactic factor. Like other chemokines, CX3CL1 is regulated at the levels of transcription and translation. Recent evidence points to additional functional regulation by cellular trafficking owing to the unique transmembrane structure. CX3CL1 is the only chemokine known to undergo constitutive internalization. To understand mechanisms governing the regulation and processing of such membrane-bound proteins, it is vital to study their subcellular distribution and transport. The methods outlined in this chapter describe (1) transfection of mammalian cells with plasmids encoding the expression of green fluorescent protein-tagged CX3CL1; (2) immunofluorescence antibody labeling as well as fluorescence recovery after photobleaching to study internalization of CX3CL1 by endocytosis; and (3) acid-stripping assays to study the recycling of internalized CX3CL1 back to the plasma membrane. Together, these methods allow for the examination of subcellular distribution and traffic of recycling membrane proteins.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Schall, T.J., Bacon, K.B. (1994) Chemokines, leukocyte trafficking, and inflammation Curr Opin Immunol 6, 865–873
Baggiolini, M. (1998) Chemokines and leukocyte traffic. Nature 392, 565–568
Bazan, J.F., Bacon, K., Hardiman, G., Wang, W., Soo, K., Rossi, D., Greaves, D.R., Zlotnick, A., and Schall, T. S. (1997) A new class of membrane-bound chemokine with a CX3C motif Nature 385, 640–644
Pan, Y., Lloyd, C., Zhou, H., Dolich, S., Deeds, J., Gonzalo, A., Vath, J., Gosselin, M., Ma, J., Dussault, B., Woolf, E., Alperin, G., Culpepper, J., Gutierrez-Ramos, J.C. and Gearing, D. (1997) Neurotactin, a membrane-anchored chemokine upregulated in brain inflammation Nature 387, 611–617
Fong, A., Robinson, L., Steeber, D., Tedder, T., Yoshie, O., Imai, T., and Patel, D. (1998) Fractalkine and CX3CR1 mediate a novel mechanism of leukocyte capture, firm adhesion and activation under physiological flow J Exp Med 188, 1413–1419
Harrison, J., Jiang, Y., Wees, E., Salafranca, M., Liang, H., Feng, L., and Belardinelli, L. (1999) Inflammatory agents regulate in vivo expression of fractalkine in endothelial cells of the rat heart J Leukocyte Biol 66, 937–944
Ludwig, A., Berkhout, T., Moores, K., Groot, P., and Chapman, G. (2002) Fractalkine Is Expressed by Smooth Muscle Cells in Response to IFN- γ and TNF-α and Is Modulated by Metalloproteinase Activity J Immunol 168, 604–612
Muehlhoefer, A., Saubermann, L., Gu, X., Luedtke-Heckenkamp, K., Xavier, R., Blumberg, R. S., Podolsky, D. K., MacDermott, R. P., and Reinecker, H. (2000) Fractalkine Is an Epithelial and Endothelial Cell-Derived Chemoattractant for Intraepithelial Lympho-cytes in the Small Intestinal Mucosa J Immunol 164, 3368–3376
Liu, G, Kulasingam, V., Alexander, R.T., Touret, N., Fong, A.M., Dhavalkumar, D.P., and Robinson, L.A. (2005) Recycling of the membrane-anchored chemokine, CX3CL1. J Biol Chem 280:19858–19866
Touret, N., Furuya, W., Forbes, J., Gros, P., and Grinstein, S. (2003) Dynamic Traffic through the Recycling Compartment Couples the Metal Transporter Nramp2 (DMT1) with the Transferrin Receptor. J Biol Chem 278: 25548–25557
Lippincott-Schwartz, J., Presley, J., Zaal, K., Hirschberg, K., Miller, C., and Ellenberg, J. (1999) Monitoring the dynamics and mobility of membrane proteins tagged with green fluorescent protein. Methods Cell Biol 58, 261–281
Reits, E., and Neefjes, J. (2001) From fixed to FRAP: measuring protein mobility and activity in living cells Nat Cell Biol 3, 145–147
Su, T., Cariappa, R., and Stanley, K. (1999) N-glycans are not a universal signal for apical sorting of secretory proteins FEBS Lett. 453, 391–394
Haller, C., Kiessling, F., and Kubler, W. (1997) Polarized expression of heterologous membrane proteins transfected in a human endothelial-derived cell line Eur J Cell Biol 75, 353–361
Huang, YW., Su, P., Liu, GY., Crow, MR., Chaukos, D., Yan, H., Robinson, LA. (2009). Constitutive Endocytosis of the Chemokine CX3CL1 Prevents Its Degradation by Cell Surface Metalloproteases. J Biol Chem 284 : 29644–29653
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Patel, S., Mukovozov, I., Robinson, L.A. (2011). Assessment of the Recycling of the Membrane-Bound Chemokine, CX3CL1. In: Rast, J., Booth, J. (eds) Immune Receptors. Methods in Molecular Biology, vol 748. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-139-0_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-139-0_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-138-3
Online ISBN: 978-1-61779-139-0
eBook Packages: Springer Protocols