Abstract
Lipid rafts were first defined as detergent-resistant membranes (DRMs) due to their relative insolubility in non-ionic detergents. Although they should not be confused with lipid rafts, DRMs are a valuable starting point for the study of these membrane domains and the interactions of proteins with rafts.
Here we describe the isolation of DRMs by ultracentrifugation on a sucrose gradient, a method we have used to study the role of lipid rafts in NKG2D-mediated signaling. We also describe raft fractionation of NK cells involving the selective solubility of β-octylglucoside (β-OG). OG is a non-ionic detergent that efficiently dissolves DRMs but does not disrupt protein associations with the cytoskeleton. Using these two techniques may yield useful information about the proteins involved in receptor recruitment into lipid rafts and the interactions of the actin cytoskeleton with lipid rafts.
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References
Brown DA, London E (2000) Structure and function of sphingolipid- and cholesterol-rich membrane rafts. J Biol Chem 275:17221–17224
Simons K, Toomre D (2000) Lipid rafts and signal transduction. Nat Rev Mol Cell Biol 1:31–39
Horejsi V (2005) Lipid rafts and their roles in T-cell activation. Microbes Infect 7:310–316
Lou Z, Jevremovic D, Billadeau DD et al (2000) A balance between positive and negative signals in cytotoxic lymphocytes regulates the polarization of lipid rafts during the development of cell-mediated killing. J Exp Med 191:347–354
Fassett MS, Davis DM, Valter MM et al (2001) Signaling at the inhibitory natural killer cell immune synapse regulates lipid raft polarization but not class I MHC clustering. Proc Natl Acad Sci U S A 98:14547–14552
Watzl C, Long EO (2003) Natural killer cell inhibitory receptors block actin cytoskeleton-dependent recruitment of 2B4 (CD244) to lipid rafts. J Exp Med 197:77–85
Riteau B, Barber DF, Long EO (2003) Vav1 phosphorylation is induced by beta2 integrin engagement on natural killer cells upstream of actin cytoskeleton and lipid raft reorganization. J Exp Med 198:469–474
Sanni TB, Masilamani M, Kabat J et al (2004) Exclusion of lipid rafts and decreased mobility of CD94/NKG2A receptors at the inhibitory NK cell synapse. Mol Biol Cell 15:3210–3223
Masilamani M, Nguyen C, Kabat J et al (2006) CD94/NKG2A inhibits NK cell activation by disrupting the actin network at the immunological synapse. J Immunol 177:3590–3596
Gomez TS, Billadeau DD (2008) T cell activation and the cytoskeleton: you can’t have one without the other. Adv Immunol 97:1–64
Chichili GR, Rodgers W (2009) Cytoskeleton-membrane interactions in membrane raft structure. Cell Mol Life Sci 66:2319–2328
Holowka D, Sheets ED, Baird B (2000) Interactions between Fc(epsilon)RI and lipid raft components are regulated by the actin cytoskeleton. J Cell Sci 113:1009–1019
Andrews NL, Lidke KA, Pfeiffer JR et al (2008) Actin restricts FcepsilonRI diffusion and facilitates antigen-induced receptor immobilization. Nat Cell Biol 10:955–963
Chichili GR, Rodgers W (2007) Clustering of membrane raft proteins by the actin cytoskeleton. J Biol Chem 282:36682–36691
Serrano-Pertierra E, Cernuda-Morollón E, Brdicka T et al (2014) L-plastin is involved in NKG2D recruitment into lipid rafts and NKG2D-mediated NK cell migration. J Leukoc Biol 96:437–445
Wessel D, Flügge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal Biochem 138:141–143
Acknowledgements
This work was supported by grants from the Spanish Fondo de Investigaciones Sanitarias-Fondos FEDER European Union (FISPI12/02587) from “Plan Nacional de I + D + I 2008–2011”, and the PCTI 2013–2017 (GRUPIN14-030) from Principado de Asturias, Spain.
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Serrano-Pertierra, E., López-Larrea, C. (2016). Using NK Cell Lipid Raft Fractionation to Understand the Role of Lipid Rafts in NK Cell Receptor Signaling. In: Somanchi, S. (eds) Natural Killer Cells. Methods in Molecular Biology, vol 1441. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3684-7_11
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DOI: https://doi.org/10.1007/978-1-4939-3684-7_11
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3684-7
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