Abstract
Group A Streptococcus (GAS) produces the pore-forming toxin, streptolysin O (SLO). SLO sequesters cholesterol and induces a plasma membrane repair process that removes the pores via a lipid raft-mediated endocytosis. The impact SLO has on membranes makes it an effective toxin for investigating the function of lipid rafts in cellular processes. Lipid rafts are essential for B-cell activation. Indeed, antigen-stimulated B-cell receptors (BCRs) require localization with lipid rafts for efficient signaling and internalization. SLO treatment impairs BCR activation by competing for lipid rafts. Here, disrupting lipid rafts using SLO and assessing the effects on BCR activation by fluorescence microscopy and flow cytometry are described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Lingwood D, Simons K (2010) Lipid rafts as a membrane-organizing principle. Science 327:46–50
Sezgin E, Levental I, Mayor S et al (2017) The mystery of membrane organization: composition, regulation and roles of lipid rafts. Nat Rev Mol Cell Biol 18:361–374
Cheng PC, Dykstra ML, Mitchell RN et al (1999) A role for lipid rafts in B cell antigen receptor signaling and antigen targeting. J Exp Med 190:1549–1560
Gupta N, DeFranco AL (2007) Lipid rafts and B cell signaling. Semin Cell Dev Biol 18:616–626
Gupta N, DeFranco AL (2003) Visualizing lipid raft dynamics and early signaling events during antigen receptor-mediated B-lymphocyte activation. Mol Biol Cell 14:432–444
Sohn HW, Pierce SK, Tzeng SJ (2008) Live cell imaging reveals that the inhibitory FcgammaRIIB destabilizes B cell receptor membrane-lipid interactions and blocks immune synapse formation. J Immunol 180:793–799
Phee H, Rodgers W, Coggeshall KM (2001) Visualization of negative signaling in B cells by quantitative confocal microscopy. Mol Cell Biol 21:8615–8625
Stoddart A, Dykstra ML, Brown BK et al (2002) Lipid rafts unite signaling cascades with clathrin to regulate BCR internalization. Immunity 17:451–462
Bhakdi S, Tranum-Jensen J, Sziegoleit A (1985) Mechanism of membrane damage by streptolysin-O. Infect Immun 47:52–60
Timmer AM, Timmer JC, Pence MA et al (2009) Streptolysin O promotes group A Streptococcus immune evasion by accelerated macrophage apoptosis. J Biol Chem 284:862–871
Uchiyama S, Dohrmann S, Timmer AM et al (2015) Streptolysin O rapidly impairs neutrophil oxidative burst and antibacterial responses to group A Streptococcus. Front Immunol 6:581
Miller H, Castro-Gomes T, Corrotte M et al (2015) Lipid raft-dependent plasma membrane repair interferes with the activation of B lymphocytes. J Cell Biol 211:1193–1205
Miller H, Song W (2018) Use of Streptolysin O-induced membrane damage as a method of studying the function of lipid rafts during B cell activation. Methods Mol Biol 1707:235–241
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Miller, H.E. (2020). Use of Streptolysin O (SLO) to Study the Function of Lipid Rafts. In: Proft, T., Loh, J. (eds) Group A Streptococcus. Methods in Molecular Biology, vol 2136. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0467-0_29
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0467-0_29
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0466-3
Online ISBN: 978-1-0716-0467-0
eBook Packages: Springer Protocols