Abstract
The B cell antigen receptor (BCR) is found to be non-randomly organized at nano-scale distances on the B cell surface. Studying the organization and relocalization of the BCR is thus likely to provide new clues to understand the activation of the BCR. Indeed, with the in situ Fab proximity ligation assay (Fab-PLA), we now obtain proofs for the dissociation activation of BCRs and start to gain insight into how the relocalization of B cell surface signaling molecules could activate the cells. This chapter describes our methods to study the nano-scale organization of B cell surface receptors and co-receptors with Fab-PLA.
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References
Metzger H (1992) Transmembrane signaling: the joy of aggregation. J Immunol 149:1477–1487
Lee S, O'Dowd B, George S (2003) Homo- and hetero-oligomerization of G protein-coupled receptors. Life Sci 74:173–180
Lillemeier BF, Pfeiffer JR, Surviladze Z, Wilson BS, Davis MM (2006) Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc Natl Acad Sci U S A 103:18992–18997
Tao R-H, Maruyama IN (2008) All EGF(ErbB) receptors have preformed homo- and heterodimeric structures in living cells. J Cell Sci 121:3207–3217
Yang J, Reth M (2010) Oligomeric organization of the B-cell antigen receptor on resting cells. Nature 467:465–469
Thompson MA, Lew MD, Moerner WE (2012) Extending microscopic resolution with single-molecule imaging and active control. Annu Rev Biophys 41:321–342
Shivanandan A, Deschout H, Scarselli M, Radenovic A (2014) Challenges in quantitative single molecule localization microscopy. FEBS Lett 588:3595–3602
Mattila PK, Feest C, Depoil D, Treanor B, Montaner B, Otipoby KL, Carter R, Justement LB, Bruckbauer A, Batista FD (2013) The actin and tetraspanin networks organize receptor nanoclusters to regulate B cell receptor-mediated signaling. Immunity 38:461–474
Avalos AM, Bilate AM, Witte MD, Tai AK, He J, Frushicheva MP, Thill PD, Meyer-Wentrup F, Theile CS, Chakraborty AK, Zhuang X, Ploegh HL (2014) Monovalent engagement of the BCR activates ovalbumin-specific transnuclear B cells. J Exp Med 211:365–379
Maity PC, Blount A, Jumaa H, Ronneberger O, Lillemeier BF, Reth M (2015) B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation. Sci Signal 8:ra93–ra93
Lee J, Sengupta P, Brzostowski J, Lippincott-Schwartz J, Pierce SK (2017) The nanoscale spatial organization of B-cell receptors on immunoglobulin M- and G-expressing human B-cells. Mol Biol Cell 28:511–523
Söderberg O, Leuchowius K-J, Gullberg M, Jarvius M, Weibrecht I, Larsson L-G, Landegren U (2008) Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45:227–232
Kläsener K, Maity PC, Hobeika E, Yang J, Reth M (2014) B cell activation involves nanoscale receptor reorganizations and inside-out signaling by Syk. elife 3:e02069
Volkmann C, Brings N, Becker M, Hobeika E, Yang J, Reth M (2016) Molecular requirements of the B-cell antigen receptor for sensing monovalent antigens. EMBO J 35:2371–2381
Acknowledgments
This work was supported by the Excellence Initiative of the German Federal and State Governments (EXC 294), by ERC-grant 322972, and by the Deutsche Forschungsgemeinschaft through TRR130.
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Kläsener, K., Yang, J., Reth, M. (2018). Study B Cell Antigen Receptor Nano-Scale Organization by In Situ Fab Proximity Ligation Assay. In: Liu, C. (eds) B Cell Receptor Signaling. Methods in Molecular Biology, vol 1707. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7474-0_12
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DOI: https://doi.org/10.1007/978-1-4939-7474-0_12
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