Abstract
Binding of antigen to the B cell receptor (BCR) triggers both BCR signaling and endocytosis simultaneously. BCR signaling pathways and their regulation have been studied extensively by both biochemical methods and flow cytometry, resulting in a comprehensive understanding of the temporal dynamics of the signaling enzymes and effector proteins. However, spatial regulation of these signaling pathways in subcellular pathways is relatively poorly understood. Here, we describe a method to study the spatio-temporal distribution of phosphorylated-kinases in antigen-activated B cells by confocal microscopy. This method can also be applied to other cell types where it is of interest to understand the spatial distribution of signaling enzymes and their effector 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 subscriptionsReferences
Cambier JC, Gauld SB, Merrell KT, Vilen BJ (2007) B-cell anergy: from transgenic models to naturally occurring anergic B cells? Nat Rev Immunol 7:633–643
Reth M, Wienands J (1997) Initiation and processing of signals from the B cell antigen receptor. Annu Rev Immunol 15:453–479
Dalporto J (2004) B cell antigen receptor signaling 101. Mol Immunol 41:599–613
Campbell KS (1999) Signal transduction from the B cell antigen-receptor. Curr Opin Immunol 11:256–264
Kurosaki T, Shinohara H, Baba Y (2010) B cell signaling and fate decision. Annu Rev Immunol 28:21–55
Clark MR, Massenburg D, Zhang M, Siemasko K (2003) Molecular mechanisms of B cell antigen receptor trafficking. Ann N Y Acad Sci 987:26–37
Lanzavecchia A (1990) Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu Rev Immunol 8:773–793
Chaturvedi A, Martz R, Dorward D, Waisberg M, Pierce SK (2011) Endocytosed BCRs sequentially regulate MAPK and Akt signaling pathways from intracellular compartments. Nat Immunol 12:1119–1126
Murphy JE, Padilla BE, Hasdemir B, Cottrell GS, Bunnett NW (2009) Endosomes: a legitimate platform for the signaling train. Proc Natl Acad Sci U S A 106:17615–17622
Sadowski L, Pilecka I, Miaczynska M (2009) Signaling from endosomes: location makes a difference. Exp Cell Res 315:1601–1609
Vieira AV, Lamaze C, Schmid SL (1996) Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274:2086–2089
Nazarewicz RR, Salazar G, Patrushev N, Martin AS, Hilenski L, Xiong S, Alexander RW (2011) Early endosomal antigen 1 (EEA1) is an obligate scaffold for angiotensin II-induced, PKC- -dependent Akt activation in endosomes. J Biol Chem 286:2886–2895
Chaturvedi A, Pierce SK (2009) How location governs toll-like receptor signaling. Traffic 10(6):621–628
Chaturvedi A, Dorward D, Pierce SK (2008) The B cell receptor governs the subcellular location of Toll-like receptor 9 leading to hyperresponses to DNA-containing antigens. Immunity 28:799–809
Delcroix J-D, Valletta JS, Wu C, Hunt SJ, Kowal AS, Mobley WC (2003) NGF signaling in sensory neurons: evidence that early endosomes carry NGF retrograde signals. Neuron 39:69–84
Wu C, Lai CF, Mobley WC (2001) Nerve growth factor activates persistent Rap1 signaling in endosomes. J Neurosci 21:5406–5416
Omerovic J, Prior IA (2009) Compartmentalized signalling: Ras proteins and signalling nanoclusters. FEBS J 276:1817–1825
Taelman VF, Dobrowolski R, Plouhinec J-L, Fuentealba LC, Vorwald PP, Gumper I, Sabatini DD, De Robertis EM (2010) Wnt signaling requires sequestration of glycogen synthase kinase 3 inside multivesicular endosomes. Cell 143:1136–1148
Chaturvedi A, Siddiqui Z, Bayiroglu F, Rao KVS (2002) A GPI-linked isoform of the IgD receptor regulates resting B cell activation. Nat Immunol 3:951–957
Acknowledgments
A.R.M. is the recipient of DST-INSPIRE fellowship. A.C. is supported by intramural funds by IISER Pune.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Mishra, A.R., Chaturvedi, A. (2018). B Cell Receptor Signaling and Compartmentalization by Confocal Microscopy. 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_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7474-0_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7473-3
Online ISBN: 978-1-4939-7474-0
eBook Packages: Springer Protocols