Abstract
Antigen-induced B cell activation requires mobilization of the Ca2+ second messenger. This process is associated with the subcellular relocalization of signal effector proteins of the B cell antigen receptor such as the adaptor protein SLP65. Here we describe a broadly applicable live cell imaging method to simultaneously visualize intracellular Ca2+ flux profiles and the translocation of cytosolic signaling proteins to the plasma membrane in real time. Our approach delineated the kinetic hierarchy of Ca2+ signaling events in B cells and revealed a timely ordered contribution of various organelles to the overall Ca2+ signal. The developed experimental setup provides a useful tool to resolve the spatiotemporal signaling dynamics in various receptor signaling systems.
Highlights
Ligation of the B cell antigen receptor (BCR) induces the mobilization of the second messenger Ca2+ that is indispensable for antibody-mediated immune responses [1,2]
For the first time we simultaneously imaged the BCR-triggered processes of SLP65 membrane recruitment and Ca2+ mobilization in real time
Our results demonstrate that translocation of SLP65 from the cytosol to the plasma membrane precedes the release of Ca2+ from the endoplasmic reticulum (ER)
Summary
Ligation of the B cell antigen receptor (BCR) induces the mobilization of the second messenger Ca2+ that is indispensable for antibody-mediated immune responses [1,2]. Emptying the ER Ca2+ store is sensed by the ER membrane residents STIM1 and STIM2 that mediate the opening of Ca2+ channels in the plasma membrane [9] Other cellular organelles such as mitochondria have been described to contribute to the Ca2+ response in antigen-stimulated T cells [10,11]. Biphasic Ca2+ fluxes can be recorded by flow cytometry upon loading of cells with Ca2+-sensitive dyes such as Indo. Biphasic Ca2+ fluxes can be recorded by flow cytometry upon loading of cells with Ca2+-sensitive dyes such as Indo1 This approach revealed that Ca2+ mobilization in B cells is not an all-or-nothing event but can be modulated in kinetic and quantitative terms depending on the developmental stage of the B cell [2,12]. It has been a puzzling observation that intracellular Ca2+
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