Spatiotemporal resolution of Ca2+ signaling events by real time imaging of single B cells

Antigen-induced B cell activation requires mobilization of the Ca(2+) 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 Ca(2+) flux profiles and the translocation of cytosolic signaling proteins to the plasma membrane in real time. Our approach delineated the kinetic hierarchy of Ca(2+) signaling events in B cells and revealed a timely ordered contribution of various organelles to the overall Ca(2+) signal. The developed experimental setup provides a useful tool to resolve the spatiotemporal signaling dynamics in various receptor signaling systems.