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

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.

► A multi-parameter approach to visualize intracellular signaling events in the live B cell.
► The subcellular translocation of signaling proteins precedes Ca2+ mobilization.
► Various organelles participate in a timely-ordered manner to Ca2+ mobilization.