Multicolor imaging of Ca2+ and protein kinase C signals using novel epifluorescence microscopy

Dynamic changes in intracellular free Ca2+ concentrations ([Ca2+](i)s) control many important cellular events, including binding of Ca2+-calmodulin (Ca2+-CaM) and phosphorylation by protein kinase C (PKC). The two signals compete for the same domains in certain substrates, such as myristoylated alanine-rich PKC-substrate (MARCKS). To observe the convergence and relative time of arrival of CaM and PKC signals at their shared domain of MARCKS, we need to image cells that are loaded with more than two fluorescent dyes at a reasonable speed. We have developed a simple and powerful multicolor imaging system using conventional fluorescence microscopy. The epifluorescence configuration uses a glass reflector and rotating filter wheels for excitation and emission paths. As it is free of dichroic (multichroic) mirrors, multiple fluorescence images can be acquired rapidly regardless of the colors of fluorophores. We visualized Ca2+ -CaM and PKC together with the dynamics of their common target, MARCKS, in single live cells. Receptor-activation resulted in translocation of MARCKS from the plasma membrane to cytosol through its phosphorylation by PKC. By observing fluorescence resonance energy transfer, we also obtained direct evidence that Ca2+-CaM binds MARCKS to drag it away from the membrane in circumstances when Ca2+ -mobilization predominates over PKC activation.