Multicolor imaging of Ca(2+) and protein kinase C signals using novel epifluorescence microscopy.
Dynamic changes in intracellular free Ca(2+) concentrations ([Ca(2+)](i)s) control many important cellular events, including binding of Ca(2+)-calmodulin (Ca(2+)-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 Ca(2+)-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 Ca(2+)-CaM binds MARCKS to drag it away from the membrane in circumstances when Ca(2+)-mobilization predominates over PKC activation.
Sawano
Asako
A
Laboratory for Cell Function and Dynamics, Advanced Technology Development Center, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako-city, Saitama, 351-0198, Japan.
Hama
Hiroshi
H
Saito
Naoaki
N
Miyawaki
Atsushi
A
D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002135:Calcium-Binding Proteins; D002147:Calmodulin / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000494:pharmacology; D005959:Glucosidases; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D008027:Light; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence / Q000379:methods*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000737:chemistry* / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D008164:Luminescent Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D049452:Green Fluorescent Proteins; C087857:protein kinase C gamma; D011493:Protein Kinase C; D005959:Glucosidases; C070368:PRKCSH protein, human; D002118:Calcium