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. 2002 Feb;82(2):1076–1085. doi: 10.1016/S0006-3495(02)75467-2

Multicolor imaging of Ca(2+) and protein kinase C signals using novel epifluorescence microscopy.

Asako Sawano 1, Hiroshi Hama 1, Naoaki Saito 1, Atsushi Miyawaki 1
PMCID: PMC1301914  PMID: 11806947

Abstract

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.

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Selected References

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  1. Bastiaens P. I., Majoul I. V., Verveer P. J., Söling H. D., Jovin T. M. Imaging the intracellular trafficking and state of the AB5 quaternary structure of cholera toxin. EMBO J. 1996 Aug 15;15(16):4246–4253. [PMC free article] [PubMed] [Google Scholar]
  2. Blackshear P. J. The MARCKS family of cellular protein kinase C substrates. J Biol Chem. 1993 Jan 25;268(3):1501–1504. [PubMed] [Google Scholar]
  3. Chakravarthy B. R., Isaacs R. J., Morley P., Durkin J. P., Whitfield J. F. Stimulation of protein kinase C during Ca(2+)-induced keratinocyte differentiation. Selective blockade of MARCKS phosphorylation by calmodulin. J Biol Chem. 1995 Jan 20;270(3):1362–1368. doi: 10.1074/jbc.270.3.1362. [DOI] [PubMed] [Google Scholar]
  4. Chakravarthy B. R., Isaacs R. J., Morley P., Whitfield J. F. Ca2+ x calmodulin prevents myristoylated alanine-rich kinase C substrate protein phosphorylation by protein kinase Cs in C6 rat glioma cells. J Biol Chem. 1995 Oct 20;270(42):24911–24916. doi: 10.1074/jbc.270.42.24911. [DOI] [PubMed] [Google Scholar]
  5. Chakravarthy B., Morley P., Whitfield J. Ca2+-calmodulin and protein kinase Cs: a hypothetical synthesis of their conflicting convergences on shared substrate domains. Trends Neurosci. 1999 Jan;22(1):12–16. doi: 10.1016/s0166-2236(98)01288-0. [DOI] [PubMed] [Google Scholar]
  6. Graff J. M., Young T. N., Johnson J. D., Blackshear P. J. Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C. J Biol Chem. 1989 Dec 25;264(36):21818–21823. [PubMed] [Google Scholar]
  7. Harpur A. G., Wouters F. S., Bastiaens P. I. Imaging FRET between spectrally similar GFP molecules in single cells. Nat Biotechnol. 2001 Feb;19(2):167–169. doi: 10.1038/84443. [DOI] [PubMed] [Google Scholar]
  8. Kim J., Shishido T., Jiang X., Aderem A., McLaughlin S. Phosphorylation, high ionic strength, and calmodulin reverse the binding of MARCKS to phospholipid vesicles. J Biol Chem. 1994 Nov 11;269(45):28214–28219. [PubMed] [Google Scholar]
  9. Miyawaki A., Griesbeck O., Heim R., Tsien R. Y. Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci U S A. 1999 Mar 2;96(5):2135–2140. doi: 10.1073/pnas.96.5.2135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Miyawaki A., Llopis J., Heim R., McCaffery J. M., Adams J. A., Ikura M., Tsien R. Y. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature. 1997 Aug 28;388(6645):882–887. doi: 10.1038/42264. [DOI] [PubMed] [Google Scholar]
  11. Miyawaki A., Tsien R. Y. Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 2000;327:472–500. doi: 10.1016/s0076-6879(00)27297-2. [DOI] [PubMed] [Google Scholar]
  12. Oancea E., Meyer T. Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals. Cell. 1998 Oct 30;95(3):307–318. doi: 10.1016/s0092-8674(00)81763-8. [DOI] [PubMed] [Google Scholar]
  13. Ohmori S., Sakai N., Shirai Y., Yamamoto H., Miyamoto E., Shimizu N., Saito N. Importance of protein kinase C targeting for the phosphorylation of its substrate, myristoylated alanine-rich C-kinase substrate. J Biol Chem. 2000 Aug 25;275(34):26449–26457. doi: 10.1074/jbc.M003588200. [DOI] [PubMed] [Google Scholar]
  14. Pepperkok R., Squire A., Geley S., Bastiaens P. I. Simultaneous detection of multiple green fluorescent proteins in live cells by fluorescence lifetime imaging microscopy. Curr Biol. 1999 Mar 11;9(5):269–272. doi: 10.1016/s0960-9822(99)80117-1. [DOI] [PubMed] [Google Scholar]
  15. Toullec D., Pianetti P., Coste H., Bellevergue P., Grand-Perret T., Ajakane M., Baudet V., Boissin P., Boursier E., Loriolle F. The bisindolylmaleimide GF 109203X is a potent and selective inhibitor of protein kinase C. J Biol Chem. 1991 Aug 25;266(24):15771–15781. [PubMed] [Google Scholar]
  16. Tsien R. Y., Miyawaki A. Seeing the machinery of live cells. Science. 1998 Jun 19;280(5371):1954–1955. doi: 10.1126/science.280.5371.1954. [DOI] [PubMed] [Google Scholar]
  17. Tsien R. Y. The green fluorescent protein. Annu Rev Biochem. 1998;67:509–544. doi: 10.1146/annurev.biochem.67.1.509. [DOI] [PubMed] [Google Scholar]

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