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박형주,백찬기,Masataka Kinjo,강봉균 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.1
We employed dual color Fluorescence Cross Correlation Spectroscopy (FCCS) to measure the interaction between PKA regulatory (RII) and catalytic subunits (CAT) in living cells. Elevation of intracellular cAMP with forskolin decreased the cross-correlation amplitude between RFP-fused RII (RII-mRFP) and GFP-fused CAT (CAT-EGFP) by 50%, indicating that cAMP elevation leads to dissociation of RII-CAT complexes. Moreover, diffusion coefficient analysis showed that the diffusion rate of CAT-EGFP was significantly increased, suggesting that the decreased RII-CAT association caused by cAMP generated free CAT subunits. Our study demonstrates that in vivo FCCS measurements and their quantitative analysis permit one not only to directly quantify protein-protein interactions but also to estimate changes in the intracellular cAMP concentration.
Park, Hyungju,Pack, Changi,Kinjo, Masataka,Kaang, Bong-Kiun Korean Society for Molecular Biology 2008 Molecules and cells Vol.26 No.1
<P>We employed dual color Fluorescence Cross Correlation Spectroscopy (FCCS) to measure the interaction between PKA regulatory (RII) and catalytic subunits (CAT) in living cells. Elevation of intracellular cAMP with forskolin decreased the cross-correlation amplitude between RFP-fused RII (RII-mRFP) and GFP-fused CAT (CAT-EGFP) by 50%, indicating that cAMP elevation leads to dissociation of RII-CAT complexes. Moreover, diffusion coefficient analysis showed that the diffusion rate of CAT-EGFP was significantly increased, suggesting that the decreased RII-CAT association caused by cAMP generated free CAT subunits. Our study demonstrates that in vivo FCCS measurements and their quantitative analysis permit one not only to directly quantify protein-protein interactions but also to estimate changes in the intracellular cAMP concentration.</P>
Ito, Toshiyuki,Oshita, Shugo,Nakabayashi, Takakazu,Sun, Fan,Kinjo, Masataka,Ohta, Nobuhiro Korean Society of Photoscience 2009 Photochemical & photobiological sciences Vol.8 No.6
Fluorescence lifetime images of HeLa cells expressing enhanced green fluorescent protein (EGFP) have been measured as apoptosis is induced by tumor necrosis factor-$\alpha$ (TNF-$\alpha$) in combination with cycloheximide. The fluorescence lifetime of EGFP is found to decrease after the induction of apoptosis, indicating that the change in environment occurs around the chromophore of EGFP with the apoptosis process. The fluorescence lifetime imaging technique can be used to perform in vivo observation of cell death processes. Fluorescence lifetime measurements are useful to examine the induction of the apoptosis process, even when a morphological change of each cell cannot be observed because of a low spatial resolution.