Direct detection of caspase-3 activation in single live cells by cross-correlation analysis.
Dual color fluorescence cross-correlation spectroscopy (FCCS) provides information about the coincidence of spectrally well-defined two fluorescent molecules in a small observation area at the single-molecule level. To evaluate the activity of caspase-3 in vivo directly, FCCS was applied to single live cells. We constructed chimeric proteins that consisted of tandemly fused enhanced green FP (EGFP) and monomeric red FP (mRFP). In control experiments, the protease reaction was monitored in solution, where a decrease in cross-correlation amplitude was observed due to specific cleavage of the amino acid sequence between EGFP and mRFP. Moreover, a decrease in cross-correlation amplitude could be detected in a live cell, where caspase-3 activation was induced by apoptosis. This is the first report of FP-based in vivo cross-correlation analysis. FP-based FCCS may become the most versatile method for analysis of protein-protein interactions in live cells.
Laboratory of Supramolecular Biophysics, Research Institute for Electronic Science, Hokkaido University, N12W6, Kita-ku, Sapporo 060-0812, Japan.
Kenta
K
Saito
Ikuo
I
Wada
Mamoru
M
Tamura
Masataka
M
Kinjo
D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism*; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D015233:Models, Statistical; D010447:Peptide Hydrolases / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D000097573:Red Fluorescent Protein
D053148:Caspase 3; D020169:Caspases; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D010447:Peptide Hydrolases; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C505407:CASP3 protein, human