Simultaneous imaging of phosphatidyl inositol metabolism and Ca2+ levels in PC12h cells.
To elucidate the spatial and temporal relationships between phosphatidyl inositol metabolism and changes in intracellular calcium levels, we developed a simultaneous imaging system using green fluorescent protein fused with the pleckstrin homology domain, and the fluorescent calcium indicator, FuraRed. The redistribution of the fusion protein, which represents the phosphatidyl inositol metabolism process, was quantified by calculating the coefficient of variance of the fluorescence over the entire cytosolic region, excluding the nucleus. This calculation increased the reproducibility, compared to the normalized fluorescence changes in arbitrarily selected cytosolic regions used in conventional analysis. The application of this method to analyzing the response of PC12h cells to a number of pharmacological stimuli showed that the extent of the phosphatidyl inositol metabolism was related to the calcium level, but not induced by calcium alone.
Morita
Mitsuhiro
M
Laboratory of Cellular Neurobiology, School of Life Science, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, 192-0392 Tokyo, Japan. moritam@ls.toyaku.ac.jp
Yoshiki
Fumito
F
Kudo
Yoshihisa
Y
D000818:Animals; D001572:Benzofurans / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007093:Imidazoles / Q000494:pharmacology*; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D016716:PC12 Cells; D010716:Phosphatidylinositols / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D013997:Time Factors
D001572:Benzofurans; D007093:Imidazoles; D008164:Luminescent Proteins; D010716:Phosphatidylinositols; D049452:Green Fluorescent Proteins; C082877:fura red; D004247:DNA; D002118:Calcium