USE OF TIME-RESOLVED FLUORESCENCE TO DETERMINE INTRACELLULAR ION GRADIENTS

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The fluorescent probe Quin-2 has been used extensively to determine intracellular calcium ion concentrations by steady-state fluorescence measurements. Using nanosecond time-resolved pulse and phase fluorometric instrumentation we have determined that this probe exhibits unique decay characteristics upon binding various cations including Ca++, Mg++, and Zn++; specifically, it shows a five- to ten-fold increase in intensity as well as alterations in anisotropy. Measurements to study the interactions between Quin-2 and cation complexes in solution demonstrate that concentrations for free probe and several different ion complexes can be resolved. We have obtained spectral and time-resolved data from Quin-2- loaded A431 cells to examine intracellular compartmentalization as a function of signal transductory events induced by cytokine binding. From our data which support the idea that Quin-2 exists in heterogeneous cellular microenvironments we are developing a growth factor-dependent cytoplasmic functional map of the cell.