Investigation of factors involved in the uptake velocity of fluorescein diacetate and intracellular fluorescence polarization value. I. Physiological aspects in lymphoblastoid cells.

Abstract

Measurement of cellular fluidity by the fluorescence polarization method with a fluorogenic dye, fluorescein diacetate (FDA), is used widely. But values obtained with this method may be affected greatly by unknown factors. To determine the various factors that affect fluorescence polarization (FP-) measurements and intrinsic cellular fluidity, we investigated the uptake of FDA into cells, cellular esterases, fluorescein-binding protein, microtubules and osmolarity. Our results show that some reagents inhibit FDA-uptake without affecting esterases and that several types of esterases are involved in FDA-hydrolysis.The dye-binding experiment indicated that dye-binding protein is absent from the cells. In addition, to determine whether there is involvement of microtubules in cellular fluidity, we treated cells with Ca2+ antagonists, calmodulin inhibitors and low temperature, and verified that there is little evidence for that hypothesis. In other experiments, cells were brought into a hypertonic condition, which resulted in osmolysis and higher intracellular concentrations, primarily of biopolymers, and higher viscosity. The effect of this viscosity on the FP value was investigated. Results showed a parallel relationship between osmolarity and therefore, intracellular viscosity and FP value.In conclusion, the concentrations of intracellular proteins and other biopolymers appear to be a major factor that determines cellular fluidity, but the role of microtubules which undergo assembly-disassembly appears to be insignificant.