IN-SITU DETERMINATION OF INTRACELLULAR MEMBRANE PHYSICAL STATE HETEROGENEITY IN RENAL EPITHELIAL-CELLS USING FLUORESCENCE RATIO MICROSCOPY

6-Lauroyl-2-dimethylaminonaphtalene (laurdan) shows a spectral sensiti vity to the lipid phase state with a 50 nm red shift of the emission m aximum when passing from the gel to the liquid crystalline phase. This spectral sensitivity allows one to determine the membrane physical st ate using Generalized Polarization (GP). In the present experiments, w e used fluorescence ratio imaging microscopy to determine the laurdan GP in living kidney cells. Two renal epithelial cells lines, MDCK and LLC-PK1 cells, and CV-1 cells, a fibroblast-like renal cell line were investigated. In these cells, laurdan labels both the plasma membrane and intracellular membranes. Comparison of spectrofluorimetry and fluo rescence ratio imaging data obtained from liposomes and cells suspensi ons labeled with laurdan demonstrates that the GP can be accurately de termined using common fluorescence microscopy equipment. The GP mean v alues determined from individual cells varied from 0.2 to 0.4 for the epithelial cells as compared to 0.0-0.1 for CV1 cells. Using living MD CK cells grown as a monolayer, the GP maps indicated that, within a si ngle cell, the intracellular GP values varied from 0.0 to 0.6, i. e., from the equivalent of a liquid-crystalline state to a gel or a lipid- ordered state, and that there was a marked heterogeneity in the spatia l distribution of the GP values. To further characterize this intracel lular heterogeneity, co-localization experiments with specific organel le markers were undertaken. The results strongly suggest that in intac t cells at physiological temperature, GP values decrease in the follow ing order: plasma membranes > endosomes > mitochondria > Golgi apparat us.