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 sensitivity to the lipid phase state with a 50 nm red shift of the emission maximum when passing from the gel to the liquid crystalline phase. This spectral sensitivity allows one to determine the membrane physical state using Generalized Polarization (GP). In the present experiments, we 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 fluorescence ratio imaging data obtained from liposomes and cells suspensions labeled with laurdan demonstrates that the GP can be accurately determined using common fluorescence microscopy equipment. The GP mean values 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 MDCK cells grown as a monolayer, the GP maps indicated that, within a single 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 spatial distribution of the GP values. To further characterize this intracellular heterogeneity, co-localization experiments with specific organelle markers were undertaken. The results strongly suggest that in intact cells at physiological temperature, GP values decrease in the following order: plasma membranes > endosomes > mitochondria > Golgi apparatus.