Fluorescence Probes as Monitors of Surface Membrane Fluidity Gradients in Murine Fibroblasts

Abstract

Fluorescence probe molecules were utilized in conjunction with quenching agents to investigate the possibility that mammalian cell surface membranes may display vertical asymmetry of physico‐chemical properties. Two different approaches indicated that the outer monolayer of a murine fibroblast surface membrane was more fluid than the inner monolayer, First, a fluorescent glucosamine derivative of trans‐parinaric acid did not penetrate intact LM cells (a strain of transformed murine fibroblasts) or phagosomes derived from these cells. This probe molecule was easily accessible to nonpenetrating quenching agents such as trinitrophenyl‐glycine, indicating that it resided in the exposed membrane monolayer. The fluorescence polarization, P, of glucosamine trans‐parinarate in intact LM cells was 0.218 ± 0.009 while in phagocytosed latex bead membranes the polarization was 0.248 0.006. Second, trans‐parinaric acid, 1,6‐diphenyl‐1,3,5‐hexatriene, and N‐phenyl‐l‐naphthylamine were used as fluorescent membrane probes in isolated plasma membrane vesicles. Their fluorescence in the plasma membrane was quenched by either covalently linking trinitrophenyl groups to exposed NH2 constituents on the membrane surface or by adding the water‐soluble non‐penetrating quenching agent, trinitrophenyl‐glycine. Trinitrophenyl‐amino groups have an absorption maximum at 415 nm and can therefore chemically quench the fluorescence of trans‐parinarate, 1,6‐diphenyl‐1,3,5‐hexatriene, and N‐phenyl‐1‐naphthylamine which have fluorescence emission maxima near 415 nm. With both methods, when only the outer monolayer amino groups were trinitrophenylated, the absorption‐corrected fluorescence emission and the relative fluorescence efficiency of trans‐parinarate in the plasma membrane were decreased by 45 ± 5% and 44 ± 6% respectively. Neither quenching method altered the fluorescence lifetime of trans‐parinarate. In contrast, when both sides of the plasma membrane had covalently linked trinitrophenyl groups, these parameters were diminished by 90–95%. Similar results were obtained with 1,6‐diphenyl‐1,3,5‐hexatriene and N‐phenyl‐1‐naphthylamine. Polarization measurements of trans‐parinarate indicated that the inside monolayer of the membrane was more rigid (P= 0.364 ± 0.005) than the whole membrane (P= 0.323 ± 0.005). Similar results were obtained with 1,6‐diphenyl‐l,3,5‐hexatriene. However, N‐phenyl‐1‐naphthylamine which resides near the polar interface of the bilayer showed no difference in polarization upon quenching by either method. Thus, results obtained with glucosamine trans‐parinarate in the absence of quenching agents or with trans‐parinarate and 1,6‐diphenyl‐1,3,5‐hexatriene in the presence of quenching agents indicated that a vertical asymmetry or gradient of certain physicochemical parameters may exist in LM cell plasma membranes. Preliminary data indicated that this asymmetry could be altered by lipid polar head group manipulation. Copyright © 1980, Wiley Blackwell. All rights reserved