Two-Photon Fluorescence Microscopy Studies of Bipolar Tetraether Giant Liposomes from Thermoacidophilic Archaebacteria Sulfolobus acidocaldarius

Abstract

The effects of temperature and pH on Laurdan (6-lauroyl-2-(dimethylamino)naphthalene) fluorescence intensity images of giant unilamellar vesicles (GUVs) (∼20–150 μm in diameter) composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius have been studied using two-photon excitation. PLFE GUVs made by the electroformation method were stable and well suited for microscopy studies. The generalized polarization (GP) of Laurdan fluorescence in the center cross section of the vesicles has been determined as a function of temperature at pH 7.23 and pH 2.68. At all of the temperatures and pHs examined, the GP values are low (below or close to 0), and the GP histograms show a broad distribution width (> 0.3). When excited with light polarized in the y direction, Laurdan fluorescence in the center cross section of the PLFE GUVs exhibits a photoselection effect showing much higher intensities in the x direction of the vesicles, a result opposite that previously obtained on monopolar diester phospholipids. This result indicates that the chromophore of Laurdan in PLFE GUVs is aligned parallel to the membrane surface. The x direction photoselection effect and the low GP values lead us to further propose that the Laurdan chromophore resides in the polar headgroup region of the PLFE liposomes, while the lauroyl tail inserts into the hydrocarbon core of the membrane. This unusual L-shaped disposition is presumably caused by the unique lipid structures and by the rigid and tight membrane packing in PLFE liposomes. The GP exhibited, at both pH values, a small but abrupt decrease near 50°C, suggesting a conformational change in the polar headgroups of PLFE. This transition temperature fully agrees with the d-spacing data recently measured by small-angle x-ray diffraction and with the pyrene-labeled phosphatidylcholine and perylene fluorescence data previously obtained from PLFE multilamellar vesicles. Interestingly, the two-photon Laurdan fluorescence images showed snowflake-like lipid domains in PLFE GUVs at pH 7.23 and low temperatures (<20°C in the cooling scan and <24°C in the heating scan). These domains, attributable to lipid lateral separation, were stable and laterally immobile at low temperatures (<23°C), again suggesting tight membrane packing in the PLFE GUVs.