Pressure-Tuning of the Morphology of Heterogeneous Lipid Vesicles - a Two-Photon-Excitation Fluorescence Microscopy Study
Chiara Nicolini 1, Anna Celli 2, Enrico Gratton 2 and Roland Winter 1*
1 University of Dortmund
2 University of Illinois
* To whom correspondence should be addressed. E-mail: roland.winter{at}uni-dortmund.de.
Submitted on May 9, 2006
Revised on June 12, 2006
Accepted on 17 July 2006
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Abstract |
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We used a technique that allows us to visualize local and morphological changes of the membrane of giant more-component unilamellar vesicles (GUVs) due to high pressure perturbation. Under these conditions, thermally induced processes are largely suppressed, and the bending rigidity and line tension are influenced by pressure-induced changes in lipid molecular packing and shape, only. We studied the effect of pressure on the lateral organization and morphology of the model raft system DOPC/sphingomyelin/cholesterol as well as of the fluid mixture POPC/DLPC by two-photon excitation fluorescence microscopy. The pressure dependent experiments were carried out using a sample cell made from thin fused silica capillary. The use of Laurdan as fluorescence label allowed us to follow also the lipid phase state by calculating the generalized polarization (GP) values of the vesicles and extracting their average value. During the compression cycle, a reduction in the volume of the vesicles is observed, accompanied by an increase of the average GP value, indicating an increasingly tighter packing of the lipids. Interestingly, the two systems studied show phenomena of budding and fission, and these at surprisingly low pressures of 200-300 bar, already. Moreover, theses budding processes are not directly related to phase transitions to an overall ordered conformational state of the lipid membrane, which occur at much higher pressures. The topological changes of the lipid vesicles are irreversible and exhibit a different behavior depending if the pressure is increased or decreased. The results are discussed in light of the various contributions to the free energy functional of lipid vesicles. Finally, the biological relevance of these studies in highlighted.
Key Words:
fluorescence microscopy, phase transitions, phospholipid mixtures, pressure, topological changes
