Fluorescence Microscopy to Study Pressure Between Lipids in Giant Unilamellar Vesicles

Fluorescence Microscopy to Study Pressure Between Lipids in Giant Unilamellar Vesicles Anna Celli, Claudia Y. C. Lee, and Enrico Gratton Summary The authors developed a technique to apply high hydrostatic pressure to giant unilamellar vesicles and to directly observe the consequent structural changes with two-photon fluorescence microscopy imaging using high numerical aperture oil-immersion objectives. The data demonstrate that high pressure has a dramatic effect on the shape of the vesicles, and both fluidity and homogeneity of the membrane. Key Words: Microscopy; pressure; vesicles. 1. Introduction Lipid membranes are the envelopes that separate the interior of the cell and its organelles from external media. Extensive research in the past two decades has shown that the lipids forming the matrix of the cell membranes do not merely play a structural role but have a cru- cial function in regulating many vital processes (1,2). Recently, much attention has been paid to the dynamics of lipid interaction and organization in the membrane. In particular, the lipid phase determines the fluidity of the membrane, and thus, regulates the diffusion of membrane proteins. Many studies have pointed out that microdomains of different fluidity and lipid organization (rafts) exist under physiological conditions in biomembranes. For this reason, the direct visualization of membrane heterogeneity has received particular attention in the past few years. Fluorescence microscopy has proved to be a powerful tool to directly study biological membranes noninvasively under physiological conditions. Among different artificial membranes, giant unilamellar vesicles (GUV) are particularly good models of cell membranes because of their similarity to cells in terms of their dimen- sions (10–100 µm), radius of curvature, and lamellarity. Moreover, GUV can be visualized individually under the microscope, allowing researchers to study the local properties of the membrane. The study of the physics involved in lipid–lipid interactions requires perturbation of the system and the eventual observation of how the system reacts to the perturbation. In this chapter, a technique is presented that allows to visualize the local changes in GUV mem- branes caused by high-pressure perturbation. The response of biomembranes to pressure has been studied for a variety of reasons: from the need to understand the adaptation of deep-sea organisms to high pressures (up to a 1 Kbar) to the effects of pressure on anesthesia (3–5). Moreover, pressure offers a very powerful means to perturb the biophysical conditions of the membranes, and therefore, can be used to investigate the physics of the interactions of biomolecules contained in the bilayer. Many From: Methods in Molecular Biology, vol. 400: Methods in Membrane Lipids Edited by: A. M. Dopico © Humana Press Inc., Totowa, NJ