Quantitative Measurements of Membrane Lipid Order in Yeast and Fungi

Membrane lateral heterogeneity, historically referred to as the lipid raft hypothesis, has been extensively investigated through physiochemical experiments on model membranes. Currently, the basic principles are well understood; however, the physiological relevance of these structures in living organisms is still not clear. Thus, studying membrane organization in vivo is extremely important and elucidates the role of such structures in various membrane-associated processes. This is particularly true when a whole single-celled organism can be studied rather than an isolated cell. The ordered and disordered membrane phases are characterized by the degree of acyl chain packing in the lipid bilayer. Polar water molecules can penetrate into the low-density lipid packing of the disordered phase, but are more excluded from the tightly packed ordered phase. Here, polarity-sensitive probes, embedded in the lipid bilayer, are used to report on membrane organization and to quantitate this parameter via 2-channel fluorescence microscopy. Coupling genetic approaches, which are easily accessible in yeast model organisms, with the imaging approach described here provides a great opportunity to investigate how membrane heterogeneity impacts physiology.