Optical Techniques Illuminate Phase Behavior In Model Membranes

Abstract

The lipid raft hypothesis, which describes active role of lipid heterogeneities in the organization and function of mammalian cell plasma membranes, has produced an enormous body of research since its inception in the late 1980's. Much of this research employs model systems, with the goal of elucidating the thermodynamics governing lateral heterogeneity in membrane lipids. This dissertation describes an optical approach for investigating the behavior of one model system, a ternary lipid system composed of dioleoylphosphatidylcholine (DOPC), N-stearoyl-sphingomyelin (SSM), and cholesterol. Measurements included miscibility transitions of the coexistence regions of this system and an all-optical method for determining the acyl chain order parameters in each phase and the tie directions of coexisting liquid-ordered (Lo) and liquid disordered (Ld) phases. A variety of fluorescent probes were examined for their partitioning behavior in the different phases of the SM/DOPC/cholesterol system (Chapter 2). This resulted in the selection of PAH probes with similar partitioning to cholesterol for use in subsequent studies: for the imaging of temperature-dependent phase behavior (Chapter 3) and for the characterization of the lipid acyl chain order as a function of composition (Chapter 5). The detailed phase behavior as a function of temperature and composition is mapped out and a quasi-ternary phase diagram is presented (Chapter 3). The phase diagram depicts the miscibility behavior of optically resolvable coexisting lipid phases as determined by fluorophore partitioning. Specific perturbations during sample preparation and imaging that affect the observed lipid phase behavior are discussed in the general context of model membrane research. This is important because these perturbations affect the validity of analogies drawn between model and biological systems. During the course of this research, an apparatus for ultra-stable (mK) control of the sample temperature during imaging was developed and it is the first of its kind. The details of the hardware necessary for such fine temperature control are related in Chapter 4. Finally, a new technique that uses mass spectrometry in conjunction with membrane patch excision is discussed in Chapter 6. The results of these experiments are preliminary but promising as a method of determining the absolute compositions of each phase.