Biological plasma membranes are essential for a cell’s proper function. They play key roles in cell-communication, structural support, and are involved in key processes, such as inducing anesthesia. They are composed of hundreds of distinct molecules with the majority being phospholipids, followed by proteins and carbohydrates. We take advantage of this well-crafted system by creating artificial bilayer models, whose parameters are well-controlled. Ultimately, the goal of this research is to understand the complex interplay of molecules in a model bilayer through translational diffusion constants and connect the results to live plasma membranes. The model bilayer composition was simplified by focusing on major lipids, namely cholesterol, 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), sphingomyelin, and ceramide on mica substrates. Various applications of artificial membranes exist, and we choose to focus on the area of anesthesiology. Though anesthesia use continues to be vital, from minor to major surgical processes, molecular mechanisms are not clear and lack of widespread consensus on theories cause debate among researchers. The main contenders are anesthetics acting directly on proteins or indirectly by dissolving in lipids and affecting transmembrane protein functions. Drawing from previous work in the Blanchard research group, we apply ethanol and n-butanol, on our models to understand interactions of general anesthetics with the bilayer. With perylene or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) as fluorescent probes (sensitive to phospholipid headgroup or tail mobility, respectively), the fluorescent recovery after photobleaching (FRAP) technique results in fluorescence recovery curves of the supported lipid bilayers, from which translational diffusion constants are extracted. The values are related directly to fluidity and, as predicted, application of the short-chain alcohols led to higher diffusion coefficients overall. However, high alcohol concentrations resulted in lower diffusion coefficients potentially due to bilayer interdigitation. On both the macroscopic and microscopic scale, ceramide rigidified the system. As a whole, further investigations pointed to the heterogeneous morphology of the models. Size-dependent FRAP measurements led to an important observation that anomalous diffusion is occurring. Ultimately, results from the models need to be connected with those on live cell membranes and we had this experience with a collaborative project with the Busik laboratory in the physiology department at Michigan State University. Their group works on various projects, one of which involves treatment options for retinal-based degradation in diabetic subjects. Increased cholesterol generally decreases membrane fluidity, and related to our research goals, the drug N, N-dimethyl-3β-hydroxy-cholenamide (DMHCA), a selective LXR agonist, rejuvenated circulating angiogenic cell (CAC) membrane fluidity which has potential in vascular repair of people afflicted with the diabetic retinopathy complication.
Read
