Lipid raft formation and lipid-protein interactions in model membranes

The biological membranes of eukaryotic organisms contain functional, highly dynamic nano-domains called "lipid rafts" (LRs) which are enriched in cholesterol, sphingolipids and GPI-anchor proteins. They are involved in several biological processes which implicate or are mediated by the plasma membrane. Moreover, LRs seem to have a critical role in the onset of some neurodegenerative diseases such as the Alzheimer’s disease (AD), Parkinson’s disease (PD) and Prion protein disorders. In the last two decades, the complexity of studying such domains in living cells has caused a growing interest in the use and design of artificial membrane models, which mimic the structure and composition of biological membranes. In this context, I promoted the formation and investigated the properties of lipid raft domains in artificial lipid bilayers by exploiting Atomic Force Microscopy (AFM). I compared two different fabrication methods for the production of artificial lipid bilayers, the drop-casting and the direct vesicle fusion techniques. I started from one-component lipid membranes and I progressively moved towards more complex models, as binary and ternary lipid compositions, in order to study the main LRs features in relation to specific biological phenomena, such as protein-lipid interactions involved in particular pathological diseases. The direct vesicle fusion method appeared to be the most suitable approach in term of reproducibility, stability and control of lipid composition. I took advantage from this method for carrying out a morphological characterization of raft-like model membranes composed by phosphocoline (DOPC), sphingomyelin (SM) and cholesterol focusing in particular on lipid phase behavior. Membranes exhibited the coexistence of two lipid phases, the fluid phase made by DOPC, and the solid-ordered phase made by SM and cholesterol, the latter resembling raft-like domains. With selected 3-component lipid systems, I then investigated the distribution of GM1 ganglioside, a LR marker, into my system, demonstrating its preferential localization in the nano-domains and highlighting the feasibility and versatility of model membrane technology. For the first time, I studied the binding of synthetic full-length Prion protein (PrPc), carrying a C-terminal membrane anchor (MA), to LRs domains. The conversion of PrPc into the scrapie isoform PrPsc, which displays high propensity to aggregate leading to cytotoxicity, has been reported to take place into LRs and to be influenced by lipid-anchors. I demonstrated with this study the propensity of this protein to specifically target LR domains of my artificial systems, observing an aggregation process occurring even at low protein concentrations. A comparative analysis with PrPc lacking of MA is however required to assess the role of lipid-anchor into the protein distribution and aggregation. Finally, in the last part of my research I focused on the study of the role of iron ions in the interaction between alpha synuclein (αS) and lipid membranes. αS is the central protein of PD and the presence of amyloid αS fibrils is the main pathological hallmark of the disease. By AFM in combination with attenuated total reflectance infrared (ATR-IR) spectroscopy, I compared the structural behavior of the wild-type (wt) and a mutant form of αS (A53T) in presence of Fe2+ ions and the effect of the iron ions on the interaction with my artificial membrane, and specifically with LRs.