The Molecular Structure of Human Red Blood Cell Membranes from Highly Oriented, Solid Supported Multi-Lamellar Membranes

Abstract

There are various techniques that are used to study the interactions between drugs and cells. However, the interaction between a drug and the cell membrane – the barrier between inter- and intracellular medium - is difficult to determine and is the focus of current research. Various common drugs, such as aspirin and cortisone, have been shown to have a significant effect on the membrane structure. While those investigations study the interaction between drugs and artificial membranes, i.e. simple lipid bilayers, their impact on native cell membranes is challenging. Due to the complex structure of a cell membrane and the cell itself, it is difficult to separate cellular lipid bilayers from other cell compartments and the cytoskeleton. This thesis presents a new protocol that allows to extract the membranes from red blood cells (RBCs) and to prepare highly ordered multi-lamellar stacks of these membranes on silicon wafers. Human RBC ghosts were prepared by hemolysis. The name of these empty RBC vesicles originates from their appearance under the microscope. Although the original preparation protocol by Dodge, Mitchell and Hanahan was a remarkable step in the development of membrane proteomics and lipidomics, modifications were necessary. The morphology of the vesicles, the hemoglobin concentration and the presence of the cytoskeleton were analyzed using fluorescence microscopy and Ultraviolet spectroscopy. Due to the observed complex structure and the present actin filaments, the ghost solution was sonicated before applied onto silicon wafers and slowly dried and annealed over several days. The vesicles fuse on the silicon substrate, resulting in highly ordered multi-lamellar stacks of RBC membranes. X-ray diffraction was used to analyze the structure of these membranes. Multiple series of well-developed Bragg peaks were observed indicating a high degree of orientation of the membranes. These series can be identified as signals from liquid ordered (lo), liquid disordered (ld) lipids and coiled-coils peptide domains. There was evidence that the lipids form nanometer sized domains and the collected data allows presenting a detailed picture of the domain- and membrane structure. The RBC ghosts allows to analyze the effect of drugs on the structure of a native membrane. The effect of aspirin, which is one of the most common drugs from the class of non-steroidal anti-inflammatory drugs (NSAID's), was investigated. NSAID's are known to interact with cell membranes. However, the data presented in this thesis indicates that aspirin incorporates preferably into the head group region of the liquid ordered lipid domains leading to a fluidification of these membrane domains.