Spectrin-Lipid Interactions and their Effect on the Membrane Mechanical Properties

Abstract

This thesis presents the experimental work performed on the spectrin protein. The aim of the work was to study the direct interactions of spectrin, the cytoskeleton of RBCs, with membrane lipid to determine its effects on the mechanical properties of the lipid bilayer. Motivation for this work came from a lack of unanimity in the field of spectrin, and the hypothesized potential of the protein to perforate giant unilamellar vesicles. The work aimed to investigate and determine how spectrin-lipid interactions influence membrane mesoscopic morphology and biophysics in ways that could ultimately be important to cellular function. For this purpose, a protocol was implemented to take into account the different aspects of the binding. Direct visualisation of the spectrin-lipid interaction and distribution was achieved using confocal fluorescence microscopy. Changes in the mechanical properties of the membrane were investigated using the micropipette aspiration technique. Finally the thermodynamics of the interaction were considered with isothermal titration calorimetry experiments. This allowed evaluation of the protein-lipid interaction in a complete and coherent manner. Experiments were also performed on another elastic protein, alpha-elastin, for comparison. In addition to its similarities with spectrin (both possess hydrophobic domains and entropy elasticity), elastin is auto-fluorescent which makes it an attractive model protein. Elastin was also used as a sample model to implement new techniques using nonlinear optics microscopy.