Study of lipid bilayer behaviour modified by substrate interactions

Abstract

Biological membranes rarely exist as free-floating structures but are often confined and supported by various cellular assemblies such as the cytoskeleton and the extracellular matrix. It has already been shown that biological and polymeric substrates can modulate the morphology and response to various stimuli of supported lipid bilayers significantly. The interaction between such structures and the membrane are obviously important yet remain poorly understood even in minimal or synthetic systems.
The work of this thesis utilises a variety of fluorescence microscopy and atomic force microscopy (AFM) techniques to investigate the behaviour and structure of supported lipid bilayers, in particular how interfacial features of their support substrate influence and modulate their morphology and biophysical properties. First, surface modification of polydimethylsiloxane is systematically explored, in particular how the interfacial properties of such a polymer substrate can be modified to create fully and partially plasma-treated interfaces that stably support lipid bilayers. Lipid patch formation on such substrates is then investigated, revealing that the membrane undergoes significant morphological reorganisation after vesicle fusion has completed forming a lipid patch. The underlying mechanisms can be altered by substrate interactions following different pathways for fully and partially plasma-treated PDMS substrates. Furthermore, partially plasma-treated substrates are demonstrated to be capable of specifically depleting cholesterol from supported lipid membranes, while stably supporting the other remaining phospholipid species. Studies of cholesterol depletion of lipid patches possessing liquid-ordered and disordered domains reveal a disruption in domains structure, with the partitioning of fluorescent dyes into regions from which they were previously excluded. This structure perturbation was found to be reversible upon the reinsertion of cholesterol into the bilayer.
Many of the discussed mechanisms are only observed in the presence of a substrate, emphasising the importance of substrate interactions in both functional biomembranes and the development of supported membrane technologies