To investigate, the interaction of fibronectin with lipids, simplified models of the cell membrane that take into account elements of its' lipid raft structure were examined. Atomic force microscopy (AFM) was employed to characterize the biomolecule adsorption. For binary lipid mixtures that display phase separation, AFM revealed that fibronectin preferentially and almost exclusively interacts with the gel phase domains. Single molecule force spectroscopy confirmed the location of the protein with respect to the lipid topography and allowed mechanical, thermodynamic and kinetic parameters concerning protein unfolding to be determined. Force distance curves also allowed characteristic features of the lipid-protein interaction to be measured in addition to the thickness of fibronectin layers on top of the gel phase domains. The effect of the adsorption of the fibronectin molecules at a lower concentration and at a temperature close to the glass transition temperature of the lipids was also investigated. Gel phase domains were found to self assemble into ring like structures in the presence of low concentrations of fibronectin. Force spectroscopy located the protein on the rings and together with topographic analysis allowed a model for the self assembly process to be proposed. The discovery of these unusual rings suggests that fibronectin has the ability to trigger lipid organisation. Finally α5β1 integrins were reconstituted into supported lipid bilayers in order to create a more biologically relevant model membrane. Topographic analysis demonstrated that the integrins had reconstituted into the bilayer with their head regions protruding from the bilayer as would be expected in a real cell membrane. Moreover the integrin head regions were shown to be functional by subsequent binding of fibronectin molecules specifically onto these head regions. All of these observations with respect to the adsorption behaviour of fibronectin molecules onto lipid bilayers have implications for understanding cell behaviour as fibronectin is more selective and has more of an effect on lipid organisation than previously thought.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:577177 |
| Date | January 2012 |
| Creators | Bin Nordin, Darman |
| Publisher | University of Newcastle Upon Tyne |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0062 seconds