| Fluorescence techniques are widely used to study membrane dynamics. Specifically, fluorescence microscopy is an excellent tool for the detection of phase separation on lipid vesicles. Fluoresce resonance energy transfer (FRET) is commonly used to monitor interactions and binding events It can also be implemented to detect phase separation down to the nanometer range, past the limitations of confocal microscopy. Avidin-biotin cross-linking on the vesicle surface is used to perturb the phase dynamics. This dissertation utilized microscopy to investigate phase distribution and separation on cross-linked systems. It will also focus on the development and implementation of FRET.;The first part of this dissertation focuses on cross-linking two-phase vesicles. Along with cross-linking, quenching, temporal, and non-specific binding effects are also important. Using microscopy, sample populations of vesicles are categorized and any global shifts due to perturbations are documented. Cross-linking, the binding of avidin to membrane bound biotin, caused a vesicle population shift from the majority two-phase two-domain to a majority two-phase multiple domain. Analysis of our cross-linking data suggests that avidin forms clusters on the surface of the fluid-disordered domains resulting in a large immobile fraction and restricted diffusion.;Quenching of the vesicles had its strongest effect on the one-phase vesicles rather than the two-phase vesicle population. The sample imaging times were sufficiently long enough to allow vesicles to reach thermodynamic equilibrium. Non-specific binding experiments verified the specific binding of avidin to biotin caused the multiple domain vesicles.;The second part of this dissertation focuses on cross-linking one-phase vesicles and the development of FRET to detect nano-sized domains. Avidin and two of its analogs, neutravidin and avidin horseradish peroxidase (avdin-HRP) were used as the cross-linking proteins. Structure differences between the three proteins effects the protein-protein interaction and clustering on the membrane. Avidin was the only biotin binding protein that induced phase separation with both microscopy and FRET techniques.;Common activities, such as ligand--receptor coupling, contribute to lateral heterogeneity and membrane protein clustering, adding to cell membrane complexity. Fundamental studies into subtle shifts such as cross-linking events, which induce global cellular response, are pertinent to understanding membrane activities and effects of external stimuli. |
