Browsing by Subject "fluorescence correlation spectroscopy"

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    Molecular and mechanical manipulation of membrane domains in planar supported bilayers
    (2015-08) Warner, Rochelle
    Biomembranes are dynamic, two-dimensional fluids that actively participate in biological functions such as signaling, membrane trafficking, endocytosis and exocytosis. They are composed of thousands of lipid species and hundreds of proteins in cells, and the membrane itself is constantly remodeling. Nano-membrane domains are hypothesized to play an integral role in many cell signaling pathways. Their transient nature and biocomplexity underlies a myriad of fundamental questions about lipid-lipid and lipid-protein interactions and their roles in cellular functions. As a result, there is a need for innovative approaches for understanding different biophysical aspects of membrane assemblies and their underlying, multiscale dynamics. Membranes in living cells are very complex and highly dynamic making it difficult to manipulate crosslinking at the molecular level. We overcome this issue by using light (optical trapping) to crosslink proto-domains in a well-controlled, yet non-invasive manner and quantitative fluorescence microscopy to follow the subsequent dynamics. To simplify the investigations of specific molecular interactions and their dynamics, we use biomimetic, or model, membranes that are chemically well-defined; that is, they are composed of only a few molecular species. The goal is to integrate dynamic holographic optical trapping and fluorescence imaging with fluorescence correlation spectroscopy to characterize membrane domain nucleation in biomimetic planar supported bilayers. Our hypothesis is that by trapping multiple microsphere-bound receptors, the associated heterogeneous lipid domains will nucleate a larger domain upon interaction in a manner that depends on the lipid type, cholesterol and protein content.