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Can localized impurities exert global effects on lipid model membranes?

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Thesis type
(Thesis) Ph.D.
Date created
The currently accepted model for cell membrane organization involves "lipid rafts"', which differ in composition from the surrounding lipid sea. The existence of these nano-scale heterogeneities is supported by observation of coexisting ordered and disordered lipid phases in lipid model membranes. Fluorescence is a popular family of techniques that can provide dynamic and structural information about membranes. With any probe method, characterization of the effects of fluorescent probe addition on the systems they are used to study is important for the interpretation of experimental data. Comparison can be made between labelled and unlabelled samples using a non-perturbing method, such as deuterium nuclear magnetic resonance spectroscopy (2H NMR). This thesis used 2H NMR to study the effects of an equipartitioning probe, Laurdan, and a non-equipartitioning probe, naphthopyrene, on a well-studied three-component lipid membrane system (35:35:30 dioleoyl-sn-glycero-3-phosphocholine (DOPC)/dipalmitoyl-sn-glycero-3-phosphocholine-D62 (DPPC-D62)/cholesterol) with a miscibility phase transition. In phase-separated membranes, 0.03-0.6\% naphthopyrene disordered lipid chains of DPPC-D62 in the liquid-disordered phase, but not of those in the liquid-ordered phase. 0.1-2% Laurdan did not affect the DPPC-D62 in either phase in these membranes. Above the miscibility transition temperature (22 degrees Celsius), there is a single homogeneous liquid phase that is not perturbed by the addition of either probe. Laurdan is particularly well suited to the study of phase separation in lipid membranes. It partitions equally well into ordered and disordered lipid phases and displays a polarity-dependent emission spectral shift. Laurdan general polarization (GP) parameter, which characterizes said spectral shift, has been used to characterize membrane fluidity. Two-photon excitation microscopy Laurdan GP images were acquired for membranes with 0.2-2% Laurdan, and Laurdan GP values were found to be strongly correlated with \dnmr methylene order parameters of DPPC-D62 in the liquid-ordered phase. Finally, photo-induced phase separation is known to occur in fluorescence microscopy experiments; however, our 2H NMR experiments showed that naphthopyrene can alter membrane properties in the absence of light. The fact that trace amounts of probe (e.g., 0.03-0.6% naphthopyrene) affect lipid molecular order has biological implications: biomolecules present in very small amounts are known to have important functions in cells.
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Supervisor or Senior Supervisor
Thesis advisor: Thewalt, Jenifer
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