Date of Award

2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Chemistry

First Advisor

Tobias Baumgart

Abstract

The composition and morphology of cellular membranes are highly dynamic. Potential parameters modulating protein and lipid distributions in different organelles include membrane shapes and the structures of lipids and proteins. Moreover, the concept of "lipid rafts" provides a prevailing view where nanodomains serve as centers for signal transduction, membrane trafficking, and cytoskeletal organization. In this contribution, we first investigated the lipid and protein organizations as a function of membrane curvature. To this end, a system consisting of solid-supported wavy membranes that exhibits a continuous curvature distribution with positive and negative curvature ranges was fabricated. Spatial distributions of ENTH (epsin N-terminal homology) domain and N-BAR (Bin-Amphiphysin-Rvs) domains derived from the proteins Endophilin and BIN-1 were found to vary approximately linearly with membrane curvature. In contrast, streptavidin and fluorescent lipid analogues exhibited homogenous distributions on wavy membranes. Fluorescence recovery after photobleaching and single-molecule tracking experiments revealed that protein domains remain laterally fluid in the curved regions. We next studied the membrane organization with respect to lipid structures, more specifically, the length and degree of saturation of acyl chains of lipids. The ganglioside GM1 binds cholera toxin (CT) on host cells and carries it retrograde from the plasma membrane (PM) through endosomes, the trans-Golgi network (TGN), and the endoplasmic reticulum (ER) to induce toxicity. To elucidate how a membrane lipid can specify trafficking in these pathways, GM1 isoforms with alternate ceramide domains were synthesized and their partitioning between liquid-ordered (Lo) and liquid-disordered (Ld) phases in GUVs was imaged. GM1 with differing ceramides showed distinct phase-partitioning behaviors. Furthermore, crosslinking of GM1 by cholera toxin subunit B (CTB) was found to drive phase partitioning shift from less preferential phase preference to exclusively Ld or Lo phases. To shed light on the stability of lipid domains, factors qwhich affect line tension were discussed and potential line-active molecules were examined. We found that the presence of cone-shaped diacylglycerol decreases line tension, while the commonly used fluorescent lipid, Texas-Red DHPE tends to increase line tension. Additionally, to bridge the connection between thermodynamics to highly dynamic cellular environments, we developed a single liposome-based kinetics system which allowed us to examine membrane binding kinetics of proteins as a function of membrane curvature. Overall, these measurements help provide an integrated view of biophysical and structural parameters underlying organizations of lipids and proteins.

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Biophysics Commons

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