Date of Award

Fall 2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

First Advisor

Christopher Naumann

Committee Chair

Christoph Naumann

Committee Member 1

Steve Wassall

Committee Member 2

Garth Simpson

Committee Member 3

Christine Hrycyna

Abstract

It is now widely recognized that lipid rafts, which are membrane domains enriched in cholesterol (CHOL) and sphingolipids (SL), play a significant functional role in the plasma membrane. Raft domains particularly affect membrane functionality by causing sequestering of membrane proteins. Underlying mechanisms of raft-associated membrane protein sequestration remain elusive, due to the complexity, transient nature, and small size of raft domains in cellular membranes. To address these challenges, this dissertation unveils the relationship between lipid raft composition and membrane protein sequestration and function using raft-mimicking model membrane mixtures comprised of coexisting liquid-ordered (lo) and liquid-disordered (ld) domains with reconstituted membrane proteins. In particular, we address the potentially important, but poorly understood role of membrane asymmetry in membrane protein sequestration and function. A sensitive experimental method comprised of confocal fluctuation spectroscopy and photon counting histogram (PCH) analysis is utilized to analyze the sequestration and oligomerization state of αvβ3 and α5β1 integrins in raft-mimicking lipid mixtures. In asymmetric bilayers, coexisting lo-ld phase separations are located in the top leaflet, while the bottom leaflet exhibits a homogeneous ld phase. The comparison of symmetric

bilayers with bilayer-spanning lo-ld phase separations results revealed that αvβ3 and α5β1 show lo phase preference in asymmetric bilayers, but ld phase affinity in symmetric bilayers. Previously it has been shown that integrins translocate from the ld to lo phase upon addition of their respective ligands in symmetric bilayers, while there was no notable translocation of integrins in response to addition of native ligands in asymmetric bilayers. These interesting results indicate that integrin sequestration is dependent on lo and ld differences in lipid packing density, hydrophobic mismatch of integrin transmembrane and lipid bilayer regions, as well as the interaction between bilayer and integrin extracellular region. Next we investigated the influence of CHOL content on integrin sequestration because CHOL concentration influences lipid packing density, bilayer thickness, and line tension between lo and ld domains. Importantly, our data show that CHOL plays a substantial role in integrin sequestration in raft-mimicking lipid mixtures. These findings highlight the important role of bilayer asymmetry, distinct lipid densities and bilayer thicknesses in lo and ld regions of the bilayer for the regulation of membrane protein sequestration.

Changes in lipid packing density may also impact membrane elastic properties and lateral stress within the bilayer. Previously it has been shown that phospholipid monolayers with elevated concentration of lipopolymers are able to respond to increasing lateral stress by inducing membrane buckling, a stress relaxation phenomena. As part of the current dissertation, we established that membrane buckling can also be induced by gradually increasing CHOL concentration in polymer-tethered membranes of low lipopolymer content. Further analysis using quantitative epifluorescence and atomic force microscopy, combined with buckling theory for a thin elastic sheet confirmed that CHOL causes buckling due to the increase in biaxial stress within the membrane. These findings are intriguing in light of the important role of CHOL in membrane functionality.

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