Ternary And Quaternary Lipid Phase Diagrams To Investigate Membrane Raft Behavior

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Nonrandom mixing of lipids and proteins in the plasma membrane plays an important role in many cellular processes, including immune signaling, protein sorting, endocytosis, and virus entry and exit, yet the mechanisms governing the coalescence of these small domains are still not completely understood. Functional domains ("lipid rafts") that arise from nonrandom mixing of membrane components are believed to be important in governing the spatial organization of lipids and proteins. Model systems comprising chemically simplified lipid mixtures have played a central role in studies aimed at elucidating the mechanisms responsible for forming and regulating the size and lifetime of membrane domains. In this study we report the first solved phase diagram for the four-component mixture DSPC/DOPC/POPC/Chol (distearoylphosphatidylcholine/ dioleoylphosphatidylcholine/1-palmitoyl,2-oleoylphosphatidylcholine/cholesterol), which exhibits a domain size transition from nanoscopic to macroscopic, including a regime of spatially modulated domains within the region of coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. I also describe a novel FRET method used to determine boundaries. This phase diagram clearly shows that all phase boundaries determined for the 3-component mixture containing DOPC transition smoothly into the boundaries for the 3-component mixture containing POPC, which has iii nanoscopic phase domains of Ld + Lo. Together, our studies show that cells could control domain size and morphology by merely changing lipid composition. Phospholipids having a polyunsaturated acyl chain are abundant in biological membranes, but their behavior in lipid mixtures is difficult to study. In this work we elucidate the nature of such mixtures with this report of the first ternary phase diagram containing the polyunsaturated lipid SDPC in mixtures of BSM/SDPC/Chol (brain sphingomyelin/1-stearoyl-2-docosahexaenoylsn-glycero-3phosphocholine/ cholesterol). These mixtures show macroscopic Ld + Lo phase separation, with phase boundaries determined by FRET and by fluorescence microscopy imaging of giant unilamellar vesicles (GUVs). Surprisingly, SDPC mixes with BSM/Chol similarly to how DOPC and POPC mix with BSM/Chol. In addition, modulated phases are observed within the Ld + Lo liquid-liquid immiscibility region upon addition of fourth component POPC. We find mixtures of BSM/SDPC/POPC/Chol to exhibit nanoscopic Ld + Lo domains over a very large volume of composition space. In this work we have successfully accomplished the following goals: 1) solved the first ternary phase diagram containing a PUFA lipid; 2) solved the first phase diagram for a fourcomponent lipid bilayer mixture, including developing a new FRET method for accurate location of boundaries; and 3) synthesized a novel phosphorescent lipid-probe analog. iv
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phase diagrams; lipids; lipid rafts
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Feigenson, Gerald W
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Zipfel, Warren R.
Sondermann, Holger
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Ph. D., Biophysics
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Doctor of Philosophy
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Government Document
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dissertation or thesis
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