Model Systems For Studies At The Plasma Membrane: Investigating Lipid Phase Behavior And Protein-Mediated Membrane Remodeling

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Abstract
The plasma membrane is a multicomponent mixture of lipids and proteins. 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. Proper compartmentalization of lipids and proteins is vital to facilitate cellular processes, such as signaling, endocytosis and trafficking. While evidence has shown that lipids play an integral role in protein-mediated processes, the chemical complexity of the plasma membrane and the dynamic nature of intermolecular interactions pose challenges for systematic investigations that aim to determine the interplay between lipids and proteins. Model membrane mixtures provide chemically simplified systems for studies of both lipid-lipid, and lipid-protein interactions. In four-component lipid mixtures that model the outer leaflet of the plasma membrane, we observed a nanoscopic-to-macroscopic transition of domain size and morphology by tuning lipid composition. Using fluorescence microscopy imaging of giant unilamellar vesicles (GUVs), we found that this nano-to-macro transition exhibits a regime of patterned fluid domains within the liquid coexistence region of this four-component system. Temperature-dependent FRET and microscopy studies strongly suggest that the patterned domains are thermodynamically stable, lending support for the existence of nanoscopic domains with possibly complex morphology in cellular plasma membranes. Together, our studies allude to a possible mechanism for cells to control domain size and morphology by merely changing lipid composition. The importance of lipids in facilitating cellular processes is evident from examining protein-mediated membrane remodeling events. Using in vitro liposome deformation and liposome binding assays, we examined the activation mechanism of pacsin-1, an F-BAR domain protein enriched in neurons, by dynamin-1 PRD (prolinerich domain). While key basic residues in the PRD were vital to the activation of pacsin-1, we found that pacsin-1's membrane sculpting potential depends on membrane properties such as curvature and bending rigidity. In separate in vitro investigations of HIV-1 Gag-membrane associations, we found that lipid composition strongly affects Gag membrane affinity. These results highlighted the complex nature of protein-mediated membrane remodeling processes, which requires understanding both protein function and lipid phase behavior.
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2013-05-26
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model membranes; BAR domain; patterned phases
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Feigenson, Gerald W
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Sondermann, Holger
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Baird, Barbara Ann
Brown, William J
Degree Discipline
Biochemistry
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Ph. D., Biochemistry
Degree Level
Doctor of Philosophy
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Government Document
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dissertation or thesis
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