Lipid Bilayer Membrane Organization
Open Access
- Author:
- Heetderks, Julia J.
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- May 30, 2007
- Committee Members:
- Paul S Weiss, Committee Chair/Co-Chair
Andrew Ewing, Committee Member
David Lawrence Allara, Committee Member
William O Hancock, Committee Member
Anne M Andrews, Committee Member - Keywords:
- FRET
membrane
bilayer
lipid
fluorescent - Abstract:
- Lipid membrane structure affects many biological processes; understanding the structure and organization of lipid bilayers is important to the development of new medical treatments and for understanding how cell membranes function. Giant unilamellar vesicles (GUVs) are utilized as lipid membrane models. Fluorescence microscopy is used to study the inherent order of lipids in membranes, the effects of dyes on the perceived lipid structures, and the movement of particles through flow cells. Ultrafast time-resolved measurements of fluorescence intensity are also used to probe membrane dynamics and dye/lipid interactions in the bilayers. Complex membrane structures were found in dilauroylphosphatidylcholine/ dipalmitoylphosphatidylcholine GUVs. Multiple labels used in combination highlighted gradients of lipid phase properties through the gel phase of dual-phase vesicles. This is in conflict with the previous research suggesting that phases are uniform, with homogeneous gel and fluid domains. Dyes may also affect the kinetics of lipid reorganization during domain formation in GUVs. The effects of several dye combinations were measured through determination of gel-phase melting temperatures at different times after GUV formation, as lipid organization was changing. Fluorescently labeled lipid N-(fluorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine had a larger effect on lipid rearrangement kinetics than carbocyanine dyes. Fluorescence lifetime and fluorescence polarization anisotropy measurements also probe dynamics of lipid and dye movement within dual-phase membranes. Spectroscopic measurements of carbocyanine dyes within fluid and gel domains were determined; these will aid later research with the same dyes in cell membranes. The values also indicated heterogeneity of gel-phase domains, and the differences in dye and lipid behavior at elevated dye concentrations. A new design for a flow cell was demonstrated, to be used in later research of adhesion properties between cells or GUVs and different chemically functionalized surfaces. This design utilizes multiple parallel flow channels of different sizes, which allows rapid testing of the effects of several different shear forces on the adhesion of GUVs (or cells) to surfaces.