Sculpting membranes into dynamic, curved shapes is central to intracellular cargo trafficking and other cellular functions. However, generation of membrane curvature during trafficking involves lipids and membrane-associated proteins; current mechanisms focus on creating rigid cages, curved scaffolds, or membrane surface area changes by proteins. This dissertation provides an alternative mechanistic example for the control of membrane deformations, involving modulation of membrane material properties. Sar1, a GTPase of the COPII family, regulates vesicle trafficking from the endoplasmic reticulum. We find that Sar1p lowers the rigidity of the lipid bilayer membrane to which it binds. We examine the behavior of Saccharomyces cerevisiae Sar1 (Sar1p) and Homo sapiens paralogs of Sar1 (Sar1A and Sar1B). Like Sar1p, human Sar1s lower membrane rigidity. Unlike Sar1p, the rigidity is not a monotonically decreasing function of concentration. At high concentrations, we find increased bending rigidity and decreased protein mobility. These features imply a model in which human Sar1 clustering governs membrane mechanical properties.
Membrane rigidity measurements remain rare, however, and show a large variance, a situation that can be addressed by improving techniques and comparing disparate techniques applied to the same systems. I introduce applying selective plane illumination microscopy (SPIM) to image thermal fluctuations of giant vesicles. SPIM's optical sectioning enables high-speed fluorescence imaging of freely suspended vesicles and quantification of edge localization precision, yielding robust fluctuation spectra and rigidity estimates. For lipid-only membranes and membranes bound by the intracellular trafficking protein Sar1p, we show rigidity values from giant
unilamellar vesicle fluctuations in close agreement with those derived from our independent assay based on membrane tether pulling. We also show that a model of homogeneous quasi-spherical vesicles poorly fits fluctuation spectra of vesicles bound by Sar1A at high concentrations, suggesting that SPIM-based analysis can offer insights into spatially inhomogeneous properties.
I conclude by discussing our current work on amphipathic alpha helices, their ability to reduce membrane rigidity, and our hopes to create artificial helical structures capable of mimicking trafficking systems. Supplemental videos represent membrane disintegration with Sar1p and fluctuations of membrane only and Sar1p incubated vesicles.
This dissertation contains previously published co-authored material.
| Identifer | oai:union.ndltd.org:uoregon.edu/oai:scholarsbank.uoregon.edu:1794/18311 |
| Date | 29 September 2014 |
| Creators | Loftus, Andrew |
| Contributors | DeRose, Victoria |
| Publisher | University of Oregon |
| Source Sets | University of Oregon |
| Language | en_US |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Rights | All Rights Reserved. |
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