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Access Type

WSU Access

Date of Award

January 2018

Degree Type

Dissertation

Degree Name

Ph.D.

Department

Physics and Astronomy

First Advisor

Christopher V. Kelly

Abstract

The curvature of biological membranes at the nanometer scale is critically important for vesicle trafficking, organelle morphology, and disease propagation. Many proteins and lipids interact with diverse curvature sensing and curvature generating mechanisms. Deciphering the molecular mechanisms of toxin-membrane interactions has been limited by the resolution and drawbacks of conventional experimental techniques. This study reveals the inherent membrane bending capability of cholera toxin subunit B (CTxB) through the development and implementation of Polarized Localization Microscopy (PLM). PLM is a pointillist optical imaging technique for the detection of nanoscale membrane curvature in correlation with single-molecule dynamics and molecular sorting.

PLM combines polarized total internal reflection fluorescence microscopy and fluorescence localization microscopy to reveal membrane orientation without reducing localization precision by point spread function manipulation. Further, membrane curvature detection with PLM requires ≤19% of the localization density required with 3D fluorescence localization microscopy (e.g., PALM or STORM). Engineered hemispherical membrane curvature with varying radii of 24, 51, and 70 nm were detected with PLM while surrounded by planar supported lipid bilayers. Nanoscale membrane bud growth was spontaneously induced by CTxB on otherwise planar, quasi-one component lipid bilayers, revealing a mechanism of cholera immobilization and cellular internalization. The single lipid and single protein trajectories further quantified the effects of nanoscale membrane curvature and protein-lipid interactions. CTxB sorting to high membrane curvatures was detected and quantified.

Nanoscale membrane budding and tubulation was mainly driven by CTxB valency and structure. We demonstrated that varying either GM1 or CTxB concentrations on the membrane affects the budding structures. The number of crosslinked GM1s to a single CTxB affected the toxin behavior and mechanism on the membrane. Changing the lipid structure altered the bending mechanism and the eventual size and density of induced buds. Through future incorporation of single-particle tracking and live cells, PLM is poised to image the diverse molecular mechanisms that regulate nanoscale membrane bending.

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