Studies of Mechanisms of Lipid Binding by the Adaptor Protein Complex AP-3 using Single and Bulk Molecule Fluorescence in vitro Assays. Open Access

Gans, Jared Henry (2010)

Permanent URL: https://etd.library.emory.edu/concern/etds/g732d9285?locale=en
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Abstract

Studies of Mechanisms of Lipid Binding by the Adaptor Protein Complex AP-3 using Single and Bulk Molecule Fluorescence in vitro Assays.
By Jared Henry Gans

The mechanism behind guidance of intracellular vesicles is an active field of study in Cell Biology. Protein-lipid binding serves an important method in this guidance. Specifically, adaptor proteins complexes (APs) such as AP-3 and the phospholipids they bind and concentrate in vesicles have been shown to be important in the specificity of this mechanism. However, the dynamics of vesicle budding and the role of the different components, in particular lipids, on this process have not been directly studied. Reported here is the study of these protein-lipid interactions through in vitro model systems. The interactions of streptavidin with biotinylated phosphoethanolamine and of AP-3 with fluorescently labeled phosphatidylinositol 4-phosphate were tracked at the single molecule and bulk levels within solid supported lipid bilayers of phosphocholine on quartz slides. Their interactions with their respective proteins were characterized by changes in their diffusion coefficients as measured by Single Particle Tracking and Fluorescence Correlation Spectroscopy. Additionally, an antibody immobilization assay for protein binding imaged by Total Internal Reflection Fluorescence Microscopy was developed. While no change in phosphatidylinositol diffusion was observed in the presence of AP-3 for any of the systems, a distinct and unexpected drop in diffusion was found for the biotin-streptavidin system. This effect proved dependent on solution viscosity and illustrates a novel drag force on the bound protein slowing down lipid diffusion within the bilayer.

Table of Contents

Table of Contents
I. Introduction...1

a. Model Membranes...1
b. Single Molecule and Bulk Studies of Lipid Supports...3
c. Adapter Complexes...7

II. Materials and Methods...10

a. Lipid Materials...10
b. AP-3 Purification...10
c. Slides...11
d. SUV Formation...12
e. SLB Formation...12
f. Biotinylated SLBs...13
g. TIRFM...13
h. FCS...14
i. Viscosity Tests of Biotinylated SLBs...14
j. AP-3 Immobilization...15
k. Data Analysis...16

III. Results...16

a. TIRFM of Biotinylated Bilayers...16
b. FCS of Biotinylated Bilayers and Effects of Viscosity...17
c. TIRFM of PIP systems with and without AP-3...17
d. FCS of PI4P, RhPE...18
e. Nonspecific Binding Check for Antibody Immobilization...18
f. AP-3 Binding with Antibodies...18

IV. Discussion...19

a. TIRFM and FCS of Biotinylated SLBs...19
b. TIRFM of Labeled Phosphoinositides PI4P and PI(4,5)P2...20
c. TIRFM of AP-3 and Labeled PI4P...21
d. AP-3 Immobilization...21

V. Conclusions...22
VI. References...24
VII. Figures and Tables...29


List of Tables and Figures
I. Figure 1...29
II. Figure 2...30
III. Figure 3...31
IV. Figure 4...32
V. Figure 5...33
VI. Figure 6...34
VII. Figure 7...35
VIII. Figure 8...36
IX. Figure 9...37
X. Figure 10...38
XI. Figure 11...39
XII. Figure 12...40
XIII. Figure 13...41
XIV. Figure 14...42
XV. Figure 15...43
XVI. Figure 16...44
XVII. Figure 17...45
XVIII. Table 1...46
XIX. Figure 18...47

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