Date Available

12-6-2020

Year of Publication

2018

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Arts and Sciences

Department/School/Program

Chemistry

First Advisor

Dr. Christopher I. Richards

Abstract

Single molecule studies can provide information of biological molecules which otherwise is lost in ensemble studies. A wide variety of fluorescence-based techniques are utilized for single molecule studies. While these tools have been widely applied for imaging soluble proteins, single molecule studies of transmembrane proteins are much more complicated. A primary reason for this is that, unlike membrane proteins, soluble proteins can be easily isolated from the cellular environment. One approach to isolate membrane proteins into single molecule level involves a very low label expression of the protein in cells. However, cells generate background fluorescence leading to a very low signal to noise ratio. An alternative approach involves isolating membrane proteins in artificial membrane derived vesicles. This approach is limited to proteins which can be solubilized or stabilized in detergent solution. This intermediate step endangers the structural integrity of proteins with multiple subunits. Hence, we isolated transmembrane proteins into cell-derived vesicles which maintain the proteins in their physiological membrane without compromising their functional integrity. We studied the stoichiometric assembly of α3β4 nicotinic receptors which are pentameric receptor with possible stoichiometry of (α3)2(β4)3 and (α3)3(β4)2. We found that (α3)2(β4)3 is the predominant stoichiometry, and we have verified our finding with both single and double color experiments. We have also demonstrated that cell-derived vesicles can be utilized to study ligand receptor interactions.

Cell-derived vesicles generated from cellular preparations provide a method to study the overall structural and functional properties of membrane proteins. However, organelle specific information is not available in this approach. Alternatively, separating vesicles based on their original organelle could provide information on the assembly and trafficking of membrane proteins. For example, it has been hypothesized that nicotine acts as a pharmacological chaperone of α4β2 nicotinic receptors and nicotine alters the assembly of the nicotinic receptors towards the high sensitivity isoform in the ER. To validate this hypothesis, we isolated α4β2 nicotinic receptors located on vesicles derived from the ER and plasma membrane origins and utilized single molecule studies to determine the stoichiometric assembly of the receptor. The data suggested that the ER has a higher percentage of the low sensitivity isoform ((α4)3(β2)2) than the plasma membrane indicating that the high sensitivity isoform trafficked more efficiently to the cell surface. When nicotine was added, the distribution of nicotinic receptors changes in those compartments. In both the ER and plasma membrane, the percentage of high sensitivity isoform was greater than the sample without the presence of nicotine. The results suggested that nicotine altered the assembly of nicotinic receptors to form the high sensitivity isoform in the ER and the altered assembly trafficked to the plasma membrane efficiently increasing the ratio of this isoform in the plasma membrane.

The cell derived vesicles we utilized to isolate single receptors are structurally similar to liposomes, an FDA approved drug delivery system, which is spherical vesicles composed of at least one lipid bilayer. Hence, cell-derived vesicles possess potential to be utilized as drug delivery vehicles. I explored the applicability of cell-derived vesicles as general delivery vehicles to cultured cells. Additionally, we implanted xenografts into immune compromised nude mice and prepared cell derived vesicles labeled with dye molecules. The vesicles were injected in a mouse containing a xenograft to monitor whether these vesicles can reach to the xenograft. Our data suggested that cell-derived vesicles can successfully reach the xenograft and thus have potential to be utilized as a drug delivery vehicle.

Digital Object Identifier (DOI)

https://doi.org/10.13023/ETD.2018.217

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