Date of Award
Doctor of Philosophy (PhD)
The fluorescent toolbox is expanding, and Fluorogen Activating Protein Technology (FAP) is contributing by providing tools for collecting direct real-time, live cell protein trafficking measurements. Chapter I includes a brief overview of current fluorescent and fluorogenic labeling approaches. After the review of labeling methods, FAP Technology is presented as a well suited tool for cell surface protein trafficking investigations. Core strengths of the developed fluorogenic dyes are highlighted. This chapter is a discussion on using FAP labeling strategies to quantify cell surface protein translocation, particularly addressing the needs for a direct, quick, and high-throughput adaptable protein trafficking detection assay for GPCRs and Ion channels. Chapter I concludes with introducing new methods for assessing intracellular trafficking of protein pools in the cell. Chapter II entails the development of a new pH sensor tool that can quantify pH related changes associated with protein trafficking from the surface to the endosomal network, and how it was applied to observe B2AR differential agonist induced trafficking behavior. Chapter III describes the development of a high-throughput assay for identifying potential kinase targets that enhance VX-809 corrector ability to rescue the mutant ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR). The screen was based on a FAP approach that uses both surface and total protein fluorescence measurements collected from a plate reader. Chapter IV presents the current work on creating a lysosomal FAP construct that will function as a test-bed for monitoring protein trafficking related to lysosomal dysfunction.
Perkins, Lydia, "Built for the Dye-Way: Fluorogenic Tools for Protein Traffic" (2018). Dissertations. 1203.
Available for download on Saturday, May 30, 2020