Date of Award

Spring 1-1-2014

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Amy E. Palmer

Second Advisor

Hang Hubert Yin

Abstract

Zinc is a micronutrient that is required for cellular growth, proliferation, and differentiation. Dozens of zinc transporters and zinc-buffering proteins regulate the intracellular concentration of zinc, and dysregulation of zinc homeostasis is implicated in the pathogenesis of several human diseases, including diabetes, cancer, and neurodegeneration. Even though zinc is important for many critical cellular processes, it is still unclear whether and how cells redistribute zinc in response to stimuli, and how changes in zinc affect signaling pathways. Our laboratory has developed several genetically-encoded, ratiometric, fluorescent sensors of zinc that can quantitatively report zinc concentrations in different organelles of individual living cells. In this work, I optimized and characterized genetically-encoded zinc sensors for two major applications: the estimation of mitochondrial zinc in multiple cell types and quantitative imaging of cytosolic zinc in insulin-secreting cells. I found that zinc is buffered in the mitochondrial matrix at sub-picomolar concentrations, which can vary significantly among different cell types. I also made the observation that zinc can modulate calcium signaling in glucose-stimulated insulin-secreting cells. In addition, I present a set of protocols for optimizing the use of genetically-encoded sensors in quantitative imaging, refined throughout the progress of experiments that generated the results of this thesis work. The development and application of new genetically-encoded zinc sensors enabled us to make novel observations that improve our understanding of the role of zinc in biology.

Included in

Biochemistry Commons

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