Targeted sensors for investigating mobile Zinc in biology

Abstract

Chapter 1. Sensing Strategies for Detection of Mobile Zinc. Mobile zinc plays important physiological roles in areas such as the hippocampus, prostate, and pancreas. A better understanding of the distribution of intracellular mobile zinc could provide insight into the pathology of diseases including prostate cancer and Alzheimer's disease, both of which have been linked to abnormal mobile zinc levels. Accordingly, a palette of spatially-specific mobile zinc sensors is needed to investigate mobile zinc in important areas such as the mitochondria or zinc vesicles in neurons. Instead of repeating the de novo sensor design process for each target, a modular approach was developed to take advantage of the existing library of sensors and enable rapid creation of targeted probes. Chapter 2. Targeting Fluorescent Zinc Sensors to the Mitochondria Using Triphenylphosphonium Ions. Mitochondrial mobile zinc plays an important, although poorly understood, role in prostate cancer. To investigate the biology of zinc in the mitochondria, constructs incorporating fluorophores and the mitochondria-targeting triphenylphosphonium (TPP) moiety were tested and used to study sensor uptake and localization to the mitochondria. Chapter 3. Peptide-based Targeting of Fluorescent Zinc Sensors to the Plasma Membrane and Intracellular Targets in Live Cells. Peptide-sensor constructs were explored as a modular method of targeting sensors to specific locations. Taking advantage of the synthetic flexibility and targeting advantages of peptide-sensor constructs, mobile zinc sensors were directed to the exterior of the plasma membrane and to intracellular targets. A series of peptide-sensor constructs were created to further investigate fluorophore effects on sensor uptake and localization. Chapter 4. Improvement of Sensor Uptake, Localization, and Photophysics Through Acetylation of Fluorescein-based Sensors. A generalizable strategy for improving the uptake and photophysics of mobile zinc probes was explored. The ZPI-TPP sensor construct was modified by one-step acetylation of phenolic oxygen atoms. This modification dramatically improved photophysics and eliminated problems with membrane impermeability that would otherwise result in endosomal sequestration. The resulting DA-ZP1-TPP sensor was highly selective for zinc, resilient against cellular esterases, and, most importantly, was specifically targeted to the mitochondria.