Detection of Osmotic Shock-Induced Extracellular Nucleotide Release with a Genetically Encoded Fluorescent Sensor of ADP and ATP

Purinergic signals, such as extracellular adenosine triphosphate (ATP) and adenosine diphosphate (ADP), mediate intercellular communication and stress responses throughout mammalian tissues, but the dynamics of their release and clearance are still not well understood. Although physiochemical methods provide important insight into physiology, genetically encoded optical sensors have proven particularly powerful in the quantification of signaling in live specimens. Indeed, genetically encoded luminescent and fluorescent sensors provide new insights into ATP-mediated purinergic signaling. However, new tools to detect extracellular ADP are still required. To this end, in this study, we use protein engineering to generate a new genetically encoded sensor that employs a high-affinity bacterial ADP-binding protein and reports a change in occupancy with a change in the Förster-type resonance energy transfer (FRET) between cyan and yellow fluorescent proteins. We characterize the sensor in both protein solution studies, as well as live-cell microscopy. This new sensor responds to nanomolar and micromolar concentrations of ADP and ATP in solution, respectively, and in principle it is the first fully-genetically encoded sensor with sufficiently high affinity for ADP to detect low levels of extracellular ADP. Furthermore, we demonstrate that tethering the sensor to the cell surface enables the detection of physiologically relevant nucleotide release induced by hypoosmotic shock as a model of tissue edema. Thus, we provide a new tool to study purinergic signaling that can be used across genetically tractable model systems

Engineering Fluorescent Protein Biosensors for Interrogating Biologically Relevant Chemical Species

Fluorescent proteins and the biosensors created with them have been used extensively to monitor chemical species inside and outside of the cell. They have been used to increase our knowledge of cellular function in normal and diseased states. Fluorescent biosensors are advantageous because they can be genetically encoded, do not require exogenous reagents, and can be quantitative. Fluorescent biosensors are also able to measure analytes with high spatial and temporal resolutions, enabling measurements at the scale of physiological events. In this thesis efforts have made to increase the available fluorescent biosensor tools for imaging cellular events. This work includes creation of new sensors for two molecules not yet detectable via fluorescent protein biosensor, acetylcholine and adenosine diphosphate. Efforts were also made to improve the current available biosensors for adenosine triphosphate and cellular redox, to make them more compatible with multiplex and deep tissue imaging. Here I present my work to design, characterize and utilize these fluorescent biosensors

Cancer and pH Dynamics: Transcriptional Regulation, Proteostasis, and the Need for New Molecular Tools

An emerging hallmark of cancer cells is dysregulated pH dynamics. Recent work has suggested that dysregulated intracellular pH (pHi) dynamics enable diverse cancer cellular behaviors at the population level, including cell proliferation, cell migration and metastasis, evasion of apoptosis, and metabolic adaptation. However, the molecular mechanisms driving pH-dependent cancer-associated cell behaviors are largely unknown. In this review article, we explore recent literature suggesting pHi dynamics may play a causative role in regulating or reinforcing tumorigenic transcriptional and proteostatic changes at the molecular level, and discuss outcomes on tumorigenesis and tumor heterogeneity. Most of the data we discuss are population-level analyses; lack of single-cell data is driven by a lack of tools to experimentally change pHi with spatiotemporal control. Data is also sparse on how pHi dynamics play out in complex in vivo microenvironments. To address this need, at the end of this review, we cover recent advances for live-cell pHi measurement at single-cell resolution. We also discuss the essential role for tool development in revealing mechanisms by which pHi dynamics drive tumor initiation, progression, and metastasis