Abstract
The fluorescence imaging toolkit has continuously accelerated biomedical research. While a number of fluorescent biosensors are now available for studying the spatiotemporal regulation of signaling networks, it remains challenging to achieve multiplexed sensor imaging and imaging in more complex biological systems such as live animals.
Although most Zn2+ ions in our bodies are immobilized within proteins, less tightly bound Zn2+ has emerged as a key modulator in biological systems. In the brain, Zn2+ is concentrated into presynaptic vesicles and released into synaptic clefts along with other neurotransmitters such as glutamate. Synaptically released Zn2+ modulates brain excitability through its interaction with surface receptors of neurons. In the pancreas, Zn2+ secretion from β-cells in islets is an indicator of insulin secretion and the functional potency of islets. Previous studies have reported a number of genetically encoded Zn2+ indicators (GEZIs), which have been widely used to monitor Zn2+ in the cytosol and intracellular organelles. However, it is challenging to localize existing GEZIs to the extracellular space to detect secreted Zn2+. In the second chapter, I describe two photostable, green fluorescent protein (GFP) based indicators, ZIBG1 and ZIBG2, which respond to Zn2+ selectively and have affinities suited for detecting Zn2+ secretion from intracellular vesicles. In particular, ZIBG2 can be effectively targeted to the extracellular side of plasma membrane. We applied ZIBG2 to monitoring glucose-induced dynamic Zn2+ secretion from human pancreatic islets. In addition, we developed novel GEZIs with fluorescence excitation and emission in the far-red region of the visible spectrum. We expect these new tools will be particularly useful for in vivo imaging due to reduced phototoxicity and increased deep tissue penetration.
Imaging of serotonin, a crucial signaling monoamine playing important roles both within and outside the central nervous system, has been difficult. We fused a circularly permuted green fluorescent protein (cpGFP) with a serotonin-binding protein. Structure-guided mutagenesis and library screening yielded Green Genetically Encoded Serotonin Sensors (G-GESSs). G-GESS1 has proven to be a robust indicator for visualizing serotonin dynamics in vitro as well as in live cells and tissue. To validate the G-GESS fluorescent biosensors in complex biological systems, we applied G-GESS1 in brain tissue imaging and live mice imaging. We were able to capture the transient release of serotonin utilizing G-GESS1. Research is ongoing to continue the characterization of G-GESS1.
Compared to green fluorescent protein (GFP) based biosensors, red fluorescent protein (RFP) based biosensors are inherently advantageous because of reduced phototoxicity, decreased autofluorescence, and enhanced tissue penetration. However, there is a limited choice of RFP-based biosensors and development of each biosensor requires significant effort. In the fourth chapter, we describe a general and convenient method, which uses the genetically encoded amino acid, 3-aminotyrosine (aY), to convert GFPs and GFP- based biosensors into red.
