Abstract
The fate of a cell is intricately hinges upon a complex network of intertwined signals, where even the slightest disruption can steer it towards a diseased state. The occurrence, location, and intensity of these signals are tightly regulated, and any slight deviation in these parameters can result in a different cellular response to the same external stimuli. Tools capable of capturing these dynamics with the spatial and temporal resolution are required for comprehensive understanding. Therefore, there is a need for better tools for unravelling the intricacies of complex chemical signals in cells.
To address this critical gap, this dissertation entails the development of improved fluorescent protein-based biochemical tools across three separate projects. In the first project, genetically encoded green fluorescent indicators were engineered for monitoring secretion of signaling chemicals. HP-GRISZ, a high-performance genetically encoded green fluorescent indicator for monitoring secreted zinc, was successfully developed. This tool offers a reliable approach to image the secretion of zinc into the extracellular space in real time in live cells with single-cell resolution. The development of this tool will advance the knowledge of the functional role of extracellular unbound free zinc in the brain, pancreas, and other zinc-secreting cells.
In the second project, I engineered and characterized several red-shift circular-permutated fluorescent proteins (cpFPs) incorporated with 3-aminotyrosine (aY), a nonconical amino acid (ncAA). By fine-tuning expression conditions and introducing strategic mutations, I have improved the expression and brightness of these red-emitting proteins. This study builds upon previous work and offers further guidance on utilizing aY as a strategy for red-shifting fluorescent protein (FP) and FP-based sensor development.
In my third project, I have uncovered four optimal donor-dark acceptor FP pairs with different color palettes. These FP pairs are suitable for constructing pseudo-single-color fluorescence lifetime imaging microscopy probes (FLIMPs). Leveraging these pairs, novel sensors targeting key kinases in the insulin resistance pathway, such as Erk, Akt, and AMP kinase, were developed. This suite of sensors will contribute to our understanding of the interplay between the kinases in insulin resistance pathway.
Collectively, these projects created FP-based tools with improved photoproperties, enhanced dynamic range, and expanded color spectra. By providing higher performance and resolution, these tools open new avenues for unraveling the complexities of cell biology and advancing our understanding of the cell biology and disease.
