Development And Application Of Genetically Encoded Fluorescent Indicators For Metabolite And Ions

Cell metabolism is the foundation of all vital processes.However,it has always been difficult to systematacially elaborate the complex integration of intracellular metabolic pathway and the underlying regulation mechanism.Genetically encoded fluorescent indicators have become the efficient approach to solve this problem due to its powerful ability to real-time visualization of cell metabolism.In this thesis,we developed three series of novel fluorescent reporters using different design strategies and illustrated their utilization in individual intact cells and in vivo.NADPH provides the reducing equivalents for biosynthetic reactions and antioxidant functions;however,spatiotemporal detection of NADPH metabolism in living cells remains technically challenging.Herein,we develop and characterize ratiometric,pH-resistant,genetically encoded fluorescent indicators for NADPH(iNap sensors)with various affinities and wide dynamic range.The iNap sensors permitted quantification of cytosolic and mitochondrial NADPH pools that were controlled by cytosolic NAD+ kinase levels,and revealed unexpected cellular NADPH dynamics under oxidative stress depending on glucose availability.We found that mammalian cells have a strong tendency to maintain physiological NADPH homeostasis,which is regulated by glucose-6-phosphate dehydrogenase(G6PD)and AMP kinase(AMPK).Unexpectedly,a physiological steroid hormone,dehydroepiandrosterone(DHEA),a well-known G6PD inhibitor,actually protected against oxidative stress-induced NADPH oxidation and cell death by activating the AMPK pathway and rerouting NADPH consumption to antioxidant functions,but did so only in the glucose-fed state;in the absence of glucose,DHEA inhibition of G6PD depleted the NADPH pool.In addition,the iNap sensors have been demonstrated to be valuable tools for monitoring NADPH fluctuations during the activation of macrophage cells or wound response in vivo,which would gain new insights into cell metabolism.Compartmentalized distribution of pH is a vital parameter for analyzing the activities of live cells.In this study,we present pHluorin3,an ultra-sensitive,ratiometric pH indicator with enhanced brightness and expanded dynamic range.This indicator allows the quantification of local pH in different subcellular organelles and the visualization of rapid reversible acidification and alkalization in HeLa cells at high spatiotemporal resolutions.In conjunction with the red hydrogen peroxide biosensor,we found the onset of intracellular H2O2 clearance is correlated with the onset of pH acidification when the cells suffer from oxidation crisis.This ultra-sensitive optical tool provides additional details of pH fluctuation in previously inaccessible sites.Engineered fluorescent indicators for visualizing mercury ion are powerful tools to illustrate the serious intracellular toxicity.However,it is difficult for the sensitive and specific detection of mercury in live cells.Here,we developed two improved green ratiometric indicators,as well as the first red intensiometric indicator by inserting circularly permuted fluorescent protein(cpFP)into a mercury highly specific repressor(MerR).These chimeras,named GEIMs,enable real-time detect mercury dynamics in solutions,bacteria,subcellular organelles of mammalian cells and zebrafish.In conjunction with HyPer,we found the entry of mercury would trigger oxidative stress in cytosol and mitochondria at single cell level.GEIMs would paint the landscape of mercury toxicity to cell functions.In summary,we described the development and application of three kinds of robust optical biosensors,which allowed to assess spatiotemporal metabolic dynamics and unravel the elaborate regulation mechanism.We hope that this dissertation would draw some inspiration to the research of fluorescent probes and cell metabolism area.