GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this ...GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this study, we determined crystal structures of CaZ＋-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its par- ent version GCaMP5G. Our analyses reveal the structural basis for the outperformance of this newly developed Ca2＋ indicator. Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and in- creased fluorescence intensity common to all three versions of GCaMP6. Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity, maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling. Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.展开更多
The complex signaling mechanisms in red blood cells(RBCs)enable them to adapt to physiological stresses such as exposure to low O_(2)levels,metabolic demands,oxidative stress,and shear stress.Since Ca^(2+)is a crucial...The complex signaling mechanisms in red blood cells(RBCs)enable them to adapt to physiological stresses such as exposure to low O_(2)levels,metabolic demands,oxidative stress,and shear stress.Since Ca^(2+)is a crucial determinant of RBC fate,various ion channels,pumps,and exchangers regulate the delicate balance of Ca^(2+)influx and efflux in RBCs.Elevated intracellular Ca^(2+)can activate processes such as membrane phospholipid scrambling and alter RBC deformability,which is essential for effective capillary transit.However,the dynamic information about Ca^(2+)regulation in RBCs is limited.Although static mapping and bioanalytical methods have been utilized,the absence of a nucleus and the presence of hemoglobin create challenges for real-time probing of RBC signaling,necessitating innovative approaches.This work introduces a synthetic chemistry−recombinant protein-based strategy to assemble sensors at genetically intact healthy human RBC surfaces for measuring dynamic signaling.Using this approach,we measured autocrine regulation of RBC Ca^(2+)influx in response to low O_(2)tension-induced ATP release.The study also explores the utilization of synthetic glycosylphosphatidylinositol(GPI)anchor mimics and sortagging for targeting sensors to the surfaces of primary as well as immortalized cells.This demonstrated the wide applicability of this approach to probe dynamic signaling in intact cells.展开更多
基金supported in part by an International Early Career Scientist grant from the Howard Hughes Medical Institute to Shao Fenggrant from the National Basic Research Program of China (2011CB910304 and 2011CB911103) to Wang DaChengNational Natural Science Foundation of China (31100535) to Ding JingJin
文摘GCaMP is one of the most widely used calcium indicators in neuronal imaging and calcium cell biology. The newly developed GCaMP6 shows superior brightness and ultrasensitivity to calcium concentration change. In this study, we determined crystal structures of CaZ＋-bound GCaMP6 monomer and dimer and presented detailed structural analyses in comparison with its par- ent version GCaMP5G. Our analyses reveal the structural basis for the outperformance of this newly developed Ca2＋ indicator. Three substitution mutations and the resulting changes of local structure and interaction explain the ultrasensitivity and in- creased fluorescence intensity common to all three versions of GCaMP6. Each particular substitution in the three GCaMP6 is also structurally consistent with their differential sensitivity and intensity, maximizing the potential of using GCaMP6 in solving diverse problems in neuronal research and calcium signaling. Our studies shall also be beneficial to further structure-guided optimization of GCaMP and facilitate the design of novel calcium indicators.
文摘The complex signaling mechanisms in red blood cells(RBCs)enable them to adapt to physiological stresses such as exposure to low O_(2)levels,metabolic demands,oxidative stress,and shear stress.Since Ca^(2+)is a crucial determinant of RBC fate,various ion channels,pumps,and exchangers regulate the delicate balance of Ca^(2+)influx and efflux in RBCs.Elevated intracellular Ca^(2+)can activate processes such as membrane phospholipid scrambling and alter RBC deformability,which is essential for effective capillary transit.However,the dynamic information about Ca^(2+)regulation in RBCs is limited.Although static mapping and bioanalytical methods have been utilized,the absence of a nucleus and the presence of hemoglobin create challenges for real-time probing of RBC signaling,necessitating innovative approaches.This work introduces a synthetic chemistry−recombinant protein-based strategy to assemble sensors at genetically intact healthy human RBC surfaces for measuring dynamic signaling.Using this approach,we measured autocrine regulation of RBC Ca^(2+)influx in response to low O_(2)tension-induced ATP release.The study also explores the utilization of synthetic glycosylphosphatidylinositol(GPI)anchor mimics and sortagging for targeting sensors to the surfaces of primary as well as immortalized cells.This demonstrated the wide applicability of this approach to probe dynamic signaling in intact cells.
