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
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.
