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Chemogenetic tools are recombinant enzymes that can be targeted to specific organelles and tissues. The provision or removal of the enzyme substrate permits control of its biochemical activities. Yeast-derived enzyme D-amino acid oxidase (DAAO) represents the first of its kind for a substrate-based chemogenetic approach to modulate H2O2 concentrations within cells. Combining these powerful enzymes with multiparametric imaging methods exploiting genetically encoded biosensors has opened new lines of investigations in life sciences. In recent years, the chemogenetic DAAO approach has proven beneficial to establish a new role for (patho)physiological oxidative stress on redoxdependent signaling and metabolic pathways in cultured cells and animal model systems. This mini-review covers established or emerging methods and assesses newer approaches exploiting chemogenetic tools combined with genetically encoded biosensors. [ABSTRACT FROM AUTHOR]

In this study, we introduce a new separation of phases-based activity reporter of kinase (SPARK) for AMP-activated kinase (AMPK), named AMPK-SPARK, which reports the AMPK activation by forming bright fluorescent clusters. Furthermore, we introduce a dual reporter system, named GCaMP-AMPK-SPARK, by incorporating a single-fluorescent protein (FP)-based Ca 2+ biosensor, GCaMP6f, into our initial design, enabling simultaneous monitoring of Ca 2+ levels and AMPK activity. This system offers the essential quality of information by single-channel fluorescence microscopy without the need for coexpression of different biosensors and elaborate filter layouts to overcome spectral limitations. We used AMPK-SPARK to map endogenous AMPK activity in different cell types and visualized the dynamics of AMPK activation in response to various stimuli. Using GCaMP-AMPK-SPARK, we revealed cell-to-cell heterogeneities in AMPK activation by Ca 2+ mobilization. We anticipate that this dual reporter strategy can be employed to study the intricate interplays between different signaling networks and kinase activities.

Chemogenetic tools are recombinant enzymes that can be targeted to specific organelles and tissues. The provision or removal of the enzyme substrate permits control of its biochemical activities. Yeast-derived enzyme D-amino acid oxidase (DAAO) represents the first of its kind for a substrate-based chemogenetic approach to modulate H2O2 concentrations within cells. Combining these powerful enzymes with multiparametric imaging methods exploiting genetically encoded biosensors has opened new lines of investigations in life sciences. In recent years, the chemogenetic DAAO approach has proven beneficial to establish a new role for (patho)physiological oxidative stress on redox-dependent signaling and metabolic pathways in cultured cells and animal model systems. This mini-review covers established or emerging methods and assesses newer approaches exploiting chemogenetic tools combined with genetically encoded biosensors., (© 2022 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)