Gasotransmitter Regulation of Phosphatase Activity in Live Cells Studied by Three‐Channel Imaging Correlation

Abstract

Enzyme activity in live cells is dynamically regulated by small‐molecule transmitters for maintaining normal physiological functions. A few probes have been devised to measure intracellular enzyme activities by fluorescent imaging, but the study of the regulation of enzyme activity via gasotransmitters in situ remains a long‐standing challenge. Herein, we report a three‐channel imaging correlation by a single dual‐reactive fluorescent probe to measure the dependence of phosphatase activity on the H2S level in cells. The two sites of the probe reactive to H2S and phosphatase individually produce blue and green fluorescent responses, respectively, and resonance energy transfer can be triggered by their coexistence. Fluorescent analysis based on the three‐channel imaging correlation shows that cells have an ideal level of H2S to promote phosphatase activity up to its maximum. Significantly, a slight deviation from this H2S level leads to a sharp decrease of phosphatase activity. The discovery further strengthens our understanding of the importance of H2S in cellular signaling and in various human diseases.

Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents

“…With this desire in mind, Yu et al reported a new FRET system in 2019 that could be used for monitoring DC m levels through readout of a ratiometric fluorescence signal. 40 The FRET system was constructed using a carbazole linked to a benzyl chloride functionalized pyridinium group (48) as the fluorescent donor and a quinoline derivative providing for an extended D-p-A structure (49) as the fluorescence acceptor (Fig. 30).…”

“…30). Initially, the fluorescent donor (48) was immobilized to the mitochondria by means of covalent bonds formed between the benzyl chloride moiety of 48 and endogenous protein thiol groups localised in the mitochondria. In this state, 48 displayed a fluorescence signal (green channel, 510-550 nm) under excitation at 405 nm, irrespective of changes in the DC m .…”

“…37) that was designed to allow investigations of how phosphatase activity in cells depend on H 2 S levels. 48 In analogy to other dualresponsive FRET systems, a coumarin/rhodol dyad exists at the core of this FRET system. Probe 57 incorporated an azide group as the recognition unit for H 2 S. It also contains a phosphate group that was expected to serve as a recognition site for the phosphatase enzyme being targeted for study.…”

Small-molecule fluorescent probes for imaging gaseous signaling molecules: current progress and future implications

“…In 2019, Zhang constructed a two-response site uorescent probe for simultaneously sensing H 2 S and phosphatase via two independent uorescence signal channels (probe 60). 79 The H 2 S-responsive uorophore coumarin derivative (emission at 445 nm) was linked to rhodol (emission at 545 nm) which served as a phosphate-reactive dye. More importantly, through combination with the FRET signal between rhodol and coumarin uorescence signals, the authors established a three-channel strategy to detect and image H 2 S and phosphatase activity and investigate the correlation between them in living cells.…”

A Multi‐responsive Fluorescent Probe Reveals Mitochondrial Nucleoprotein Dynamics with Reactive Oxygen Species Regulation through Super‐resolution Imaging

“…[6] More recently, super-resolution nanoscopy aided by membrane-penetrating inorganic carbon dots has achieved dynamical tracking of nucleic acids in cells and C. elegans. [7] In parallel, al arge number of fluorescent probes have been developed to detect ROS, [8] enzymes [9] and other biological analytes [10] in live cells, but most of them exhibit optical properties undesirable for multicolor super-resolution imaging such as limited by small Stokes shift and photostability. [11] Furthermore,these imaging agents all have different chemical properties and intracellular biodistributions,m aking multi-analyte imaging in the same cell compartment extremely difficult.…”