Single‐Molecule Imaging of Active Mitochondrial Nitroreductases Using a Photo‐Crosslinking Fluorescent Sensor

Abstract

Many biomacromolecules are known to cluster in microdomains with specific subcellular localization. In the case of enzymes, this clustering greatly defines their biological functions. Nitroreductases are enzymes capable of reducing nitro groups to amines, and play a role in detoxification and pro‐drug activation. Although nitroreductase activity has been detected in mammalian cells, the subcellular localization of this activity remains incompletely characterized. Here, we report a fluorescent probe that enables super‐resolved imaging of pools of nitroreductase activity within mitochondria. This probe is activated sequentially by nitroreductases and light to give a photo‐crosslinked adduct of active enzymes. In combination with a general photoactivatable marker of mitochondria, we performed two‐color, three‐dimensional, single‐molecule localization microscopy. These experiments allowed us to image the sub‐mitochondrial organization of microdomains of nitroreductase activity.

Rapid differentiation between bacterial infections and cancer using a near-infrared fluorogenic probe

“…This makes this enzyme family an attractive diagnostic target to detect the hypoxic status of cancer cells and potential bacterial infections. 17,18 Even though the detection of NTR activity within hypoxic tumours in vitro and in vivo using uorogenic methods has already been well described, chemical tools for the exploitation of NTRs as diagnostic markers for localizing bacterial infections are still scarce. 19 Moreover, NTR-activated uorogenic probes that can specically image live bacteria in vivo and do not accumulate at tumour sites have not been investigated thus far.…”

An Activatable and Switchable Nanoaggregate Probe for Detecting H2S and Its Application in Mice Brains

“…[8] Accordingly, investigators have paid an increasing attention to the activatable probes in recent years. [9][10][11][12] On the other hand, as the third multifunctional gasotransmitter, H 2 S has been recognized as an important signaling molecule in the cardiovascular and nervous systems. [13,14] There is not much to debate anymore about the significance on monitoring the internal level of H 2 S, [15][16][17][18] however, when the investigations nowadays come to the tissues or models in vivo, near-infrared (NIR) implements seem more favoured due to the strong penetrability and photothermal/photoacoustic effect.…”

Evolutionary aspects of the Viridiplantae nitroreductases

“…Up to date, not much is known about localization of the nitroreductase activity in mammalian cells. The recently developed fluorescent sensor allows to image submitochondrial localization of nitroreductase activity in live HEK 293 cells (human embryonic kidney cells) [53]. Similarly, bacterial nitroreductases, transformed into plants, have higher activity in case of chloroplast and mitochondrial localization [65].…”