A modular probe can be programmed to travel to a precise cellular destination.
Diphtheria bacteria infecting Caernohabditis elegans worms (pictured) co-opt one of the worms’ enzymes to make a toxin, according to a technology that can pick out even low levels of enzymes in cells. Credit: Sinclair Stammers/SPL
A self-guided fluorescent probe can reveal minuscule amounts of enzymes hidden in the farthest reaches of a living cell.
The adaptable probe — designed by Yamuna Krishnan and her colleagues at the University of Chicago in Illinois — includes both ‘sensing’ and ‘targeting’ modules. In the team’s prototype probe, the sensor glows when it encounters a chemical reaction called a disulfide exchange. The targeting module consists of a particular DNA segment; this guides the probe to cell structures known as endosomes that function as internal cargo compartments.
When added to the cells of Caenorhabditis elegans worms, the probe ferreted out two enzymes that catalyse disulfide exchange inside endosomes. The researchers deployed the probe in worms infected with the diphtheria bacterium (Corynebacterium diphtheria) and found that the bacterium hijacks one of these enzymes to produce its powerful toxin.
The probe’s sensing and targeting modules can be customized to hunt for various enzymes in a vast assortment of cellular structures, the scientists say.
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DNA nanodevices map enzymatic activity in organelles
Abstract
Cellular reporters of enzyme activity are based on either fluorescent proteins or small molecules. Such reporters provide information corresponding to wherever inside cells the enzyme is maximally active and preclude minor populations present in subcellular compartments. Here we describe a chemical imaging strategy to selectively interrogate minor, subcellular pools of enzymatic activity. This new technology confines the detection chemistry to a designated organelle, enabling imaging of enzymatic cleavage exclusively within the organelle. We have thus quantitatively mapped disulfide reduction exclusively in endosomes in Caenorhabditis elegans and identified that exchange is mediated by minor populations of the enzymes PDI-3 and TRX-1 resident in endosomes. Impeding intra-endosomal disulfide reduction by knocking down TRX-1 protects nematodes from infection by Corynebacterium diphtheriae, revealing the importance of this minor pool of endosomal TRX-1. TRX-1 also mediates endosomal disulfide reduction in human cells. A range of enzymatic cleavage reactions in organelles are amenable to analysis by this new reporter strategy.
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