A molecular sensor to quantify the localization of proteins, DNA and nanoparticles in cells

Abstract

Intracellular trafficking governs receptor signaling, pathogenesis, immune responses and fate of nanomedicines. These processes are typically tracked by observing colocalization of fluorescent markers using confocal microscopy. However, this method is low throughput, limited by the resolution of microscopy, and can miss fleeting interactions. To address this, we developed a localization sensor composed of a quenched SNAP-tag substrate (SNAP _Switch) that can be conjugated to biomolecules using click chemistry. SNAP _Switch enables quantitative detection of trafficking to locations of interest within live cells using flow cytometry. Using SNAP _Switch, we followed the trafficking of DNA complexes from endosomes into the cytosol and nucleus. We show that antibodies against the transferrin or hyaluronan receptor are initially sorted into different compartments following endocytosis. In addition, we can resolve which side of the cellular membrane material was located. These results demonstrate SNAP _Switch is a high-throughput and broadly applicable tool to quantitatively track localization of materials in cells.

Abstract

Determining the trafficking of intracellular material is commonly done by colocalisation analysis using microscopy. Here the authors monitor trafficking of select cargo by measuring the conversion of quenched SNAP-tag substrates by subcellularly-localised SNAP-tag and detection by flow cytometry.