Engineering the ChlorON Series: Turn-On Fluorescent Protein Sensors for Imaging Labile Chloride in Living Cells

Abstract

Beyond its role as the "queen of electrolytes", chloride can also serve as an allosteric regulator or even a signaling ion. To illuminate this essential anion across such a spectrum of biological processes, researchers have relied on fluorescence imaging with genetically encoded sensors. In large part, these have been derived from the green fluorescent protein found in the jellyfish Aequorea victoria. However, a standalone sensor with a turn-on intensiometric response at physiological pH has yet to be reported. Here, we address this technology gap by building on our discovery of the anion-sensitive fluorescent protein mNeonGreen (mNG). The targeted engineering of two non-coordinating residues, namely K143 and R195, in the chloride binding pocket of mNG coupled with an anion walking screening and selection strategy resulted in the ChlorON sensors: ChlorON-1 (K143W/R195L), ChlorON-2 (K143R/R195I), and ChlorON-3 (K143R/R195L). In vitro spectroscopy revealed that all three sensors display a robust turn-on fluorescence response to chloride (20-to 45-fold) across a wide range of affinities (K d ≈ 30−285 mM). We further showcase how this unique sensing mechanism can be exploited to directly image labile chloride transport with spatial and temporal resolution in a cell model overexpressing the cystic fibrosis transmembrane conductance regulator. Building from this initial demonstration, we anticipate that the ChlorON technology will have broad utility, accelerating the path forward for fundamental and translational aspects of chloride biology.

White Light Emissive Tetraphenylethene Molecular Cage-Based Hybrid Nanoparticles for Intracellular Long-Term Imaging

“…Fluorescence bioimaging is one of the main driving forces in determining the dynamics and functions of bioprocesses in live cells, due to its advantages of low toxicity, high spatiotemporal resolution, and utilization of an inexpensive instrument. − To date, many types of fluorescent probes, such as fluorescent organics, − fluorescent proteins, , inorganic nanoparticles, , and semiconductor polymer nanoparticles, have been developed and widely used in bioimaging. Comparatively, among these fluorescent probes, fluorescent organic probes, including the small organic probes − and fluorescent organic nanoparticles, − attracted more attention because of their controllable synthesis, stable luminescence, good biocompatibility, sensitive responsiveness, and high signal-to-noise ratio.…”