Illuminating anions in biology with genetically encoded fluorescent biosensors

Abstract

Anions are critical to all life forms. Anions can be absorbed as nutrients or biosynthesized. Anions shape a spectrum of fundamental biological processes at the organismal, cellular, and subcellular scales. Genetically encoded fluorescent biosensors can capture anions in action across time and space dimensions with microscopy. The firsts of such technologies were reported more than 20 years for monoatomic chloride and polyatomic cAMP anions. However, the recent boom of anion biosensors illuminates the unknowns and opportunities that remain for toolmakers and end users to meet across the aisle to spur innovations in biosensor designs and applications for discovery anion biology. In this review, we will canvas progress made over the last three years for biologically relevant anions that are classified as halides, oxyanions, carboxylates, and nucleotides.

Figure 1.

The uptake, storage, and metabolism of anions is essential to all living systems. (a) Halides and oxyanions (green) are nutrients, signaling molecules, and regulators of homeostatic functions such as electrolyte balance and post-translational modifications. The carboxylates (blue) are metabolic and biosynthetic precursors serving as energy reservoirs, signaling anions, and potential biomarkers of disease states. The nucleotides (red) exist in an intricate balance, and are key energy storage molecules, secondary messengers for signal transduction, biosynthesis, and indicators of the metabolic state of the cell. Genetically encoded fluorescent biosensors can intercept and illuminate anions across these processes at the subcellular, cellular, and organismal scales. Representative biosensors discussed in this review are listed. (b) Overall β-barrel structure of avGFP (λ_ex = 395 nm, λ_em = 509 nm, chromophore pK_a = 4.5, Φ = 0.79, ε = 25,000 M⁻¹ cm⁻¹) from the jellyfish Aequorea victoria (PDB ID: 1GFL) with the chromophore equilibrium [21,92]. (c) Common biosensor designs and sensing mechanisms for intrinsic and extrinsic biosensors.

Figure 2.

Representative strategies used to engineer FP biosensors for anions. (a) Iterative rounds of directed evolution guided by coevolutionary modeling generates GR2-CFP. (b) Structure-guided selection and engineering of the non-coordinating residues K143 and R195 in mNeonGreen (PDB IDs: 5LTP, 5LTR, [53]) unlocked chloride sensitivity at physiological pH. Abbreviations: CRO, chromophore. (c) Improved iGluSnFR variants for in vivo brain imaging were generated through screening and selection in E. coli lysate, followed by primary rat neurons. (d) The BeadScan system to discover LiLac for 2pFLIM allowed for multi-parameter screening of individual library variants encapsulated in gel-shell beads.

Figure 3.

Representative applications of FP biosensors for subcellular, multiplex, and 2P imaging of anions. (a) Localization of the FLIPE glutamate (Glu) sensor to the cytosol or plasma membrane of A. thaliana impacted plant growth. (b) Cytosolic localization of UGAcS and endoplasmic reticulum localization of bapaUGAC enabled UDP-GlcNAc imaging in HEK293T cells. (c) Multiplex imaging of glutamate with iGluSnFR and calcium (Ca²⁺) with Cal-590 in sinoatrial node pacemaker cardiac cells. (d) Laser-induced injury (inset) to neurons in the visual cortex of zebrafish larvae triggered ATP release as visualized by GRAB_ATP1.0, and recruitment of DsRed-labeled astrocytes. (e) cAMPFIRE-L expressed in neurons in the somatosensory cortex of mice that were awake, anesthetized, or during locomotion revealed distinct cAMP dynamics. (f) Expression of SClm in neurons from mice indicated intracellular chloride (Cl⁻) changes in neuronal development. Abbreviations: GlcN, glucosamine, ROS, reactive oxygen species.