A general method for the development of multicolor biosensors with large dynamic ranges

Hellweg, Lars; Edenhofer, Anna; Barck, Lucas; Huppertz, Magnus-Carsten; Frei, Michelle S.; Tarnawski, Mirosław; Bergner, Andrea; Koch, Birgit; Johnsson, Kai; Hiblot, Julien

doi:10.1101/2022.11.29.518186

Cited by 5 publications

(6 citation statements)

References 63 publications

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“…Given the modularity of the extrinsic biosensor design, it will come as no surprise when a biosensor for virtually every anion becomes available. Moreover, it is foreseeable that current trends in chemigenetic and bioluminescent biosensor designs and machine learning-guided engineering will also be adopted [85][86][87]. While not explicitly discussed, rigorous in vitro photophysical characterization and in silico methods can provide deep mechanistic insights [29,88].…”

Section: Discussionmentioning

confidence: 99%

Illuminating Anions in Biology with Genetically Encoded Fluorescent Biosensors

Cook,

Phelps,

Tutol

et al. 2024

Preprint

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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 ago 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 a first-of-its-kind review, we will canvas progress made over the last three years for biologically relevant anions that can be classified as halides, oxyanions, carboxylates, and nucleotides.

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Section: Discussionmentioning

confidence: 99%

Illuminating Anions in Biology with Genetically Encoded Fluorescent Biosensors

Cook,

Phelps,

Tutol

et al. 2024

Preprint

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“…Furthermore, modifications within the fluorescent domains may benefit the sensor performance in the cellular environments. Here, fastmaturating fluorescent proteins may be introduced (Höfig et al, 2018), but also orthogonal moieties, such as HaloTag and SNAP-Tag (Hellweg et al, 2023;Zhang et al, 2023), which ensure higher photostability and brightness, and would reduce the sensor propensity to form oligomers at the membrane.…”

Section: Discussionmentioning

confidence: 99%

Probing macromolecular crowding at the lipid membrane interface with genetically‐encoded sensors

Löwe,

Hänsch,

Hachani

et al. 2023

Protein Science

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Biochemical processes within the living cell occur in a highly crowded environment, where macromolecules, first of all proteins and nucleic acids, occupy up to 30 % of the volume. The phenomenon of macromolecular crowding is not an exclusive feature of the cytoplasm and can be observed in the densely protein‐packed, nonhomogeneous cellular membranes and at the membrane interfaces. Crowding affects diffusional and conformational dynamics of proteins within the lipid bilayer, alters kinetic and thermodynamic properties of biochemical reactions, and modulates the membrane organization. Despite its importance, the non‐invasive quantification of the membrane crowding is not trivial. Here, we developed the genetically‐encoded fluorescence‐based sensor for probing the macromolecular crowding at the membrane interfaces. Two sensor variants, both composed of fluorescent proteins and a membrane anchor, but differing by the flexible linker domains were characterized in vitro, and the procedures for the membrane reconstitution were established. Steric pressure induced by membrane‐tethered synthetic and protein crowders altered the sensors’ conformation, causing increase in the intramolecular Förster's resonance energy transfer. Notably, the effect of protein crowders only weakly correlated with their molecular weight, suggesting that other factors, such as shape and charge contribute to the crowding via the quinary interactions. Finally, measurements performed in inner membrane vesicles of E. coli validated the crowding‐dependent dynamics of the sensors in the physiologically relevant environment. The sensors offer broad opportunities to study interfacial crowding in a complex environment of native membranes, and thus add to the toolbox of methods for studying membrane dynamics and proteostasis.This article is protected by copyright. All rights reserved.

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“…), similar to previously reported measurements with chemicalgenetic hybrid ChemoX sensors (320 nM Δ[Ca 2+ ]). 44 Imaging Ca 2+ in neurons and cardiomyocytes isoCaRed-1Me can visualize Ca 2+ transients in other contexts besides immortalized cell lines, where the transfection required for expression of genetically encoded Ca 2+ indicators is inefficient, including hippocampal neurons isolated from rats and cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). Rat hippocampal neurons stained with isoCaRed-1Me AM show strong intracellular fluorescence, when excited with either violet or cyan light (Figure 4a-b).…”

Section: Live Cell Imaging In Hela Cellsmentioning

confidence: 99%

Ratio-based indicators for cytosolic Ca2+ with visible light excitation

Zhou,

Belavek,

Navarro

et al. 2024

Preprint

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Calcium ions (Ca2+) play central roles in cellular physiology. Fluorescent indicators for Ca2+ ions revolutionized our ability to make rapid, accurate, and highly parallel measurement of Ca2+ concentrations in living cells. The use of ratio-based imaging with one particular indicator, fura-2, allowed practitioners to correct for a number of experimental confounds, including dye bleaching, variations in sample thickness, and fluctuations in illumination intensity. Ratio-based imaging with fura-2 was the most accurate and reliable method for measuring Ca2+ concentrations. Two drawbacks to fura-2 exist. First, it requires ultraviolet (UV) excitation, which is more toxic to living cells than visible light. Second, our ability to use fura-2 for accurate, stable, ratio-based determinations of Ca2+ concentration in living cells is fast becoming a method of the past. This is due, in part, because modern microscopes are phasing out the use of mercury arc lamps that provide the UV excitation needed for fura-2 imaging. To address this problem, we describe the design, synthesis, and cellular application of benzo[b]phosphole-based fluorescent Ca2+ indicators for ratio-based imaging of Ca2+ in living cells that can be used with modern light emitting diode (LED)-equipped fluorescence microscopes. We show that isoCaRed-1Me has absorbance spectra in the visible range, shows Ca2+ selectivity over Mg2+, and displays a Ca2+-dependent excitation spectra. These unique properties enable ratio-based imaging in immortalized cell lines, primary mammalian hippocampal neurons, and human induced pluripotent stem cell-derived cardiomyocytes. These data show that isoCaRed-1Me will be useful for ratio-based Ca2+ imaging using modern microscopes.

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A general method for the development of multicolor biosensors with large dynamic ranges

Cited by 5 publications

References 63 publications

Illuminating Anions in Biology with Genetically Encoded Fluorescent Biosensors

Illuminating Anions in Biology with Genetically Encoded Fluorescent Biosensors

Probing macromolecular crowding at the lipid membrane interface with genetically‐encoded sensors

Ratio-based indicators for cytosolic Ca2+ with visible light excitation

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