Dataset Information

In vivo visualization of nitrate dynamics using a genetically encoded fluorescent biosensor.

ABSTRACT: Nitrate (NO₃^-) uptake and distribution are critical to plant life. Although the upstream regulation of NO₃^- uptake and downstream responses to NO₃^- in a variety of cells have been well studied, it is still not possible to directly visualize the spatial and temporal distribution of NO₃^- with high resolution at the cellular level. Here, we report a nuclear-localized, genetically encoded fluorescent biosensor, which we named NitraMeter3.0, for the quantitative visualization of NO₃^- distribution in Arabidopsis thaliana. This biosensor tracked the spatiotemporal distribution of NO₃^- along the primary root axis and disruptions by genetic mutation of transport (low NO₃^- uptake) and assimilation (high NO₃^- accumulation). The developed biosensor effectively monitors NO₃^- concentrations at the cellular level in real time and spatiotemporal changes during the plant life cycle.

SUBMITTER: Chen YN

PROVIDER: S-EPMC9581486 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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In vivo visualization of nitrate dynamics using a genetically encoded fluorescent biosensor.

Chen Yen-Ning YN Cartwright Heather N HN Ho Cheng-Hsun CH

Science advances 20221019 42

Nitrate (NO3-) uptake and distribution are critical to plant life. Although the upstream regulation of NO3- uptake and downstream responses to NO3- in a variety of cells have been well studied, it is still not possible to directly visualize the spatial and temporal distribution of NO3- with high resolution at the cellular level. Here, we report a nuclear-localized, genetically encoded fluorescent biosensor, w ...[more]

PMID: 36260665

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Project description:Hydrogen peroxide (H2O2) plays an important role in modulating cell signaling and homeostasis in live organisms. The HyPer family of genetically encoded indicators allows the visualization of H2O2 dynamics in live cells within a limited field of view. The visualization of H2O2 within a whole organism with a single cell resolution would benefit from a slowly reducible fluorescent indicator that integrates the H2O2 concentration over desired time scales. This would enable post hoc optical readouts in chemically fixed samples. Herein, we report the development and characterization of NeonOxIrr, a genetically encoded green fluorescent indicator, which rapidly increases fluorescence brightness upon reaction with H2O2, but has a low reduction rate. NeonOxIrr is composed of circularly permutated mNeonGreen fluorescent protein fused to the truncated OxyR transcription factor isolated from E. coli. When compared in vitro to a standard in the field, HyPer3 indicator, NeonOxIrr showed 5.9-fold higher brightness, 15-fold faster oxidation rate, 5.9-fold faster chromophore maturation, similar intensiometric contrast (2.8-fold), 2-fold lower photostability, and significantly higher pH stability both in reduced (pKa of 5.9 vs. ≥7.6) and oxidized states (pKa of 5.9 vs.≥ 7.9). When expressed in the cytosol of HEK293T cells, NeonOxIrr demonstrated a 2.3-fold dynamic range in response to H2O2 and a 44 min reduction half-time, which were 1.4-fold lower and 7.6-fold longer than those for HyPer3. We also demonstrated and characterized the NeonOxIrr response to H2O2 when the sensor was targeted to the matrix and intermembrane space of the mitochondria, nucleus, cell membranes, peroxisomes, Golgi complex, and endoplasmic reticulum of HEK293T cells. NeonOxIrr could reveal endogenous reactive oxygen species (ROS) production in HeLa cells induced with staurosporine but not with thapsigargin or epidermal growth factor. In contrast to HyPer3, NeonOxIrr could visualize optogenetically produced ROS in HEK293T cells. In neuronal cultures, NeonOxIrr preserved its high 3.2-fold dynamic range to H2O2 and slow 198 min reduction half-time. We also demonstrated in HeLa cells that NeonOxIrr preserves a 1.7-fold ex vivo dynamic range to H2O2 upon alkylation with N-ethylmaleimide followed by paraformaldehyde fixation. The same alkylation-fixation procedure in the presence of NP-40 detergent allowed ex vivo detection of H2O2 with 1.5-fold contrast in neuronal cultures and in the cortex of the mouse brain. The slowly reducible H2O2 indicator NeonOxIrr can be used for both the in vivo and ex vivo visualization of ROS. Expanding the family of fixable indicators may be a promising strategy to visualize biological processes at a single cell resolution within an entire organism.

Dataset Information

In vivo visualization of nitrate dynamics using a genetically encoded fluorescent biosensor.

Publications

In vivo visualization of nitrate dynamics using a genetically encoded fluorescent biosensor.

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