Effects of tritiated water on locomotion of zebrafish larvae: a new insight in tritium toxic effects on a vertebrate model species.

Tritium (3H), a radioactive isotope of hydrogen, is ubiquitously present in the environment. In a previous study, we highlighted a mis-regulation of genes involved in muscle contraction, eye transparency and response to DNA damages after exposure of zebrafish embryo-larvae from 3 hpf to 96 hpf at 0.4 and 4 mGy/h of tritiated water (HTO). The present study aimed to link this gene mis-regulation to responses observed at higher biological levels. Analyses on spontaneous tail movement, locomotor activity and heart rate were performed. Histological sections of eyes were made to evaluate the impact of HTO on eye transparency and whole embryo immunostainings were realized to assess DNA double strand breaks repair using gamma-H2AX foci. We found a decrease of basal velocity as well as a decrease of response in 96 hpf larvae exposed at 0.4 mGy/h after a tactile stimulus as compared to controls. Histological sections of larvae eyes performed after the exposure to 4 mGy/h did not show obvious differences in lens transparency or retinal development between contaminated and control organisms. Gamma-H2AX foci detection revealed no differences in the number of foci between contaminated organisms and controls, for both dose rates. Overall, results highlighted more detrimental effects of HTO exposure on locomotor behavior in 96 hpf larvae exposed at the lowest dose rate. Those results could be linked to mis-regulation of genes involved in muscle contraction found in a previous study at the same dose rate. It appears that not all effects found at the molecular scale were confirmed using higher biological scales. These results could be due to a delay between gene expression modulation and the onset of physiological disruption or homeostatic mechanisms to deal with tritium effects. However, crossing data from different scales highlighted new pathways to explore, i.e. neurotoxic pathways, for better understanding HTO effects on organisms.

A systems biology approach reveals neuronal and muscle developmental defects after chronic exposure to ionising radiation in zebrafish.

Contamination of the environment after the Chernobyl and Fukushima Daiichi nuclear power plant (NPP) disasters led to the exposure of a large number of humans and wild animals to radioactive substances. However, the sub-lethal consequences induced by these absorbed radiological doses remain understudied and the long-term biological impacts largely unknown. We assessed the biological effects of chronic exposure to ionizing radiation (IR) on embryonic development by exposing zebrafish embryo from fertilization and up to 120 hours post-fertilization (hpf) at dose rates of 0.5 mGy/h, 5 mGy/h and 50 mGy/h, thereby encompassing the field of low dose rates defined at 6 mGy/h. Chronic exposure to IR altered larval behaviour in a light-dark locomotor test and affected cardiac activity at a dose rate as low as 0.5 mGy/h. The multi-omics analysis of transcriptome, proteome and transcription factor binding sites in the promoters of the deregulated genes, collectively points towards perturbations of neurogenesis, muscle development, and retinoic acid (RA) signaling after chronic exposure to IR. Whole-mount RNA in situ hybridization confirmed the impaired expression of the transcription factors her4.4 in the central nervous system and myogenin in the developing muscles of exposed embryos. At the organ level, the assessment of muscle histology by transmission electron microscopy (TEM) demonstrated myofibers disruption and altered neuromuscular junctions in exposed larvae at 5 mGy/h and 50 mGy/h. The integration of these multi-level data demonstrates that chronic exposure to low dose rates of IR has an impact on neuronal and muscle progenitor cells, that could lead to motility defects in free swimming larvae at 120 hpf. The mechanistic understanding of these effects allows us to propose a model where deregulation of RA signaling by chronic exposure to IR has pleiotropic effects on neurogenesis and muscle development.

Chloroplast-Specific in Vivo Ca2+ Imaging Using Yellow Cameleon Fluorescent Protein Sensors Reveals Organelle-Autonomous Ca2+ Signatures in the Stroma.

In eukaryotes, subcellular compartments such as mitochondria, the endoplasmic reticulum, lysosomes, and vacuoles have the capacity for Ca(2+) transport across their membranes to modulate the activity of compartmentalized enzymes or to convey specific cellular signaling events. In plants, it has been suggested that chloroplasts also display Ca(2+) regulation. So far, monitoring of stromal Ca(2+) dynamics in vivo has exclusively relied on using the luminescent Ca(2+) probe aequorin. However, this technique is limited in resolution and can only provide a readout averaged over chloroplast populations from different cells and tissues. Here, we present a toolkit of Arabidopsis (Arabidopsis thaliana) Ca(2+) sensor lines expressing plastid-targeted FRET-based Yellow Cameleon (YC) sensors. We demonstrate that the probes reliably report in vivo Ca(2+) dynamics in the stroma of root plastids in response to extracellular ATP and of leaf mesophyll and guard cell chloroplasts during light-to-low-intensity blue light illumination transition. Applying YC sensing of stromal Ca(2+) dynamics to single chloroplasts, we confirm findings of gradual, sustained stromal Ca(2+) increases at the tissue level after light-to-low-intensity blue light illumination transitions, but monitor transient Ca(2+) spiking as a distinct and previously unknown component of stromal Ca(2+) signatures. Spiking was dependent on the availability of cytosolic Ca(2+) but not synchronized between the chloroplasts of a cell. In contrast, the gradual sustained Ca(2+) increase occurred independent of cytosolic Ca(2+), suggesting intraorganellar Ca(2+) release. We demonstrate the capacity of the YC sensor toolkit to identify novel, fundamental facets of chloroplast Ca(2+) dynamics and to refine the understanding of plastidial Ca(2+) regulation.

Analyses of Ca2+ accumulation and dynamics in the endoplasmic reticulum of Arabidopsis root cells using a genetically encoded Cameleon sensor.

In planta, very limited information is available about how the endoplasmic reticulum (ER) contributes to cellular Ca(2+) dynamics and homeostasis. Here, we report the generation of an ER-targeted Cameleon reporter protein suitable for analysis of Ca(2+) accumulation and dynamics in the lumen of the ER in plant cells. Using stably transformed Arabidopsis (Arabidopsis thaliana) plants expressing this reporter protein, we observed a transiently enhanced accumulation of Ca(2+) in the ER in response to stimuli inducing cytosolic Ca(2+) rises in root tip cells. In all experimental conditions, ER Ca(2+) dynamics were substantially different from those monitored in the cytosol. A pharmacological approach enabled us to evaluate the contribution of the different ER-resident Ca(2+)-ATPase classes in the regulation of the ER Ca(2+) homeostasis. Taken together, our results do not provide evidence for a role of the ER as a major source that releases Ca(2+) for stimulus-induced increases in cytosolic Ca(2+) concentration. Instead, our results show that the luminal ER Ca(2+) elevations typically follow cytosolic ones, but with distinct dynamics. These findings suggest fundamental differences for the function of the ER in cellular Ca(2+) homeostasis in plants and animals.

Ca2+ imaging in plants using genetically encoded Yellow Cameleon Ca2+ indicators.

Temporally and spatially defined changes in cellular calcium (Ca(2+)) concentration represent stimulus-specific signals and regulate a myriad of biological processes. The development of ratiometric Ca(2+) reporter proteins like Yellow Cameleons (YCs) has greatly advanced our ability to analyze Ca(2+) dynamics in vivo with unprecedented spatial and temporal resolution. In plants, the application of these Ca(2+) reporter proteins has been pioneered for the investigation of Ca(2+) dynamics in guard cells, and recently their use has been extended to other single-cell models like growing pollen tubes and root hairs. However, in plants, the use of YC reporter proteins has largely remained restricted to the investigation of cytoplasmic alterations of Ca(2+) concentrations. Here, we provide an introduction to current methods for imaging Ca(2+) dynamics with increasing sophistication.

Imaging of mitochondrial and nuclear Ca2+ dynamics in Arabidopsis roots.

Here we report a method for analyzing mitochondrial Ca(2+) dynamics in plant root cells by means of the newly generated cameleon probe 4mt-YC3.6. The use of plants expressing both nuclear- and mitochondrial-targeted cameleon, along with the resolution of confocal laser scanning microscopy (CLSM), allow simultaneous recordings of mitochondrial and nuclear Ca(2+) dynamics within the same cell.

Targeting of Cameleons to various subcellular compartments reveals a strict cytoplasmic/mitochondrial Ca²âº handling relationship in plant cells.

Here we describe use of a mitochondrial targeted Cameleon to produce stably transformed Arabidopsis plants that enable analyses of mitochondrial Ca²âº dynamics in planta and allow monitoring of the intra-mitochondrial Ca²âº concentration in response to physiological or environmental stimuli. Transgenic plants co-expressing nuclear and mitochondrial targeted Cameleons were also generated and analyzed. Here we show that mitochondrial Ca²âº accumulation is strictly related to the intensity of the cytoplasmic Ca²âº increase, demonstrating a tight association between mitochondrial and cytoplasmic Ca²âº dynamics. However, under all experimental conditions, mitochondrial Ca²âº dynamics were substantially different from those monitored in the cytoplasm, demonstrating that mitochondria do not passively sense cytosolic Ca²âº, but actively modulate the intra-mitochondrial level of the cation. In particular, our analyses show that the kinetics of Ca²âº release from mitochondria are much slower than in the cytoplasm and nucleus. The mechanisms and functional implications of these differences are discussed.