RESUMEN
Zebrafish Mate3 is one of six co-orthologs of human multidrug and toxin extrusion proteins. It is highly expressed in the kidneys, intestine, testes, and brain of males. Initial interaction studies showed its interaction with xenobiotic compounds, suggesting a role in the efflux of toxic compounds. In this study, we aimed to test various environmental contaminants for their interaction with zebrafish Mate3. We developed a stable zebrafish Mate3 cell line and optimized a high-throughput screening assay using DAPI and ASP+ as fluorescent model substrates. To gain insight into the structure and function of the Mate3 protein and relate these to the results of the DAPI and ASP+ transport measurements, we predicted its 3D structure using the AlphaFold2 algorithm. A 3D structure with high per residue confidence scores with 13 transmembrane segments (TMs) was obtained, with topology and mutual positioning characteristic of the Mate protein family in a shape open to the extracellular part. Molecular docking methods were used to identify DAPI and ASP+ binding sites on the surface and in the center of the protein cavity. Because our kinetics experiments combined with molecular docking indicated that there may be additional active sites in zebrafish Mate3, additional cytotoxicity experiments were performed and highly potent Mate3 interactors were identified from a set of 55 different environmental contaminants. Our results suggest that some of the identified interactors may be of environmental concern, as their interaction with Mate3 could lead to an impairment of its normal efflux function, making fish more sensitive to harmful substances commonly released into the aquatic environment. Finally, the quality of zebrafish Mate3 structures predicted by the AlphaFold2 algorithm opens up the possibility of successfully using this tool for in silico research on transport preferences of other Mate proteins.
RESUMEN
The increasing use of the zebrafish model in biomedical and (eco)toxicological studies aimed at understanding the function of various proteins highlight the importance of optimizing existing methods to study gene and protein expression and localization in this model. In this context, zebrafish cryosections are still underutilized compared with whole-mount preparations. In this study, we used zebrafish embryos (24-120 hpf) to determine key factors for the preparation of high-quality zebrafish cryosections and to determine the optimal protocol for (immuno)fluorescence analyses of Na+ /K+ -ATPase and F-actin, across developmental stages from 1 to 5 dpf. The results showed that the highest quality zebrafish cryosections were obtained after the samples were fixed in 4% paraformaldehyde (PFA) for 1 h, incubated in 2.5% bovine gelatin/25% sucrose mixture, embedded in OCT, and then sectioned to 8 µm thickness at -20°C. Fluorescence microscopy analysis of phalloidin-labeled zebrafish skeletal muscle revealed that 1-h-4% PFA-fixed samples allowed optimal binding of phalloidin to F-actin. Further immunofluorescence analyses revealed detailed localization of F-actin and Na+ /K+ -ATPase in various tissues of the zebrafish and a stage-dependent increase in their respective expression in the somitic muscles and pronephros. Finally, staining of zebrafish cryosections and whole-mount samples revealed organ-specific and zone-dependent localizations of the Na+ /K+ -ATPase α1-subunit. RESEARCH HIGHLIGHTS: This study brings optimization of existing protocols for preparation and use of zebrafish embryos cryosections in (immuno)histological analyses. It reveals stage-dependent localization/expression of F-actin and Na+ /K+ -ATPase in zebrafish embryos.
