Monitoring the Heavy Metal Lead Inside Living Drosophila with a FRET-Based Biosensor

Abstract

The harmful impact of the heavy metal lead on human health has been known for years. However, materials that contain lead remain in the environment. Measuring the blood lead level (BLL) is the only way to officially evaluate the degree of exposure to lead. The so-called "safe value" of the BLL seems to unreliably represent the secure threshold for children. In general, lead's underlying toxicological mechanism remains unclear and needs to be elucidated. Therefore, we developed a novel genetically encoded fluorescence resonance energy transfer (FRET)-based lead biosensor, Met-lead, and applied it to transgenic Drosophila to perform further investigations. We combined Met-lead with the UAS-GAL4 system to the sensor protein specifically expressed within certain regions of fly brains. Using a suitable imaging platform, including a fast epifluorescent or confocal laser-scanning/two-photon microscope with high resolution, we recorded the changes in lead content inside fly brains ex vivo and in vivo and at different life stages. The blood-brain barrier was found to play an important role in the protection of neurons in the brain against damage due to the heavy metal lead, either through food or microinjection into the abdomen. Met-lead has the potential to be a powerful tool for the sensing of lead within living organisms by employing either a fast epi-FRET microscope or high-resolution brain imaging.

Novel biosensor platforms for the detection of coronavirus infection and severe acute respiratory syndrome coronavirus 2

“…Förster or fluorescence resonance energy transfer (FRET) represents one additional method of recognition. 23 – 32 The concept was first proposed by Förster in 1948. Theoretically, FRET can achieve detection resolutions far beyond the limitation of optical resolution, namely, at the 1–10 nm range.…”

“…Among these SARS-CoV-2 biosensors, the viral S protein-binding peptide (derived from the corresponding domain encoded by the human ACE2 gene) will be fused with the genes of FRET pair proteins (such as enhanced cyan fluorescent protein and enhanced yellow fluorescent protein). 23 – 32 …”

Versatile Cell and Animal Models for Advanced Investigation of Lead Poisoning

“…A 96-well glass-bottom plate was first coated with 0.5% fibronectin/0.1% gelatin at 37 °C for at least 1 h. The human iPSC cardiomyocytes were transferred into the plate wells and cultured for 3 days in Nexel's Cardiomyocyte Maintenance Medium, which was then replaced with Tyrode's buffer. For FRET imaging, the human cells were seeded onto 24-mm cover-glasses coated with poly-L-lysine and then transfected with Met-lead genes using Lipofectamine (Thermo Fisher Scientific) according to the manufacturer's instructions [20,26,27]. The cells were used for lead biosensing 2 days after transfection [23].…”

“…The Drosophila melanogaster stocks were raised on a standard cornmeal medium and housed at 25 °C with 70% relative humidity in a 12 h:12 h light:dark cycle incubator. To measure the contents of Pb within the cholinergic neurons of adult brains or the larval CNS, the strain Cha-gal4 was used and was injected with UAS-Met-lead 1.44 M1 [23,26].…”

“…This offers great potential for important practical applications such as the detection of Pb in the environment (in water for drinking or irrigation), in body fluids (in cells, serum, or urine), or in other tissues (in vivo in whole animals or plants), filling a need that has been widely noted by researchers over the years. In this study, we used two different systems to demonstrate the measurement of Pb under various conditions: induced pluripotent stem cell (iPSC)-derived cardiomyocytes [24,25] with measurable functional parameters, which provide a unique in vitro tissue model, and the adult brain and larval central nervous system (CNS) of transgenic Drosophila, as an easy-to-handle in vivo model [23,26]. ) are depicted: ECFP (blue) and EYFP/Venus (yellow) led to FRET in the presence of the target (Pb, red circles), due to Pb binding by the sensing key residues (three red lines near Pb).…”