A genetically encoded fluorescent probe for imaging of oxygenation gradients in living Drosophila

Abstract

Oxygen concentrations vary between tissues of multicellular organisms and change under certain physiological or pathological conditions. Multiple methods have been developed for measuring oxygenation of biological samples and However, most require complex equipment, are laborious and have significant limitations. Here we report that oxygen concentration determines the choice between two maturation pathways of DsRed FT (Timer). At high oxygen levels, this DsRed derivate matures predominantly into a red fluorescent isoform. By contrast, a green fluorescent isoform is favored by low oxygen levels. Ratiometric analysis of green and red fluorescence after a pulse of Timer expression in larvae provides a record of the history of tissue oxygenation during a subsequent chase period, for the whole animal with single-cell precision. Tissue spreads revealed fine differences in oxygen exposure among different cells of the same organ. We expect that the simplicity and robustness of our approach will greatly impact hypoxia research, especially in small animal models.

Cumulative mitochondrial activity correlates with ototoxin susceptibility in zebrafish mechanosensory hair cells

“…While newly synthesized mitoTimer fluoresces green, the fluorescence shifts irreversibly to red over time due to dehydrogenization of the Tyr-67 residue (Verkhusha et al, 2004; Yarbrough et al, 2001). The change in fluorescence spectra and the means by which it occurs has made mitoTimer a useful tool to examine mitochondrial turnover and transport, as well as reporting cumulative redox and oxygenation history (Ferree et al, 2013; Hernandez et al, 2013; Laker et al, 2014; Stotland and Gottlieb, 2016; Laker et al, 2017; Wilson et al, 2019; Lidsky et al, 2018). We used mitoTimer to first examine changes in the state of mitochondria during neuromast maturation.…”

Genetically Encoded Tools for Research of Cell Signaling and Metabolism under Brain Hypoxia

“…Each maturation step requires an oxygen molecule that renders DsRed FT a natural oxygen sensor. In their work, Lidsky et al demonstrated that the blue-to-red conversion efficiency strongly depends on the medium oxygenation (9-21% O 2 concentrations were tested) [43]. Taking advantage of this observation, they developed the nlsTimer probe by fusing DsRed FT with a nuclear localization signal that enabled the confocal imaging of the oxygenation state of Drosophila melanogaster larvae cells during early development.…”

“…The main drawbacks of nlsTimer include its slow maturation time (days) and irreversible character of the response. In the original study, the authors implemented a system consisting of hs-GAL4 and UASt-nlsTimer constructs that allows the capture of oxygenation memory maps after heat shock in poikilothermic animal models, which reflect the average oxygen concentrations during chromophore formation rather than rapid changes [43]. The implementation of degrons could increase turnover of the probe, paving the way for repetitive imaging experiments (possible approaches are discussed in the context of HIF system-based reporters).…”

“…Therefore, it is possible to utilize this feature for the development of oxygen reporters. The nlsTimer probe [ 43 ] was engineered based on DsRed FT, a non-aggregating tetrameric form of DsRed, that demonstrates two competing pathways of chromophore formation ( Figure 2 A) [ 44 , 45 ]. The first pathway generates green species, while the second pathway produces red-emitting molecules through a blue-colored form.…”

Synthetic biology of hypoxia

“…Combination of GFP‐like proteins with O 2 ‐independent fluorophores based on flavin mononucleotides has generated a FRET biosensor in E. coli (Potzkei et al , 2012). Furthermore, a two‐color DsRed protein has been developed, whose properties of O 2 ‐dependent maturation determined a quantitative shift in FRET emission from red to green that could be used to monitor hypoxia in Drosophila (Lidsky et al , 2018). Maturation‐type ratiometric biosensors may also be obtained using tandem fluorescent protein timers (tFT).…”