Analyzing cell physiology in C. elegans with fluorescent ratiometric reporters

Abstract

Ratiometric fluorescent reporters have recently emerged a new technique to non-invasively measure aspects of cell physiology such as redox status, calcium levels, energy production, and NADH levels. These reporters consist of either a single or pair of fluorophores along with specific modifications, such as the addition of a protein domain which binds to a metabolite of interest, thereby producing gradual alterations in fluorescence in response to changes in the measured parameter. Measurement of the changes in fluorescence produces a quantitative read-out of the cellular environment. While these reporters were initially developed to easily visualize and track changes in cultured cells, several groups have adapted these reporters to use in Caenorhabditis elegans which opens a new avenue through which to explore cell physiology during development or aging, in response to changes in external environment, or in response to genetic manipulation. These reporters have the advantage of being easily targeted to any part of the worm, and because C. elegans is transparent both the reporters and changes in their fluorescence can be clearly observed in vivo. Here we discuss the application of ratiometric reporters to C. elegans, and outline a method to quantitatively measure changes in intracellular peroxide levels using the HyPer ratiometric reporter. However, these principles can be applied to alternate ratiometric reporters which are designed to measure either other chemical species or other cellular parameters.

Cell-autonomous and non-autonomous roles of daf-16 in muscle function and mitochondrial capacity in aging C. elegans

“…An important experimental advantage of C. elegans is the optical transparency of the animals which allows aspects of cell behavior to be observed in vivo via the use of fluorescent proteins [26]. To examine whether the enhanced mitochondrial mass and structure seen in the daf-2 mutants resulted in changes in muscle cell physiology, we developed transgenic worms expressing the ratiometric reporter ATeam, which shows reproducible changes in FRET-derived fluorescence in response to local ATP concentrations [18].…”

Evaluation of the antioxidant property and effects in Caenorhabditis elegans of Xiangxi flavor vinegar, a Hunan local traditional vinegar

“…Worms were cultured at 20 °C in a nematode growth medium (NGM) with E. coli OP50 as food source, as previously described (Strange, 2006). Synchronized worms were obtained by the lysis-centrifugation method (Wang et al, 2014a). Gravid hermaphrodites were lysed by a freshly prepared lysate solution (5 ml 6.4% (0.064 g/ml) sodium hypochlorite, 5 ml 1 mol/L sodium hydroxide solution).…”

Redox imaging using genetically encoded redox indicators in zebrafish and mice

“…Detailed information on the history, development, and properties of current GERIs is excellently compiled in recent reviews (Meyer and Dick, 2010;Tantama et al, 2012;Lukyanov and Belousov, 2013;Ezeriņa et al, 2014). We refer to additional publications for information on redox imaging in invertebrate systems (C. elegans and Drosophila; Albrecht et al, 2011;Barata and Dick, 2013;Lukyanov and Belousov, 2013;Wang et al, 2014).…”