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The ATP levels of an organism are an important physiological parameter that …

Biology Articles » Anatomy & Physiology » Physiology, Animal » Bridging the phenotypic gap: Real-time assessment of mitochondrial function and metabolism of the nematode Caenorhabditis elegans » Discussion

- Bridging the phenotypic gap: Real-time assessment of mitochondrial function and metabolism of the nematode Caenorhabditis elegans

In C. elegans, assessment of ATP levels has until now been carried out in vitro [7-9,23], a method which is accurate but implies the destruction of worms, limits the scalability of the analysis and cannot be carried out in real-time. We report on the construction of a luminescent transgenic C. elegans strain and have tested the hypothesis that light emitted by living C. elegans is a reflection of its ATP pools. We used two approaches: i) exposure to and recovery from sodium azide, a specific mitochondrial inhibitor, and ii) RNAi towards genes that are essential for mitochondrial function. Azide inhibits complex IV of the mitochondrial respiratory chain by binding reversibly to cytochrome c oxidase [24], this arrests the flow of electrons and leads to a decrease in ATP synthesis. Because the inhibition is reversible azide has been widely used as a C. elegans anaesthetic [25]. Changes in bioluminescence upon exposure to, and recovery from, sublethal concentrations of azide were consistent with reversible ATP depletion caused by azide. Recovery in bioluminescence occurs within 30 min of removing worms from azide. Similarly, recovery of ATP depletion resulting from anoxia occurs within 45 min of reversal of anoxia [14]. Luciferase expression and activity levels stayed constant in the azide experiments and therefore did not contribute to observed changes in bioluminescence.

The substrate luciferin has to be present intracellularly for light emission. It is not known how luciferin enters C. elegans. It is possible that it passes through the permeabilised cuticle. Alternatively, it may be ingested through the pharynx. If an active pharynx was a requirement for luciferin entry, then substrate availability could be a limiting factor for luminescence under experimental conditions that alter the pumping rate, such as toxin exposure [26]. However, in this work, although exposure to azide stopped pharynx activity, the worms were able to emit light when provided with the substrate, indicating that luciferin was able to cross the permeabilised cuticle. Luciferin has been deemed to be poorly taken up by cells [27], however, empirical data suggest that luciferin uptake occurs readily. Luciferin crossed the blood-brain barrier in mice easily, passing through endothelial cells that are amongst the least permeable cells in the mouse [28]. Studies in other organisms such as Drosophila [29], zebra fish [30] and Arabidopsis [31] also indicated that diffusion and permeability of luciferin did not limit bioluminescence. Furthermore, we have observed that luciferin was capable of crossing the shell of unhatched C. elegans embryos, considered poorly permeable to chemicals, resulting in bioluminescence. We have also captured images of widespread luminescence in the worm's tissues, contrary to what would be expected if luciferin was poorly taken up by cells.

Inhibition of respiratory chain components by RNAi provided a means of depleting ATP levels without exogenous chemicals. The response measured by bioluminescence is in agreement with in vitro data. In vitro ATP was reduced to 20–40% when cyc-1 or atp-3 were knocked down, and to 40–60% for cco-1 RNAi [7]. All experimental data were consistent with the hypothesis that C. elegans luminescence reflects its ATP pools. Additionally, the RNAi experiments illustrate bioluminescence as a phenotype that could be the basis for genetic analysis.

Wildtype firefly luciferase is targeted to peroxisomes [32] and has a 3 h half-life [27,33]. In this study, the nematodes were transformed with a modified firefly luciferase gene luc+ fused to gfp, which is not targeted to the peroxisomes. Luc+ is expressed in the cytosol and has a half-life of 10 h in human breast cancer cells [34]. The half-life of GFP is 26 h [35] and therefore should not contribute to instability of the fusion protein. The greater stability of Luc+ is an advantage for studies where ATP changes may be tracked over time, as opposed to the requirement for a short half-life when luciferase reports on gene expression.

One critical aspect that will affect the luminescence readings is the levels of firefly luciferase present, this may vary for example between different strains and developmental stages. Hence, the exact relationship between light output and ATP concentrations will depend on the experimental conditions, ruling out precise determination of ATP content. We propose this strain as a relative sensor of ATP levels which can be applied to interrogate mitochondrial function and metabolism of living worms in a non-destructive, real-time and scalable manner. This will have broad appeal as sublethal physiological parameters are often difficult to quantify, especially on a large scale. Perhaps the most exciting developments will be the identification of novel genes and pathways underlying physiological response, as well as a better understanding of classical pathways. The integrated luc+:: gfp fusion we described can be crossed into available strains carrying gene deletions or into new mutants generated by mutagenesis. Alternatively, any of the worm's genes can be targeted for inactivation by RNAi and bioluminescence will provide an easily quantifiable metabolic phenotype. The luciferase gene may also be placed under promoters that will drive its expression in specific tissues and allow for relative assessment of ATP levels in that tissue. Ballistic transformation methods will enable expression of the transgene in the germline where required. The transgenic strains described here offer a unique opportunity to explore the links between physiology and genetics of C. elegans and many other organisms with which it shares homologue genes, including humans [22].

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