Real time in vivo ATP Monitoring in C elegans

Cristina Lagido, Jonathan Pettitt, Aileen Winifred Flett, Lesley Anne Glover

Research output: Contribution to conferenceAbstract

Abstract

ATP concentrations affect how organisms function, and impaired ATP production is associated with ageing, neurodegenerative and metabolic diseases. Exposure to stress is also known to alter ATP levels. We are interested in ATP levels as an early manifestation of the metabolic and physiological changes taking place during exposure of C. elegans to stress. Determination of ATP levels is frequently carried out in vitro 1, 2, implying the destruction of the biological sample. Our aim is to do this in vivo enabling genetic analysis based on the ATP phenotype. We have generated luminescent C. elegans through ubiquitous constitutive expression of firefly luciferase, which is widely used for ATP determination. We have previously demonstrated a link between light levels and the nematodes health and viability upon exposure to environmental stress 3. Here, we describe the generation of a new strain of highly luminescent C. elegans and specifically address the hypothesis that whole animal luminescence reflects its intracellular ATP levels in vivo. To test this, we characterised the bioluminescence response of C. elegans during sublethal exposure to and recovery from azide. Azide inhibits mitochondrial respiration reversibly and causes ATP depletion. Consistent with our expectations, in vivo luminescence decreased with increasing sublethal azide levels and recovered fully when worms were removed from azide. In vitro determination of firefly luciferase activity indicated that luciferase expression levels, stability and activity did not influence the final luminescence. Results showed that C. elegans luminescence reports on ATP levels in real-time. This is the first time firefly luciferase determination of ATP levels has been applied to a whole live multicellular organism. The study of the stress response is often slowed down by laborious methodologies. Real-time ATP monitoring constitutes a unique opportunity to explore the links between physiology and genetics of C. elegans and will provide novel physiological insights in health and disease. 1. Dillin A. et al. (2002) Science 298: 2398-2401. 2. Houthoofd K. et al. (2005) Neurobiology of Aging 26: 689-696. 3. Lagido C. et al. (2001) FEBS Letters 493: 3639.
Original languageEnglish
Publication statusPublished - 2007
Event16th International C. elegans Meeting (2007) - Los Angeles, United States
Duration: 27 Jun 20071 Jul 2007

Conference

Conference16th International C. elegans Meeting (2007)
CountryUnited States
CityLos Angeles
Period27/06/071/07/07

Fingerprint

Adenosine Triphosphate
Azides
Luminescence
Firefly Luciferases
Neurobiology
Metabolic Diseases
Environmental Exposure
Luciferases
Neurodegenerative Diseases
Health Status
Respiration
Phenotype
Light
Health

Cite this

Lagido, C., Pettitt, J., Flett, A. W., & Glover, L. A. (2007). Real time in vivo ATP Monitoring in C elegans. Abstract from 16th International C. elegans Meeting (2007), Los Angeles, United States.

Real time in vivo ATP Monitoring in C elegans. / Lagido, Cristina; Pettitt, Jonathan; Flett, Aileen Winifred; Glover, Lesley Anne.

2007. Abstract from 16th International C. elegans Meeting (2007), Los Angeles, United States.

Research output: Contribution to conferenceAbstract

Lagido, C, Pettitt, J, Flett, AW & Glover, LA 2007, 'Real time in vivo ATP Monitoring in C elegans' 16th International C. elegans Meeting (2007), Los Angeles, United States, 27/06/07 - 1/07/07, .
Lagido C, Pettitt J, Flett AW, Glover LA. Real time in vivo ATP Monitoring in C elegans. 2007. Abstract from 16th International C. elegans Meeting (2007), Los Angeles, United States.
Lagido, Cristina ; Pettitt, Jonathan ; Flett, Aileen Winifred ; Glover, Lesley Anne. / Real time in vivo ATP Monitoring in C elegans. Abstract from 16th International C. elegans Meeting (2007), Los Angeles, United States.
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AU - Pettitt, Jonathan

AU - Flett, Aileen Winifred

AU - Glover, Lesley Anne

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N2 - ATP concentrations affect how organisms function, and impaired ATP production is associated with ageing, neurodegenerative and metabolic diseases. Exposure to stress is also known to alter ATP levels. We are interested in ATP levels as an early manifestation of the metabolic and physiological changes taking place during exposure of C. elegans to stress. Determination of ATP levels is frequently carried out in vitro 1, 2, implying the destruction of the biological sample. Our aim is to do this in vivo enabling genetic analysis based on the ATP phenotype. We have generated luminescent C. elegans through ubiquitous constitutive expression of firefly luciferase, which is widely used for ATP determination. We have previously demonstrated a link between light levels and the nematodes health and viability upon exposure to environmental stress 3. Here, we describe the generation of a new strain of highly luminescent C. elegans and specifically address the hypothesis that whole animal luminescence reflects its intracellular ATP levels in vivo. To test this, we characterised the bioluminescence response of C. elegans during sublethal exposure to and recovery from azide. Azide inhibits mitochondrial respiration reversibly and causes ATP depletion. Consistent with our expectations, in vivo luminescence decreased with increasing sublethal azide levels and recovered fully when worms were removed from azide. In vitro determination of firefly luciferase activity indicated that luciferase expression levels, stability and activity did not influence the final luminescence. Results showed that C. elegans luminescence reports on ATP levels in real-time. This is the first time firefly luciferase determination of ATP levels has been applied to a whole live multicellular organism. The study of the stress response is often slowed down by laborious methodologies. Real-time ATP monitoring constitutes a unique opportunity to explore the links between physiology and genetics of C. elegans and will provide novel physiological insights in health and disease. 1. Dillin A. et al. (2002) Science 298: 2398-2401. 2. Houthoofd K. et al. (2005) Neurobiology of Aging 26: 689-696. 3. Lagido C. et al. (2001) FEBS Letters 493: 3639.

AB - ATP concentrations affect how organisms function, and impaired ATP production is associated with ageing, neurodegenerative and metabolic diseases. Exposure to stress is also known to alter ATP levels. We are interested in ATP levels as an early manifestation of the metabolic and physiological changes taking place during exposure of C. elegans to stress. Determination of ATP levels is frequently carried out in vitro 1, 2, implying the destruction of the biological sample. Our aim is to do this in vivo enabling genetic analysis based on the ATP phenotype. We have generated luminescent C. elegans through ubiquitous constitutive expression of firefly luciferase, which is widely used for ATP determination. We have previously demonstrated a link between light levels and the nematodes health and viability upon exposure to environmental stress 3. Here, we describe the generation of a new strain of highly luminescent C. elegans and specifically address the hypothesis that whole animal luminescence reflects its intracellular ATP levels in vivo. To test this, we characterised the bioluminescence response of C. elegans during sublethal exposure to and recovery from azide. Azide inhibits mitochondrial respiration reversibly and causes ATP depletion. Consistent with our expectations, in vivo luminescence decreased with increasing sublethal azide levels and recovered fully when worms were removed from azide. In vitro determination of firefly luciferase activity indicated that luciferase expression levels, stability and activity did not influence the final luminescence. Results showed that C. elegans luminescence reports on ATP levels in real-time. This is the first time firefly luciferase determination of ATP levels has been applied to a whole live multicellular organism. The study of the stress response is often slowed down by laborious methodologies. Real-time ATP monitoring constitutes a unique opportunity to explore the links between physiology and genetics of C. elegans and will provide novel physiological insights in health and disease. 1. Dillin A. et al. (2002) Science 298: 2398-2401. 2. Houthoofd K. et al. (2005) Neurobiology of Aging 26: 689-696. 3. Lagido C. et al. (2001) FEBS Letters 493: 3639.

M3 - Abstract

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