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An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer.

Fourrage C, Swann K, Gonzalez Garcia JR, Campbell AK, Houliston E - Open Biol (2014)

Bottom Line: Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria.Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms).Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universités, UPMC Univ Paris 06, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Observatoire Océanologique, 06230 Villefranche-sur-mer, France.

ABSTRACT
Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

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Bioluminescence in C. hemisphaerica. Bioluminescence image of an immobilized whole adult medusa, approximately 1 cm in diameter, stimulated with 0.5 M KCl. Pixels are colour coded according to a scale in which hot colours (including white) indicate the highest levels of bioluminescence, and cold colours represent low bioluminescence. (a) Inset shows the same medusa by light microscopy indicating the position of tentacle bulbs (Tb), gonads (G) and manubrium (M). (b) Eggs (approx. 180 µm in diameter) treated first with ionomycin (b(i)) and subsequently treated with Triton X-100 to lyse the egg and discharge all clytin photoprotein luminescence (b(ii)). The images show luminescence from 200 s of integrated light collection after ionomycin and then after detergent treatment. Equivalent images taken before the treatment were completely black. Scale bars 100 µm. (c) Luminescence kinetics from one of seven similar eggs treated in the same way.
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RSOB130206F1: Bioluminescence in C. hemisphaerica. Bioluminescence image of an immobilized whole adult medusa, approximately 1 cm in diameter, stimulated with 0.5 M KCl. Pixels are colour coded according to a scale in which hot colours (including white) indicate the highest levels of bioluminescence, and cold colours represent low bioluminescence. (a) Inset shows the same medusa by light microscopy indicating the position of tentacle bulbs (Tb), gonads (G) and manubrium (M). (b) Eggs (approx. 180 µm in diameter) treated first with ionomycin (b(i)) and subsequently treated with Triton X-100 to lyse the egg and discharge all clytin photoprotein luminescence (b(ii)). The images show luminescence from 200 s of integrated light collection after ionomycin and then after detergent treatment. Equivalent images taken before the treatment were completely black. Scale bars 100 µm. (c) Luminescence kinetics from one of seven similar eggs treated in the same way.

Mentions: We visualized the sites of bioluminescence in Clytia hemisphaerica medusae (figure 1) stimulated by treatment with calcium ionophore, detergents or 0.5 M KCl to cause a rise in cytoplasmic calcium concentration [24]. Bioluminescence was detected at very restricted sites at the base of each of tentacle bulb around the bell margin (figure 1a). We cannot rule out the possibility that additional sites emit low-level bioluminescence, undetectable by our methods. As previously shown in C. gregarium [22], bioluminescence was detectable in spawned C. hemisphaerica eggs (figure 1b,c) and could be fully discharged by detergent lysis. Importantly, we found that pre-incubation in coelenterazine was required for the detection of bioluminescence in our laboratory-reared Clytia medusae and eggs, probably because this essential photoprotein substrate is normally supplied in the marine crustacean diet but not present in the artemia we used for feeding [25,26].Figure 1.


An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer.

Fourrage C, Swann K, Gonzalez Garcia JR, Campbell AK, Houliston E - Open Biol (2014)

Bioluminescence in C. hemisphaerica. Bioluminescence image of an immobilized whole adult medusa, approximately 1 cm in diameter, stimulated with 0.5 M KCl. Pixels are colour coded according to a scale in which hot colours (including white) indicate the highest levels of bioluminescence, and cold colours represent low bioluminescence. (a) Inset shows the same medusa by light microscopy indicating the position of tentacle bulbs (Tb), gonads (G) and manubrium (M). (b) Eggs (approx. 180 µm in diameter) treated first with ionomycin (b(i)) and subsequently treated with Triton X-100 to lyse the egg and discharge all clytin photoprotein luminescence (b(ii)). The images show luminescence from 200 s of integrated light collection after ionomycin and then after detergent treatment. Equivalent images taken before the treatment were completely black. Scale bars 100 µm. (c) Luminescence kinetics from one of seven similar eggs treated in the same way.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043110&req=5

RSOB130206F1: Bioluminescence in C. hemisphaerica. Bioluminescence image of an immobilized whole adult medusa, approximately 1 cm in diameter, stimulated with 0.5 M KCl. Pixels are colour coded according to a scale in which hot colours (including white) indicate the highest levels of bioluminescence, and cold colours represent low bioluminescence. (a) Inset shows the same medusa by light microscopy indicating the position of tentacle bulbs (Tb), gonads (G) and manubrium (M). (b) Eggs (approx. 180 µm in diameter) treated first with ionomycin (b(i)) and subsequently treated with Triton X-100 to lyse the egg and discharge all clytin photoprotein luminescence (b(ii)). The images show luminescence from 200 s of integrated light collection after ionomycin and then after detergent treatment. Equivalent images taken before the treatment were completely black. Scale bars 100 µm. (c) Luminescence kinetics from one of seven similar eggs treated in the same way.
Mentions: We visualized the sites of bioluminescence in Clytia hemisphaerica medusae (figure 1) stimulated by treatment with calcium ionophore, detergents or 0.5 M KCl to cause a rise in cytoplasmic calcium concentration [24]. Bioluminescence was detected at very restricted sites at the base of each of tentacle bulb around the bell margin (figure 1a). We cannot rule out the possibility that additional sites emit low-level bioluminescence, undetectable by our methods. As previously shown in C. gregarium [22], bioluminescence was detectable in spawned C. hemisphaerica eggs (figure 1b,c) and could be fully discharged by detergent lysis. Importantly, we found that pre-incubation in coelenterazine was required for the detection of bioluminescence in our laboratory-reared Clytia medusae and eggs, probably because this essential photoprotein substrate is normally supplied in the marine crustacean diet but not present in the artemia we used for feeding [25,26].Figure 1.

Bottom Line: Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria.Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms).Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution.

View Article: PubMed Central - PubMed

Affiliation: Sorbonne Universités, UPMC Univ Paris 06, Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Observatoire Océanologique, 06230 Villefranche-sur-mer, France.

ABSTRACT
Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

Show MeSH
Related in: MedlinePlus