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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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Endogenous luminescence coupling. (a) Fluorescence emission spectra for the four GFPs expressed in their endogenous tissues, collected using a Leica SP5 confocal microscope using 488 nm excitation. (b) Luminescence spectra obtained for recombined and Ca2+-stimulated aequorin compared to a group of eggs and an individual whole adult medusa following detergent stimulation [29]. Multiple spectra obtained from several individual medusae showed very little variation. Aequorin was used in this experiment rather than clytin because it was more easily available but has a very similar emission spectrum [9,15]. (c) Light emission ratios (500/470 nm) from recombined and Ca2+-stimulated aequorin or clytin2 protein was compared to those from eggs or medusae that had been preloaded with coelenterazine and then lysed with Triton to induce Ca2+-dependent luminescence. An average ratio from measurements made on six individual medusae is shown.
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RSOB130206F5: Endogenous luminescence coupling. (a) Fluorescence emission spectra for the four GFPs expressed in their endogenous tissues, collected using a Leica SP5 confocal microscope using 488 nm excitation. (b) Luminescence spectra obtained for recombined and Ca2+-stimulated aequorin compared to a group of eggs and an individual whole adult medusa following detergent stimulation [29]. Multiple spectra obtained from several individual medusae showed very little variation. Aequorin was used in this experiment rather than clytin because it was more easily available but has a very similar emission spectrum [9,15]. (c) Light emission ratios (500/470 nm) from recombined and Ca2+-stimulated aequorin or clytin2 protein was compared to those from eggs or medusae that had been preloaded with coelenterazine and then lysed with Triton to induce Ca2+-dependent luminescence. An average ratio from measurements made on six individual medusae is shown.

Mentions: To assess the extent of the BRET between clytin and GFP in vivo, we determined the GFP fluorescence emission spectra for eggs and selected tissues (figure 5a) and the luminescence spectra for groups of whole jellyfish or collected spawned eggs (figure 5b).Figure 5.


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)

Endogenous luminescence coupling. (a) Fluorescence emission spectra for the four GFPs expressed in their endogenous tissues, collected using a Leica SP5 confocal microscope using 488 nm excitation. (b) Luminescence spectra obtained for recombined and Ca2+-stimulated aequorin compared to a group of eggs and an individual whole adult medusa following detergent stimulation [29]. Multiple spectra obtained from several individual medusae showed very little variation. Aequorin was used in this experiment rather than clytin because it was more easily available but has a very similar emission spectrum [9,15]. (c) Light emission ratios (500/470 nm) from recombined and Ca2+-stimulated aequorin or clytin2 protein was compared to those from eggs or medusae that had been preloaded with coelenterazine and then lysed with Triton to induce Ca2+-dependent luminescence. An average ratio from measurements made on six individual medusae is shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB130206F5: Endogenous luminescence coupling. (a) Fluorescence emission spectra for the four GFPs expressed in their endogenous tissues, collected using a Leica SP5 confocal microscope using 488 nm excitation. (b) Luminescence spectra obtained for recombined and Ca2+-stimulated aequorin compared to a group of eggs and an individual whole adult medusa following detergent stimulation [29]. Multiple spectra obtained from several individual medusae showed very little variation. Aequorin was used in this experiment rather than clytin because it was more easily available but has a very similar emission spectrum [9,15]. (c) Light emission ratios (500/470 nm) from recombined and Ca2+-stimulated aequorin or clytin2 protein was compared to those from eggs or medusae that had been preloaded with coelenterazine and then lysed with Triton to induce Ca2+-dependent luminescence. An average ratio from measurements made on six individual medusae is shown.
Mentions: To assess the extent of the BRET between clytin and GFP in vivo, we determined the GFP fluorescence emission spectra for eggs and selected tissues (figure 5a) and the luminescence spectra for groups of whole jellyfish or collected spawned eggs (figure 5b).Figure 5.

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