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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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Clytia GFP2 and clytin2 are targeted to egg mitochondria. (a) Confocal images of eggs labelled with Mitotracker Deep Red. (a(i)) GFP fluorescence, (a(ii)) Mitotracker, (a(iii)) merge. (b) Stratified eggs comparing GFP versus Mitotracker orange distribution. (b(i)) Brightfield, (b(ii)) GFP fluorescence, (b(iii)) Mitotracker orange. (c) Stratified eggs comparing green fluorescence (c(i)) and Triton lysis-induced luminescence (c(ii)). All scale bars 100 µm.
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RSOB130206F6: Clytia GFP2 and clytin2 are targeted to egg mitochondria. (a) Confocal images of eggs labelled with Mitotracker Deep Red. (a(i)) GFP fluorescence, (a(ii)) Mitotracker, (a(iii)) merge. (b) Stratified eggs comparing GFP versus Mitotracker orange distribution. (b(i)) Brightfield, (b(ii)) GFP fluorescence, (b(iii)) Mitotracker orange. (c) Stratified eggs comparing green fluorescence (c(i)) and Triton lysis-induced luminescence (c(ii)). All scale bars 100 µm.

Mentions: The spectral comparisons revealed intriguing high-efficiency in vivo coupling between the co-expressed clytin2 and GFP2 proteins in Clytia eggs compared with medusa tissues. A further surprise came from closer examination of protein localization in the egg, where we found both fluorescence and bioluminescence localized to mitochondria (figure 6). Firstly, as reported briefly elsewhere [30], confocal microscopy showed the maternally expressed GFP2 protein to be contained within discrete vesicular structures, confirmed as mitochondria by comparison with the vital Mitotracker (figure 6a) or TMRE dyes, or use of antibodies recognizing the mitochondrial protein VDAC (not shown). Furthermore, low-speed egg centrifugation co-stratified green fluorescence with organelles stained with Mitotracker Orange (figure 6b). In such centrifuged eggs, coelenterazine-dependent, detergent-induced bioluminescence was emitted from the mitochondria-rich zone (figure 6c), indicating that the maternal clytin as well as GFP is targeted to mitochondria.Figure 6.


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)

Clytia GFP2 and clytin2 are targeted to egg mitochondria. (a) Confocal images of eggs labelled with Mitotracker Deep Red. (a(i)) GFP fluorescence, (a(ii)) Mitotracker, (a(iii)) merge. (b) Stratified eggs comparing GFP versus Mitotracker orange distribution. (b(i)) Brightfield, (b(ii)) GFP fluorescence, (b(iii)) Mitotracker orange. (c) Stratified eggs comparing green fluorescence (c(i)) and Triton lysis-induced luminescence (c(ii)). All scale bars 100 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB130206F6: Clytia GFP2 and clytin2 are targeted to egg mitochondria. (a) Confocal images of eggs labelled with Mitotracker Deep Red. (a(i)) GFP fluorescence, (a(ii)) Mitotracker, (a(iii)) merge. (b) Stratified eggs comparing GFP versus Mitotracker orange distribution. (b(i)) Brightfield, (b(ii)) GFP fluorescence, (b(iii)) Mitotracker orange. (c) Stratified eggs comparing green fluorescence (c(i)) and Triton lysis-induced luminescence (c(ii)). All scale bars 100 µm.
Mentions: The spectral comparisons revealed intriguing high-efficiency in vivo coupling between the co-expressed clytin2 and GFP2 proteins in Clytia eggs compared with medusa tissues. A further surprise came from closer examination of protein localization in the egg, where we found both fluorescence and bioluminescence localized to mitochondria (figure 6). Firstly, as reported briefly elsewhere [30], confocal microscopy showed the maternally expressed GFP2 protein to be contained within discrete vesicular structures, confirmed as mitochondria by comparison with the vital Mitotracker (figure 6a) or TMRE dyes, or use of antibodies recognizing the mitochondrial protein VDAC (not shown). Furthermore, low-speed egg centrifugation co-stratified green fluorescence with organelles stained with Mitotracker Orange (figure 6b). In such centrifuged eggs, coelenterazine-dependent, detergent-induced bioluminescence was emitted from the mitochondria-rich zone (figure 6c), indicating that the maternal clytin as well as GFP is targeted to mitochondria.Figure 6.

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