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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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Related in: MedlinePlus

Sites of CheGFP and clytin gene expression. GFP (a–p) and clytin (q–t) expression detected by in situ hybridization in adult C. hemisphaerica medusae, egg and planula larvae. GFP1 is detected in manubrium and gonad ectoderm (a,b) and in planula larva (d). GFP2 expression is strongly detected in unfertilized eggs (g), and restricted to oocytes (f) and a small proximal zone in tentacle bulb (e) in adult female tissues. GFP3 appears weakly expressed in umbrella (i), while GFP4 is strongly expressed in manubrium (m) and gonad ectoderm (n). In situ hybridization with a clytin3 antisense probe detects the combined expression of the three clytin genes in a proximal and central zone of tentacle bulbs and oocytes of gonad (r), strong expression in eggs (s) and weaker expression in planula (t). Clytin1 and clytin2 probes gave equivalent results owing to the cross-detection of highly similar sequences. m, manubrium; g, gonad; o, oocyte; tb, tentacle bulb; um, bell. Scale bars, 100 µm.
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RSOB130206F3: Sites of CheGFP and clytin gene expression. GFP (a–p) and clytin (q–t) expression detected by in situ hybridization in adult C. hemisphaerica medusae, egg and planula larvae. GFP1 is detected in manubrium and gonad ectoderm (a,b) and in planula larva (d). GFP2 expression is strongly detected in unfertilized eggs (g), and restricted to oocytes (f) and a small proximal zone in tentacle bulb (e) in adult female tissues. GFP3 appears weakly expressed in umbrella (i), while GFP4 is strongly expressed in manubrium (m) and gonad ectoderm (n). In situ hybridization with a clytin3 antisense probe detects the combined expression of the three clytin genes in a proximal and central zone of tentacle bulbs and oocytes of gonad (r), strong expression in eggs (s) and weaker expression in planula (t). Clytin1 and clytin2 probes gave equivalent results owing to the cross-detection of highly similar sequences. m, manubrium; g, gonad; o, oocyte; tb, tentacle bulb; um, bell. Scale bars, 100 µm.

Mentions: Each of the four C. hemisphaerica GFP genes was found to have a distinct stage- and tissue-specific expression profile, revealed by in situ hybridization (figure 3) and quantitative PCR (Q-PCR) (figure 4). The individual GFP gene expression profiles (figure 3a–p) covered all the sites of green fluorescence, indicating that no significantly expressed GFP genes in the Clytia genome had been overlooked. CheGFP1 expression accounted for the fluorescence in the planula larva ectoderm and it is also expressed significantly in the medusa manubrium and gonad ectoderm. CheGFP4 is strongly expressed at the same medusa sites but not in the planula. Expression of CheGFP2, the ‘maternal’ GFP, was strongly detected in developing oocytes as well as in spawned eggs, and also at the tentacle bulb photophores, but not elsewhere. CheGFP3 in situ signal was distributed mainly in the thin ectoderm of the medusa umbrella, where some CheGFP4 expression was also apparent.Figure 3.


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)

Sites of CheGFP and clytin gene expression. GFP (a–p) and clytin (q–t) expression detected by in situ hybridization in adult C. hemisphaerica medusae, egg and planula larvae. GFP1 is detected in manubrium and gonad ectoderm (a,b) and in planula larva (d). GFP2 expression is strongly detected in unfertilized eggs (g), and restricted to oocytes (f) and a small proximal zone in tentacle bulb (e) in adult female tissues. GFP3 appears weakly expressed in umbrella (i), while GFP4 is strongly expressed in manubrium (m) and gonad ectoderm (n). In situ hybridization with a clytin3 antisense probe detects the combined expression of the three clytin genes in a proximal and central zone of tentacle bulbs and oocytes of gonad (r), strong expression in eggs (s) and weaker expression in planula (t). Clytin1 and clytin2 probes gave equivalent results owing to the cross-detection of highly similar sequences. m, manubrium; g, gonad; o, oocyte; tb, tentacle bulb; um, bell. Scale bars, 100 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB130206F3: Sites of CheGFP and clytin gene expression. GFP (a–p) and clytin (q–t) expression detected by in situ hybridization in adult C. hemisphaerica medusae, egg and planula larvae. GFP1 is detected in manubrium and gonad ectoderm (a,b) and in planula larva (d). GFP2 expression is strongly detected in unfertilized eggs (g), and restricted to oocytes (f) and a small proximal zone in tentacle bulb (e) in adult female tissues. GFP3 appears weakly expressed in umbrella (i), while GFP4 is strongly expressed in manubrium (m) and gonad ectoderm (n). In situ hybridization with a clytin3 antisense probe detects the combined expression of the three clytin genes in a proximal and central zone of tentacle bulbs and oocytes of gonad (r), strong expression in eggs (s) and weaker expression in planula (t). Clytin1 and clytin2 probes gave equivalent results owing to the cross-detection of highly similar sequences. m, manubrium; g, gonad; o, oocyte; tb, tentacle bulb; um, bell. Scale bars, 100 µm.
Mentions: Each of the four C. hemisphaerica GFP genes was found to have a distinct stage- and tissue-specific expression profile, revealed by in situ hybridization (figure 3) and quantitative PCR (Q-PCR) (figure 4). The individual GFP gene expression profiles (figure 3a–p) covered all the sites of green fluorescence, indicating that no significantly expressed GFP genes in the Clytia genome had been overlooked. CheGFP1 expression accounted for the fluorescence in the planula larva ectoderm and it is also expressed significantly in the medusa manubrium and gonad ectoderm. CheGFP4 is strongly expressed at the same medusa sites but not in the planula. Expression of CheGFP2, the ‘maternal’ GFP, was strongly detected in developing oocytes as well as in spawned eggs, and also at the tentacle bulb photophores, but not elsewhere. CheGFP3 in situ signal was distributed mainly in the thin ectoderm of the medusa umbrella, where some CheGFP4 expression was also apparent.Figure 3.

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