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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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Fluorescence in C. hemisphaerica. Green fluorescence observed upon excitation with blue light under a stereomicroscope (a), fluorescence microscope (b,f) or confocal microscope with 488 nm laser excitation (c,d,e,g), superimposed on transmitted light images in (d,e,g). (a) Adult medusa immobilized in agar (courtesy of A. Amiel); M, manubrium; G, gonad; Tb, tentacle bulb. (b) Bell margin with tentacle bulb (baby medusa), (c) manubrium (baby medusa), (d) tentacle bulb, (e) tentacle, (f) planula larva (2 days post fertilization), (g) stolon. For a guide to medusa organization, see figure 4. All scale bars 100 µm.
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RSOB130206F2: Fluorescence in C. hemisphaerica. Green fluorescence observed upon excitation with blue light under a stereomicroscope (a), fluorescence microscope (b,f) or confocal microscope with 488 nm laser excitation (c,d,e,g), superimposed on transmitted light images in (d,e,g). (a) Adult medusa immobilized in agar (courtesy of A. Amiel); M, manubrium; G, gonad; Tb, tentacle bulb. (b) Bell margin with tentacle bulb (baby medusa), (c) manubrium (baby medusa), (d) tentacle bulb, (e) tentacle, (f) planula larva (2 days post fertilization), (g) stolon. For a guide to medusa organization, see figure 4. All scale bars 100 µm.

Mentions: The distribution of endogenous green fluorescence in C. hemisphaerica was readily detectable by fluorescence microscopy upon excitation with blue light, in the absence of coelenterazine (figure 2). Highly fluorescent structures included both major sites of bioluminescence (tentacle bulb spots and oocytes/eggs) but also two additional sites of fully grown adult jellyfish, the manubrium and the gonad (figure 2a,c). Closer examination of the tentacle bulb photophore revealed small masses of highly fluorescent cells adjacent to the endoderm, and individual fluorescent cells extending into the central region of each tentacle (figure 2b,d,e). In the manubrium (mouth and stomach region) and gonad, strong green fluorescence was found in both endodermal and ectodermal cells (figure 2c). Weaker fluorescence was observed across the bell (figure 2b). The planula larva showed strong fluorescence, localized to a subpopulation of epitheliomuscular ectodermal cells, largely absent from the aboral pole region (figure 2f). In polyp colonies, green fluorescence was observed in isolated cells in the endoderm of stolon, hydranth (figure 2g) and polyp tentacles as well as in some nematocytes (not shown). Fluorescence without bioluminescence in gonad somatic tissue and in planula larvae in C. hemisphaerica contrasts with the lack of green fluorescence in these tissues reported in C. gregarium [22] but has been reported in the manubrium of Obelia medusa [17,27].Figure 2.


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)

Fluorescence in C. hemisphaerica. Green fluorescence observed upon excitation with blue light under a stereomicroscope (a), fluorescence microscope (b,f) or confocal microscope with 488 nm laser excitation (c,d,e,g), superimposed on transmitted light images in (d,e,g). (a) Adult medusa immobilized in agar (courtesy of A. Amiel); M, manubrium; G, gonad; Tb, tentacle bulb. (b) Bell margin with tentacle bulb (baby medusa), (c) manubrium (baby medusa), (d) tentacle bulb, (e) tentacle, (f) planula larva (2 days post fertilization), (g) stolon. For a guide to medusa organization, see figure 4. All scale bars 100 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB130206F2: Fluorescence in C. hemisphaerica. Green fluorescence observed upon excitation with blue light under a stereomicroscope (a), fluorescence microscope (b,f) or confocal microscope with 488 nm laser excitation (c,d,e,g), superimposed on transmitted light images in (d,e,g). (a) Adult medusa immobilized in agar (courtesy of A. Amiel); M, manubrium; G, gonad; Tb, tentacle bulb. (b) Bell margin with tentacle bulb (baby medusa), (c) manubrium (baby medusa), (d) tentacle bulb, (e) tentacle, (f) planula larva (2 days post fertilization), (g) stolon. For a guide to medusa organization, see figure 4. All scale bars 100 µm.
Mentions: The distribution of endogenous green fluorescence in C. hemisphaerica was readily detectable by fluorescence microscopy upon excitation with blue light, in the absence of coelenterazine (figure 2). Highly fluorescent structures included both major sites of bioluminescence (tentacle bulb spots and oocytes/eggs) but also two additional sites of fully grown adult jellyfish, the manubrium and the gonad (figure 2a,c). Closer examination of the tentacle bulb photophore revealed small masses of highly fluorescent cells adjacent to the endoderm, and individual fluorescent cells extending into the central region of each tentacle (figure 2b,d,e). In the manubrium (mouth and stomach region) and gonad, strong green fluorescence was found in both endodermal and ectodermal cells (figure 2c). Weaker fluorescence was observed across the bell (figure 2b). The planula larva showed strong fluorescence, localized to a subpopulation of epitheliomuscular ectodermal cells, largely absent from the aboral pole region (figure 2f). In polyp colonies, green fluorescence was observed in isolated cells in the endoderm of stolon, hydranth (figure 2g) and polyp tentacles as well as in some nematocytes (not shown). Fluorescence without bioluminescence in gonad somatic tissue and in planula larvae in C. hemisphaerica contrasts with the lack of green fluorescence in these tissues reported in C. gregarium [22] but has been reported in the manubrium of Obelia medusa [17,27].Figure 2.

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