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Expression of UV-sensitive parapinopsin in the iguana parietal eyes and its implication in UV-sensitivity in vertebrate pineal-related organs.

Wada S, Kawano-Yamashita E, Koyanagi M, Terakita A - PLoS ONE (2012)

Bottom Line: Spectroscopic analysis revealed that iguana parapinopsin is a UV-sensitive pigment, similar to lamprey parapinopsin.These results strongly suggest that parapinopsin underlies the wavelength discrimination involving UV reception in the iguana parietal eye.The current findings support the idea that parapinopsin is a common photopigment underlying the UV-sensitivity in wavelength discrimination of the pineal-related organs found from lampreys to reptiles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Geosciences, Graduate School of Sciences, Osaka City University, Osaka, Japan.

ABSTRACT
The pineal-related organs of lower vertebrates have the ability to discriminate different wavelengths of light. This wavelength discrimination is achieved through antagonistic light responses to UV or blue and visible light. Previously, we demonstrated that parapinopsin underlies the UV reception in the lamprey pineal organ and identified parapinopsin genes in teleosts and frogs of which the pineal-related organs were reported to discriminate light. In this study, we report the first identification of parapinopsin in the reptile lineage and show its expression in the parietal eye of the green iguana. Spectroscopic analysis revealed that iguana parapinopsin is a UV-sensitive pigment, similar to lamprey parapinopsin. Interestingly, immunohistochemical analyses using antibodies specific to parapinopsin and parietopsin, a parietal eye green-sensitive pigment, revealed that parapinopsin and parietopsin are colocalized in the outer segments of the parietal eye photoreceptor cells in iguanas. These results strongly suggest that parapinopsin underlies the wavelength discrimination involving UV reception in the iguana parietal eye. The current findings support the idea that parapinopsin is a common photopigment underlying the UV-sensitivity in wavelength discrimination of the pineal-related organs found from lampreys to reptiles.

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The specificity of the antibodies to parapinopsin and parietopsin.The anti-iguana parapinopsin antibody (anti-PP) labels the cells expressing iguana parapinopsin (A, PP cells) but not those expressing iguana parietopsin (B, PRT cells). The anti-iguana parietopsin antibody (anti-PRT) labels PRT cells (C) but not PP cells (D). (E) Immunoblot profiles showing that anti-PP and anti-PRT antibodies specifically recognize parapinopsin and parietopsin expressed in cultured cells. Monoclonal antibody rho 1D4 stains iguana parapinopsin (arrowhead) and parietopsin (asterisk) tagged with rho 1D4 epitope, in SDS-extracts form PP and PRT cells (lanes 3 and 4) but not from mock transfected cells (lane 2). Anti-PP specifically stained ∼38 k peptide (lane 6, arrowhead), which is the same as the band stained by rho 1D4 (lane 3), in PP cells but not in mock cells (lane 5) or PRT cells (lane 7). Anti-PRT antibodies recognize specifically ∼39 k peptide (lane 10, asterisk), which is identical to the stained band by rho 1D4 (lane 4) in PRT cells, but not in mock cells (lane 8) or PP cells (lane 9). M indicates Molecular weight standard markers (lane 1) (Bio-Rad Laboratories).
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pone-0039003-g004: The specificity of the antibodies to parapinopsin and parietopsin.The anti-iguana parapinopsin antibody (anti-PP) labels the cells expressing iguana parapinopsin (A, PP cells) but not those expressing iguana parietopsin (B, PRT cells). The anti-iguana parietopsin antibody (anti-PRT) labels PRT cells (C) but not PP cells (D). (E) Immunoblot profiles showing that anti-PP and anti-PRT antibodies specifically recognize parapinopsin and parietopsin expressed in cultured cells. Monoclonal antibody rho 1D4 stains iguana parapinopsin (arrowhead) and parietopsin (asterisk) tagged with rho 1D4 epitope, in SDS-extracts form PP and PRT cells (lanes 3 and 4) but not from mock transfected cells (lane 2). Anti-PP specifically stained ∼38 k peptide (lane 6, arrowhead), which is the same as the band stained by rho 1D4 (lane 3), in PP cells but not in mock cells (lane 5) or PRT cells (lane 7). Anti-PRT antibodies recognize specifically ∼39 k peptide (lane 10, asterisk), which is identical to the stained band by rho 1D4 (lane 4) in PRT cells, but not in mock cells (lane 8) or PP cells (lane 9). M indicates Molecular weight standard markers (lane 1) (Bio-Rad Laboratories).

Mentions: To localize parapinopsin and parietopsin in the iguana parietal eye, we raised antibodies against C-terminal regions of iguana parapinopsin (49 amino acids) and parietopsin (48 amino acids) (Figure S2), because their C-terminal sequences are quite different from each other and from those of other opsins. The anti-parapinopsin and anti-parietopsin antibodies specifically labeled cultured cells expressing iguana parapinopsin and parietopsin, respectively (Figure 4A-D). They also labeled specifically ∼38 k and ∼39 k peptides in SDS-extract from transfected cells and their apparent molecular masses are similar to ones calculated based on amino acid sequences of parapinopsin and parietopsin, respectively (Figure 4E). In addition, no immunoreactivities of the antibodies were detected in the iguana pineal organ where pinopsin is expressed (Figure 2) and the retina having several kinds of visual pigments in rods and cones (Figure S3). It can be also speculated that encephalopsin (OPN3), melanopsin (OPN4), and neuropsin (OPN5) homologues exist in non-visual retinal cells according to observations for other lower vertebrates [20]–[23]. These results strongly suggested that these antibodies specifically recognized parapinopsin and parietopsin, respectively, and allowed us to investigate localization of parapinopsin and parietopsin in the parietal eye with these antibodies. The anti-parapinopsin and anti-parietopsin antibodies labeled the distal part of the photoreceptor layer of the iguana parietal eye retina, indicating that both parapinopsin and parietopsin are localized in the outer segments of the photoreceptor cells (Figure 5A-C, E, F). Remarkably, double-stained images show the colocalization of parapinopsin and parietopsin in the same photoreceptor outer segment (Figure 5D and G), suggesting that parapinopsin and parietopsin function in the same photoreceptor cells in the iguana parietal eye. The colocalization was also confirmed by confocal images, stained with the antibodies against iguana parapinopsin and side-blotched lizard parietopsin [13] (Figure S4).


Expression of UV-sensitive parapinopsin in the iguana parietal eyes and its implication in UV-sensitivity in vertebrate pineal-related organs.

Wada S, Kawano-Yamashita E, Koyanagi M, Terakita A - PLoS ONE (2012)

The specificity of the antibodies to parapinopsin and parietopsin.The anti-iguana parapinopsin antibody (anti-PP) labels the cells expressing iguana parapinopsin (A, PP cells) but not those expressing iguana parietopsin (B, PRT cells). The anti-iguana parietopsin antibody (anti-PRT) labels PRT cells (C) but not PP cells (D). (E) Immunoblot profiles showing that anti-PP and anti-PRT antibodies specifically recognize parapinopsin and parietopsin expressed in cultured cells. Monoclonal antibody rho 1D4 stains iguana parapinopsin (arrowhead) and parietopsin (asterisk) tagged with rho 1D4 epitope, in SDS-extracts form PP and PRT cells (lanes 3 and 4) but not from mock transfected cells (lane 2). Anti-PP specifically stained ∼38 k peptide (lane 6, arrowhead), which is the same as the band stained by rho 1D4 (lane 3), in PP cells but not in mock cells (lane 5) or PRT cells (lane 7). Anti-PRT antibodies recognize specifically ∼39 k peptide (lane 10, asterisk), which is identical to the stained band by rho 1D4 (lane 4) in PRT cells, but not in mock cells (lane 8) or PP cells (lane 9). M indicates Molecular weight standard markers (lane 1) (Bio-Rad Laboratories).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3375259&req=5

pone-0039003-g004: The specificity of the antibodies to parapinopsin and parietopsin.The anti-iguana parapinopsin antibody (anti-PP) labels the cells expressing iguana parapinopsin (A, PP cells) but not those expressing iguana parietopsin (B, PRT cells). The anti-iguana parietopsin antibody (anti-PRT) labels PRT cells (C) but not PP cells (D). (E) Immunoblot profiles showing that anti-PP and anti-PRT antibodies specifically recognize parapinopsin and parietopsin expressed in cultured cells. Monoclonal antibody rho 1D4 stains iguana parapinopsin (arrowhead) and parietopsin (asterisk) tagged with rho 1D4 epitope, in SDS-extracts form PP and PRT cells (lanes 3 and 4) but not from mock transfected cells (lane 2). Anti-PP specifically stained ∼38 k peptide (lane 6, arrowhead), which is the same as the band stained by rho 1D4 (lane 3), in PP cells but not in mock cells (lane 5) or PRT cells (lane 7). Anti-PRT antibodies recognize specifically ∼39 k peptide (lane 10, asterisk), which is identical to the stained band by rho 1D4 (lane 4) in PRT cells, but not in mock cells (lane 8) or PP cells (lane 9). M indicates Molecular weight standard markers (lane 1) (Bio-Rad Laboratories).
Mentions: To localize parapinopsin and parietopsin in the iguana parietal eye, we raised antibodies against C-terminal regions of iguana parapinopsin (49 amino acids) and parietopsin (48 amino acids) (Figure S2), because their C-terminal sequences are quite different from each other and from those of other opsins. The anti-parapinopsin and anti-parietopsin antibodies specifically labeled cultured cells expressing iguana parapinopsin and parietopsin, respectively (Figure 4A-D). They also labeled specifically ∼38 k and ∼39 k peptides in SDS-extract from transfected cells and their apparent molecular masses are similar to ones calculated based on amino acid sequences of parapinopsin and parietopsin, respectively (Figure 4E). In addition, no immunoreactivities of the antibodies were detected in the iguana pineal organ where pinopsin is expressed (Figure 2) and the retina having several kinds of visual pigments in rods and cones (Figure S3). It can be also speculated that encephalopsin (OPN3), melanopsin (OPN4), and neuropsin (OPN5) homologues exist in non-visual retinal cells according to observations for other lower vertebrates [20]–[23]. These results strongly suggested that these antibodies specifically recognized parapinopsin and parietopsin, respectively, and allowed us to investigate localization of parapinopsin and parietopsin in the parietal eye with these antibodies. The anti-parapinopsin and anti-parietopsin antibodies labeled the distal part of the photoreceptor layer of the iguana parietal eye retina, indicating that both parapinopsin and parietopsin are localized in the outer segments of the photoreceptor cells (Figure 5A-C, E, F). Remarkably, double-stained images show the colocalization of parapinopsin and parietopsin in the same photoreceptor outer segment (Figure 5D and G), suggesting that parapinopsin and parietopsin function in the same photoreceptor cells in the iguana parietal eye. The colocalization was also confirmed by confocal images, stained with the antibodies against iguana parapinopsin and side-blotched lizard parietopsin [13] (Figure S4).

Bottom Line: Spectroscopic analysis revealed that iguana parapinopsin is a UV-sensitive pigment, similar to lamprey parapinopsin.These results strongly suggest that parapinopsin underlies the wavelength discrimination involving UV reception in the iguana parietal eye.The current findings support the idea that parapinopsin is a common photopigment underlying the UV-sensitivity in wavelength discrimination of the pineal-related organs found from lampreys to reptiles.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Geosciences, Graduate School of Sciences, Osaka City University, Osaka, Japan.

ABSTRACT
The pineal-related organs of lower vertebrates have the ability to discriminate different wavelengths of light. This wavelength discrimination is achieved through antagonistic light responses to UV or blue and visible light. Previously, we demonstrated that parapinopsin underlies the UV reception in the lamprey pineal organ and identified parapinopsin genes in teleosts and frogs of which the pineal-related organs were reported to discriminate light. In this study, we report the first identification of parapinopsin in the reptile lineage and show its expression in the parietal eye of the green iguana. Spectroscopic analysis revealed that iguana parapinopsin is a UV-sensitive pigment, similar to lamprey parapinopsin. Interestingly, immunohistochemical analyses using antibodies specific to parapinopsin and parietopsin, a parietal eye green-sensitive pigment, revealed that parapinopsin and parietopsin are colocalized in the outer segments of the parietal eye photoreceptor cells in iguanas. These results strongly suggest that parapinopsin underlies the wavelength discrimination involving UV reception in the iguana parietal eye. The current findings support the idea that parapinopsin is a common photopigment underlying the UV-sensitivity in wavelength discrimination of the pineal-related organs found from lampreys to reptiles.

Show MeSH