Fig 1.
Light-dependent activation of transducin by parapinopsins and bovine rhodopsin in vitro.
Time courses of the ability of pufferfish, zebrafish, and lamprey parapinopsins and bovine rhodopsin to activate transducin in the dark (filled circles) and after light irradiation (open circles). The transducin activation ability of parapinopsin was measured after irradiation of parapinopsin with ultraviolet (UV) light for 30 s, followed by incubation for 30 s at 20°C for the indicated times. In addition, the bovine rhodopsin that was irradiated with >520 nm light was applied to the same assay. Note that purified parapinopsins (final concentration of 75 nM) or purified bovine rhodopsin (final concentration of 1.5 nM) was used for the assay. (Inset) The transducin activation rates of parapinopsins were compared in the dark (D), after UV-light irradiation (UV) and after additional orange light (>530 nm light) irradiation following UV-light irradiation (UV→O). Data were expressed as means of three separate experiments with standard errors.
Fig 2.
Immunoreactivity of transducins in the pufferfish retina and pineal organ.
(A, B) The antibodies against Gt1 (A) and Gt2 (B) specifically stain the outer segments of rod (ROS) and cone (COS) photoreceptor cells, respectively. (C, D) Immunoreactivities of antibody to Gt1 (C) and Gt2 (D) are observed in the pineal organ. Scale bars = 50 μm.
Fig 3.
Immunohistochemical localization of parapinopsin and transducins in the pufferfish pineal organ.
Parapinopsin (A, green) and Gt1 (B, magenta) are not colocalized (merged image in panel C), whereas parapinopsin (D, green) and Gt2 (E, magenta) are colocalized (merged image in panel F). The arrowheads show the parapinopsin immunoreactivity in the pineal organ. Scale bars = 20 μm. Low magnification images of the pufferfish pineal organ (white dotted traces) are shown in insets.
Fig 4.
Immunohistochemical localization of transducins in the zebrafish pineal organ.
(A, B) The antibodies against Gt1 (A) and Gt2 (B) immunostained the outer segments of rod (ROS) and cone (COS) photoreceptor cells, respectively. (C–E) Parapinopsin (C, green) and Gt1 (D, magenta) are not colocalized (merged image in panel E, arrowhead). (F–H) Parapinopsin (F, green) and Gt2 (G, magenta) are colocalized (merged image in panel H). The arrowheads show the parapinopsin immunoreactivity in the pineal organ. Scale bars = 20 μm.
Fig 5.
Localization of transducins in the lamprey pineal organ.
(A, B) In situ hybridizations with the antisense probes of GtS and GtL show that GtS is expressed in the photoreceptor cells of the dorsal and ventral regions (A), but GtL is not detected (B). (C) In situ hybridization with the parapinopsin antisense probe shows that parapinopsin is expressed in the photoreceptor cells of the dorsal region. (D) Parapinopsin immunoreactivity (magenta) is localized in the dorsal region of the lamprey pineal organ. (E) Transducin immunoreactivity (green) is distributed in both the dorsal and ventral regions using anti-rod/cone transducin antibody TF15. (F) Parapinopsin and transducin are colocalized in the dorsal photoreceptor cells (white in merged image). Scale bars = 50 μm.
Fig 6.
Light-induced cAMP concentration changes in HEK293 cells expressing lamprey parapinopsin or goldfish UV cone visual opsin.
Ultraviolet- (UV) light-induced decrease in luminescence signals, which represent the cAMP level, is observed in the lamprey parapinopsin-expressing (A) and the goldfish UV cone visual opsin-expressing (B) HEK293S cells. However, a green light-induced increase (recovery) of cAMP level is found in the lamprey parapinopsin-expressing (A) but not in the goldfish UV cone visual opsin-expressing (B) HEK293S cells. The arrows and vertical lines indicate forskolin treatments and UV and green light irradiations, respectively.