XB-ART-49055J Comp Neurol November 1, 2014; 522 (16): 3577-3589.
Retrograde intraciliary trafficking of opsin during the maintenance of cone-shaped photoreceptor outer segments of Xenopus laevis.
Photoreceptor outer segments (OSs) are essential for our visual perception, and take either rod or cone forms. The cell biological basis for the formation of rods is well established; however, the mechanism of cone formation is ill characterized. While Xenopus rods are called rods, they exhibit cone-shaped OSs during the early process of development. To visualize the dynamic reorganization of disk membranes, opsin and peripherin/rds were fused to a fluorescent protein, Dendra2, and expressed in early developing rod photoreceptors, in which OSs are still cone-shaped. Dendra2 is a fluorescent protein which can be converted from green to red irreversibly, and thus allows spatiotemporal labeling of proteins. Using a photoconversion technique, we found that disk membranes are assembled at the base of cone-shaped OSs. After incorporation into disks, however, Opsin-Dendra2 was also trafficked from old to new disk membranes, consistent with the hypothesis that retrograde trafficking of membrane components contributes to the larger disk membrane observed toward the base of the cone-shaped OS. Such retrograde trafficking is cargo-specific and was not observed for peripherin/rds-Dendra2. The trafficking is unlikely mediated by diffusion, since the disk membranes have a closed configuration, as evidenced by CNGA1 labeling of the plasma membrane. Consistent with retrograde trafficking, the axoneme, which potentially mediates retrograde intraflagellar trafficking, runs through the entire axis of OSs. This study provides an insight into the role of membrane reorganization in developing photoreceptor OSs, and proves that retrograde trafficking of membrane cargoes can occur there.
PubMed ID: 24855015
PMC ID: PMC4142104
Article link: J Comp Neurol
Genes referenced: cnga1 prph prph2
Article Images: [+] show captions
|Figure 1. Rod photoreceptor cells are cone-shaped at the early stages of development. A: Retinas of tadpoles expressing Opsin-Dend2 or P/rds-Dend2 in rod photoreceptors were imaged for Dend2 fluorescence at 6/7, 9, and 21–22 dpf. Rod photoreceptors are cone-shaped at the early stages of development and become rod-shaped as tadpoles age. B: Eyes of tadpoles expressing Opsin-Dend2 (green) were fixed at 6 dpf and stained with wheat germ agglutinin (WGA, blue) and an antibody against Xenopus peripherin/rds (P/rds, red). Peripherin/rds localizes to rod disk rim (arrowhead) and cone disk rim (arrow) regions that are structurally distinct. Rod and cone photoreceptor cells are indicated in the figure. Opsin-Dend2 is specifically expressed in rod cells. Scale bars = 10 μm.|
|Figure 2. Two models for formation of cone-shaped OSs. Dend2 protein is shifted from green to red fluorescent isoform by photoconversion. After photoconversion, newly synthesized Dend2 protein is green and is added to the base of the OS in of both the models. A: In model A, the cone shape is formed by the retrograde trafficking of protein from older to newer disks. Shortly after photoconversion, newly synthesized green Opsin-Dend2 is added to the evaginations formed before the photoconversion to form new disks, therefore, a few disks contain both green (new) and red (older) proteins generating yellow fluorescence (red + green = yellow). Due to the retrograde trafficking of Opsin-Dend2, the number of disks with both old and new protein increases over time after photoconversion. B: The cone shape is formed by the addition of gradually larger disks at the base of the OS. There is no trafficking between disks as indicated by a clear separation of proteins before and after photoconversion.|
|Figure 3. The renewal of rod photoreceptor OSs. Tadpoles expressing Opsin-Dend2 were photoconverted at 6 dpf. Retinas were dissected and imaged for green and red fluorescence 0 hour (immediately after photoconversion) (A), 4 hours (B), 8 hours (C), 1 day (D), and 2 days (E) after photoconversion. F: Tadpoles expressing P/rds-Dend2 were photoconverted at 6 dpf and the retinas were dissected and imaged for green and red fluorescence 2 days later. G,H: Tadpoles expressing Opsin-Dend2 were photoconverted at 21–22 dpf and the retinas were dissected and imaged for green and red fluorescence 2 days (G) and 6 days (H) later, respectively. The images in the mid row are for red-Dend2 fluorescence, and represented by multiple colors. The intensity-color relationship is as shown in the middle panel of (A). In the bottom row of each image panel, the intensity of green and red fluorescence was measured in ImageJ and the percentage of Dend2 fusion protein along the longitudinal axis of the OS was calculated by normalizing to the highest concentration (100%) on the axis. Scale bar = 10 μm.|
|Figure 5. Organization of membrane and cytoskeletal structures in OSs of developing rod photoreceptors. A: Unfixed rod photoreceptor expressing CNGA1-Dend2, a plasma membrane marker, was imaged for Dend2 fluorescence at 6 dpf. The OS is outlined by plasma membrane, indicative of disks taking closed configurations. B: Fixed rod photoreceptors of nontransgenic tadpoles were probed with wheat germ agglutinin (WGA, red) and an antibody against acetylated tubulin (green, axoneme marker). The axoneme extended the whole length of the OS when tadpoles were 6 dpf. Images are from a single x-y focal plane. Multiple photoreceptors were analyzed and the representative images are shown in (A,B). Scale bars = 5 μm.|
|Figure 6. Lack of retrograde opsin trafficking in mature rod OSs. A: Retinas were dissected and imaged for green and red fluorescence 2 days or 6 days after photoconversion at 21–22 dpf, or without photoconversion at 21–22 dpf. The intensity of green and red fluorescence was measured in ImageJ and the percentage of Opsin-Dend2 protein along the longitudinal axis of the OS was calculated by normalizing to the intensities at the crosspoint (100%). For OSs 2 days and 6 days after photoconversion, old Opsin-Dend2 protein concentrations in the disks from the crosspoint toward the base of the OS were normalized to their concentrations at the crosspoint. Likewise for nonphotoconverted OSs (0 d), the concentration distribution of Opsin-Dend2 around the bottom of the OS was normalized to the intensity closest to 50% of the maximum concentration along the OS axis. There were no significant differences among 0 day, 2 days, and 6 days in terms of the distribution of Opsin-Dend2. Thus, there was no retrograde trafficking in 21–22 dpf rod photoreceptors. For 0 and 2 days after photoconversion, n = 20 rod photoreceptors from three tadpoles were used for analysis. For 6 days after photoconversion, n = 10 rod photoreceptors from three tadpoles were used for analysis. The data points are represented by mean ± SE. B: The fluorescence intensities of Opsin-Dend2 in the rod OSs from the tadpoles at 7 (blue) and 21–22 (magenta) dpf without photoconversion were normalized to the highest intensities along the axes, and plotted along the longitudinal axis of the OS. C: The central regions (1.3 μm width) of OSs, as shown in B (light green), were used to calculate the standard deviations of normalized fluorescence intensities. Concentrations of Opsin-Dend2 along the longitudinal axis of rod photoreceptors were less variable in 7 dpf than in 21–22 dpf tadpoles. For both 7 and 21–22 dpf, n = 20 rod photoreceptors from three tadpoles were used for the calculations. ***P < 0.001 by the Mann–Whitney Rank Sum Test. Error bars represent standard deviation in C.|