Article Figures & Data
Figures
- Figure 1.
Visual cycle in photoreceptors and RPE cells. Absorption of a photon (hv) by a rhodopsin or cone-opsin pigment molecule induces isomerization of 11-cis-RAL to all-trans-RAL, which subsequently dissociates from the apo-opsin. Free all-trans-RAL is reduced to all-trans-ROL (vitamin A) by all-trans-RDH. The all-trans-ROL is released into the IPM where it binds IRBP and is subsequently taken up by the RPE. The all-trans-ROL is esterified to a fatty acid by LRAT. The resulting all-trans-RE is hydrolyzed and isomerized to 11-cis-ROL by Rpe65-isomerase. The 11-cis-ROL is oxidized by 11-cis-RDH to 11-cis-RAL. Finally, the 11-cis-RAL is released by the RPE cell into the IPM, where it binds IRBP during translocation to the OS. The 11-cis-RAL recombines with apo-rhodopsin or apo-cone opsin to form a new rhodopsin or cone-opsin pigment molecule.
- Figure 2.
Histologic analysis of wild-type and irbp −/− retinas from 5-week-old mice. A , Light microscopic analysis of retina sections. The choroid (Ch), RPE, OS, inner segment (IS), and outer-nuclear layers (ONL) are shown. Scale bars, 20 μm. B , Electron microscopy of OS and RPE. Scale bars, 2.0 μm. Note the disorganization of OS in the irbp −/− retina. Also note the reduced length,increased diameter, and irregularity of OS and the reduced number of nuclei in the ONL of the irbp −/− retina.
- Figure 3.
Retinoid dynamics in retina and RPE from wild-type and irbp −/− mice. A , 11-cis-RAL levels in retinas from mice under the lighting conditions: overnight dark-adapted (DA), immediately postbleach (BL), and at the indicated times in minutes after return to darkness. B , All-trans-RAL levels in retinas. C , All-trans-ROL levels in retinas. D , 11-cis-RAL levels in RPE. E , All-trans-RE levels in RPE. Retinoids are expressed as a percentage of total retinoids per eye. Note the lower 11-cis-RAL, higher all-trans-RAL, and higher all-trans-ROL levels in irbp −/− retinas during postbleach recovery in darkness. Also note the higher 11-cis-RAL and lower all-trans-RE levels in irbp −/− RPE during postbleach recovery. F , Lipofuscin pigments, A-500, A2PE and A2E in wild-type and irbp −/− RPE. Expressed at milli-absorbance units (mAU) per eye. G , Levels of all-trans-RAL in wild-type (WT) and irbp −/− retina explants. H , Levels of all-trans-ROL in WT and irbp −/− retina explants. Retinoids are expressed as a percentage of total retinoids per retina explant. Note the higher all-trans-RAL and all-trans-ROL in irbp −/− explants during postbleach recovery. Asterisks indicate statistically significant differences between wild-type and irbp −/− (p ≤ 0.05). Error bars indicate SDs (n = 4).
- Figure 4.
Electroretinography of wild-type and irbp −/− mice. A , Representative raw ERG tracings of rod responses in three wild-type and irbp −/− mice using stimulus-flash intensities of −2.64 log cd-s/m2. B , Representative raw ERG tracings of cone responses in four wild-type and irbp −/− mice. Data were acquired using stimulus-flash intensities of 0.70 cd-s/m2 with a rod-saturating background light. C , Rod b-wave intensity-response functions in wild-type and irbp −/− mice. D , Cone b-wave intensity-response functions in wild-type and irbp −/− mice.
- Figure 5.
Reduced cone numbers and cone-OS lengths in irbp −/− versus wild-type retinas. A , Immunocytochemistry showing M-opsin immunoreactivity (top), labeling with fluorescein-tagged PNA (middle), and S-opsin immunoreactivity plus PI staining of nuclei (bottom) in a wild-type mouse retina. B , Similar labeling in an irbp −/− mouse retina. C , Histograms showing numbers of M-cones and S-cones per 600-μm-width sections of retina from wild-type and irbp −/− mice. D , Histograms showing average lengths of M-cone and S-cone OS in wild-type and irbp −/− mice. Asterisks indicate statistically significant differences between wild-type and irbp −/− (p ≤ 0.05). Error bars indicate SDs (n = 4).
- Figure 6.
Immunocytochemistry showing mislocalization of M-opsin in irbp −/− photoreceptors. A , M-opsin immunoreactivity in a section of distal retina from a wild-type mouse. B , M-opsin immunoreactivity in a distal retina section from an irbp −/− mouse. C , M-opsin immunoreactivity in a distal retina section from an irbp −/− mouse treated with 9-cis-RAL. D , Representative differential interference contrast (DIC) image of a similar retina section from an irbp −/− mouse showing cell layers: sclera and choroid (Sc/Ch), outer and inner segment layer (OS/IS), outer nuclear layer (ONL), OPL. E , Estimates of M-opsin and S-opsin percentage-mislocalization derived by dividing OPL immunofluorescence by the sum of OPL and OS immunofluorescence. Note the restoration of M-opsin and S-opsin mislocalization in irbp −/− mice after administering 9-cis-RAL.
- Figure 7.
Immunocytochemistry showing M- and S-opsin in P1 and P3 mouse retinas. A , Retina sections from wild-type mice showing M-opsin immunoreactivity in cone cell-bodies at P1 (top) and S-opsin in cone cell-bodies at P3 (bottom). B , Retina sections from irbp −/− mice at the same ages showing M-opsin and S-opsin immunoreactivity in cone cell-bodies. C , Histograms showing numbers of M-cones and S-cones per 600-μm-width section of retina from wild-type and irbp −/− mice at P1 or P3. Error bars indicate SDs (n = 4).
- Figure 8.
Quantitation of opsin mRNA's and proteins in wild-type and irbp −/− mice. A , Relative opsin mRNA levels by qRT-PCR. Opsin mRNA levels were normalized to 18S rRNA, and the ΔCt values for each mRNA in irbp −/− retinas is shown as the percentage of the level in wild-type (WT) cyclic-light-reared mice. B , Immunoblots of opsins and Rpe65. Immunoblots were performed on the indicated amounts of total protein in retina (rhodopsin, M-opsin, and S-opsin) or RPE (Rpe65) homogenates. The relative amounts of M-opsin and S-opsin in irbp −/− retinas can be estimated by comparing the immunoblot signal strengths in lanes containing different amounts of WT retina homogenate to the immunoblot signals in lanes containing 30 μg of irbp −/− retina homogenate.
- Figure 9.
Cone ERG responses in dark-reared and 9-cis-RAL-supplemented mice. Cone b-wave responses are shown for wild-type (WT) and irbp −/− mice raised in cyclic light or total darkness. Cone b-wave responses are also shown for 9-cis-RAL-supplemented irbp −/− mice raised under cyclic light. Note the reduced cone b-wave amplitude in irbp −/− mice raised in darkness, and the improved b-wave amplitude in cyclic light-reared irbp −/− mice treated with 9-cis-RAL.
