Rhodopsin signaling and organization in heterozygote rhodopsin knockout mice

Abstract

Rhodopsin (Rho) resides within internal membrane structures called disc membranes that are found in the rod outer segments (ROS) of photoreceptors in the retina. Rho expression is essential for formation of ROS, which are absent in knockout Rho-/- mice. ROS of mice heterozygous for the Rho gene deletion (Rho+/-) may have a lower Rho density than wild type (WT) membranes, or the ROS structure may be reduced in size due to lower Rho expression. Here, we present evidence that the smaller volume of ROS from heterozygous mice is most likely responsible for observed electrophysiological response differences. In Rho+/- mice as compared with age-matched WT mice, the length of ROS was shorter by 30-40%, and the average diameter of ROS was reduced by approximately 20%, as demonstrated by transmission and scanning electron microscopy. Together, the reduction of the volume of ROS was approximately 60% in Rho+/- mice. Rho content in the eyes was reduced by approximately 43% and 11-cis-retinal content in the eye was reduced by approximately 38%, as determined by UV-visible spectroscopy and retinoid analysis, respectively. Transmission electron microscopy of negatively stained disc membranes from Rho+/- mice indicated a typical morphology apart from the reduced size of disc diameter. Power spectra calculated from disc membrane regions on such electron micrographs displayed a diffuse ring at approximately 4.5 nm(-1), indicating paracrystallinity of Rho. Atomic force microscopy of WT and Rho+/- disc membranes revealed, in both cases, Rho organized in paracrystalline and raftlike structures. From these data, we conclude that the differences in physiological responses measured in WT and Rho+/- mice are due to structural changes of the whole ROS and not due to a lower density of Rho.

Fig. 1. Single-flash ERG responses of increasing intensity for WT and Rho+/− mice

Serial responses to increasing flash stimuli were obtained for WT and Rho+/− mice under dark-adapted conditions (A) for selected intensities and plotted as a function a-wave and b-wave versus light intensities (B).

Fig. 2. Measurements of a-wave recovery with double-flash ERG

A, ERG trace from WT mouse demonstrating the double-flash technique (top). Bottom, recovery of a-wave after a test flash. The dark-adapted mice were conditioned first with the test flash (0.4 log cd s m⁻²) followed by a probe flash (1.6 log cd s m⁻²) with the delay time varied from 100 to 2000 ms. Each trace represents the average of recordings from n = 8 eyes. B, normalized a-wave recovery of the probe flash at different times after the test flash. The responses recovered more quickly in Rho+/− mice.

Fig. 3. Retina histology of WT and Rho+/− mice

A, light micrograph of the retina from WT and Rho+/− mice. B, thickness of ROS (in μm) plotted as a function of location in the retina from the optic nerve head (ONH; in mm). The age of the mice was 12 weeks, and the average from 12 eyes was used in this experiment. Note that the photoreceptor layer across the retina of Rho+/− mice is 30–40% shorter than those of WT mice. Closed circles, WT mice; open circles, Rho+/− mice. IS, inner segments; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner plexiform layer; GCL, ganglion cell layer.

Fig. 4. Retinoid analysis in Rho+/− mice

Chromatographic separation of nonpolar retinoids from WT and Rho+/− mouse eyes. Retinoids were extracted from the eye and separated on normal-phase HPLC as described under “Materials and Methods.” Inset, difference spectra calculated from spectra measured before and after bleaching of Rho in the eye extract from WT and Rho+/− mice. The homogenate from the retina of Rho+/− has ~60–70% of 11-cis-retinal or Rho compared with the age-matched retinal extract from WT mice. 1 and 1′ represent syn- and anti-11-cis-retinal oximes.

Fig. 5. TEM and SEM micrographs of retina and ROS from WT and Rho+/− mice

A, transmission electron micrographs of retina sections around the photoreceptor layer from WT and Rho+/− mice. Diameter of ROS from WT and Rho+/− mice was 1.32 ± 0.12 and 1.05 ± 0.08 μm (n = 50), respectively. B, SEM of mouse ROS attached to the retina from WT and Rho+/− mice. The average diameter of ROS from WT and Rho+/− was 1.22 ± 0.12 and 1.02 ± 0.07 μm, respectively.

Fig. 6. TEM of isolated sectioned ROS

A, isolated ROS from WT and Rho+/− mice. B, electron micrographs recorded at higher magnification of isolated ROS from WT and Rho+/− mice.

Fig. 7. AFM of native discs and disc membranes isolated from Rho+/− mice

Height (A) and deflection (B) images of an intact disc (double-layered membrane). Two different surface types are discerned: mica () and the cytoplasmic surface of the disc (). C, height image of open, flattened single-layered discs. Three additional surface types are visible: small raftlike structures (2′), larger paracrystalline Rho domains (2*), and lipid (). The area marked by the broken white box is displayed in D as a deflection image at higher magnification; lattice lines from the Rho paracrystal (2*) are visible. Inset in D, magnification of the paracrystalline area (2*). Discs and disc membranes were adsorbed on mica and imaged in buffer solution at room temperature. Scale bars, 200 nm (A and B), 500 nm (C), and 150 nm (D). Frame size of the inset in D is 267 × 143 nm. Vertical brightness ranges are 30 nm (A), 0.6 nm (B), 18 nm (C), and 0.4 nm (D).