Impact of Reduced Rhodopsin Expression on the Structure of Rod Outer Segment Disc Membranes

Abstract

Rhodopsin is the light receptor embedded in rod outer segment (ROS) disc membranes of photoreceptor cells that initiates vision via phototransduction. The relationship between rhodopsin expression and the formation of membrane structures in the ROS is unclear but important to better understand both normal function and pathological conditions. To determine the impact of reduced rhodopsin expression on the structure of ROS discs and the supramolecular organization of rhodopsin, ROS disc membrane samples from heterozygous rhodopsin knockout mice were examined by atomic force microscopy. Similar to rhodopsin in wild-type mice, rhodopsin formed nanodomains in ROS disc membranes of heterozygous knockout mice. The reduced rhodopsin expression in heterozygous knockout mice resulted in ROS disc membranes that were smaller compared to those in wild-type mice at all ages tested. Changes in ROS disc membrane properties were observed between 4 and 6 weeks of age in heterozygous knockout mice that were not present in age-matched wild-type mice. In 4 week-old mice, the number and density of rhodopsin in ROS disc membranes was lower than that in age-matched wild-type mice. In contrast, 6 and 8 week-old mice had more rhodopsin molecules present in disc membranes compared to 4 week-old mice, which resulted in rhodopsin densities similar to those found in age-matched wild-type mice. Thus, mechanisms appear to be present that maintain a constant density of rhodopsin within ROS disc membranes even when reducing the expression of the light receptor by about half. These adaptive mechanisms, however, only occur after 4 weeks of age.

Adaptations in rod outer segment disc membranes in response to environmental lighting conditions

“…Mice used in previous AFM studies were housed in a standard cyclic 12 h light and 12 h dark lighting environment [40, 50]. To examine the impact of environmental lighting on the structure of ROS disc membranes, C57Bl/6J mice were housed in constant light or constant darkness for 10 days.…”

“…ROS disc membrane samples were prepared on freshly cleaved mica and imaged by AFM as described previously [49]. Contact mode AFM imaging was performed in imaging buffer (20 mM Tris, 150 mM KCl, 25m MgCl 2 , pH 7.8) using a Veeco Multimode II atomic force microscope equipped with an E scanner and DNP-S silicon nitride cantilevers with a nominal spring constant of 0.06 N/m (Bruker Corporation, Santa Barbara, CA), as described previously [50]. Deflection AFM images were analyzed using SPIP software (version 6.4, Image Metrology A/S, Hørsholm, Denmark) to measure ROS disc membrane properties, as described previously [50].…”

“…Contact mode AFM imaging was performed in imaging buffer (20 mM Tris, 150 mM KCl, 25m MgCl 2 , pH 7.8) using a Veeco Multimode II atomic force microscope equipped with an E scanner and DNP-S silicon nitride cantilevers with a nominal spring constant of 0.06 N/m (Bruker Corporation, Santa Barbara, CA), as described previously [50]. Deflection AFM images were analyzed using SPIP software (version 6.4, Image Metrology A/S, Hørsholm, Denmark) to measure ROS disc membrane properties, as described previously [50]. Nanodomains within the lamellar region of ROS disc membranes have previously been shown to contain rhodopsin molecules by antibody labeling and single-molecule force spectroscopy [42, 51].…”

Cone-like rhodopsin expressed in the all cone retina of the colubrid pine snake as a potential adaptation to diurnality

“…A previous study used electron microscopy on the retina of T. proximus and showed that the membrane discs unique to rods in the outer segment are still present in the transmuted photoreceptor (Schott et al, 2016). Interestingly, a reduction of RH1 expression levels has been shown to reduce the size of the outer segment of rods, in addition to lowering the photosensitivity and altering the kinetics of the cell to be more cone-like (Makino et al, 2012;Rakshit and Park, 2015;Wen et al, 2009). Currently, the relative expression levels of RH1 in the retinas of colubrid snakes have not been measured.…”

Evolutionary transformation of rod photoreceptors in the all-cone retina of a diurnal garter snake

“…Recent theoretical work has proposed that the small tapering outer segments of cones may help to reduce self-screening of the visual pigments, increase signal-to-noise ratios, and allow light to more efficiently be focused on the outer segment by the ellipsoid (35). Interestingly, recent work has also suggested that reduction of RH1 expression alone can result in a more cone-like morphology, decreasing the photosensitivity of the cell and increasing the kinetics of the phototransduction cascade (34,36,37). A second striking difference in rod and cone morphology is the accessibility of the outer-segment discs to the plasma membrane.…”