Rhodopsin Mutant P23H Destabilizes Rod Photoreceptor Disk Membranes

Mutations in rhodopsin cause retinitis pigmentosa in humans and retinal degeneration in a multitude of other animals. We utilized high-resolution live imaging of the large rod photoreceptors from transgenic frogs (Xenopus) to compare the properties of fluorescently tagged rhodopsin, Rho-EGFP, and RhoP23H-EGFP. The mutant was abnormally distributed both in the inner and outer segments (OS), accumulating in the OS to a concentration of ∼0.1% compared to endogenous opsin. RhoP23H-EGFP formed dense fluorescent foci, with concentrations of mutant protein up to ten times higher than other regions. Wild-type transgenic Rho-EGFP did not concentrate in OS foci when co-expressed in the same rod with RhoP23H-EGFP. Outer segment regions containing fluorescent foci were refractory to fluorescence recovery after photobleaching, while foci in the inner segment exhibited recovery kinetics similar to OS regions without foci and Rho-EGFP. The RhoP23H-EGFP foci were often in older, more distal OS disks. Electron micrographs of OS revealed abnormal disk membranes, with the regular disk bilayers broken into vesiculotubular structures. Furthermore, we observed similar OS disturbances in transgenic mice expressing RhoP23H, suggesting such structures are a general consequence of mutant expression. Together these results show that mutant opsin disrupts OS disks, destabilizing the outer segment possibly via the formation of aggregates. This may render rods susceptible to mechanical injury or compromise OS function, contributing to photoreceptor loss

Dysfunction of Heterotrimeric Kinesin-2 in Rod Photoreceptor Cells and the Role of Opsin Mislocalization in Rapid Cell Death

Loss of kinesin-2 function causes rapid death of rod photoreceptors. The cell death is dependent on the expression of opsin, which first accumulates along the route to the outer segment, but not on signaling by opsin-arrestin complexes or by light activation; the key element appears to be the accumulation of excessive protein in the wrong place

Immunocytochemical binding of anti-opsin N-terminal-specific antibodies to the extracellular surface of rod outer segment plasma membranes. Fixation induces antibody binding.

We have examined the binding of anti-opsin antibodies to the plasma membrane of frog retinal rod outer segments (ROS) by fluorescence light microscopy and electron microscopy. Polyclonal and monoclonal antibodies specific for the N-terminal domain of opsin were observed to bind to the extracellular surface of ROS plasma membrane of aldehyde-fixed but not of unfixed retinas. This reaction was found regardless of whether purified ROS, rhodopsin, opsin, or an N-terminal peptide of opsin was used as the immunogen. The fixation-induced binding of these antibodies contrasts with the more frequently noted loss of antigenicity upon fixation. Concanavalin A, however, binds to unfixed ROS plasma membranes. Its binding sites in the plasma membrane may be oligosaccharides in the N-terminal region of opsin. These results suggest that the N-terminal domain of opsin is latent in the native membrane and that changes in conformation may account for its detectability in fixed membranes. </jats:p

Preembedding labeling with biotinylated antibodies and subsequent visualization of the biotin groups exposed on thin sections.

The feasibility of labeling cell membranes with biotinylated ligands and detecting the biotin groups on thin sections was investigated. Fixed retinal tissue was incubated with biotinyl- antiopsin . Half of the biotinyl-antibody labeled retinal tissue was incubated with avidin-ferritin (AvF) and embedded in Epon (preembedding reaction). The second half was embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Thin sections of this preparation were incubated with AvF to detect biotinyl-antibodies exposed by the sectioning (postembedding reaction). Biotin groups on the thin section surface could be readily visualized with AvF. Stereoscopic images demonstrated that the ferritin particles were localized only on the exposed surface of the thin section. The labeling was highly specific, with a very low background. Quantitative analysis was employed in order to determine the optimal reaction conditions for maximizing the labeling density with minimizing nonspecific binding. The possibility of using biotinylated molecules in the study of dynamic cellular events and for the subsequent intracellular localization of biotin on thin sections is suggested. </jats:p

Rapid embedding of tissues in Lowicryl K4M for immunoelectron microscopy.

Lowicryl K4M (K4M) was recently introduced as an embedding medium for immunocytochemistry at the electron microscope level (BL Armbruster, E Carlemalm, R Chiovetti, RM Garavito, JA Hobot, E Kellenberger, W Villiger (1982):J Microsc 126:77 and E Carlemalm, M Garavito, W Villiger (1982):J Microsc 126:123). While earlier protocols of fixation and embedding required 4-6 days, the present method has reduced the processing time by accelerating both dehydration of tissues and polymerization of K4M so that tissues can be prepared for sectioning within 4 hr. The immunocytochemical labeling density was quantitated in order to determine relative antigen preservation in tissues embedded by the accelerated protocol as compared to slower K4M embedding techniques and to tissues embedded in glutaraldehyde-cross-linked bovine serum albumin (BSA). Thin sections of Bufo marinus kidney were labeled with rabbit antibody to Na+,K+ATPase alpha chain catalytic subunit isolated from B. marinus kidney microsomes (M Girardet, K Geering, JM Frantes, D Geser, BC Rossier, JP Kraehenbuhl, C Bron (1981):Biochemistry 20:6684). B. marinus retinas were labeled with rabbit anti-opsin. After fixation in paraformaldehyde(3%)-glutaraldehyde(3%), tissues were washed in buffer, dehydrated in 50, 75, and 90% dimethyl-formamide (DMF, 10 min each); K4M:DMF, 1:2 (15 min); K4M:DMF, 1:1, (20 min); K4M (25 min); K4M (30 min) at room temperature and transferred in fresh K4M to BEEM capsules for exposure to ultraviolet light (GE 15 watt, Black-lite, 10 cm, 45 min or less) at 4 degrees C. Thin sections were labeled successively with antibody, biotinylated sheep anti-rabbit F(ab')2 and avidin-ferritin. Ferritin labeling densities were determined by point counting. High labeling densities were observed with both antibodies, equaling or exceeding levels of labeling by slower protocols or embedment in BSA. </jats:p

Biosynthesis and vectorial transport of opsin on vesicles in retinal rod photoreceptors.

Retinal rod photoreceptor cells absorb light at one end and establish synaptic contacts on the other. Light sensitivity is conferred by a set of membrane and cytosol proteins that are gathered at one end of the cell to form a specialized organelle, the rod outer segment (ROS). The ROS is composed of rhodopsin-laden, flattened disk-shaped membranes enveloped by the cell's plasma membrane. Rhodopsin is synthesized on elements of the rough endoplasmic reticulum and Golgi apparatus near the nucleus in the inner segment. From this synthetic site, the membrane-bound apoprotein, opsin, is released from the Golgi in the membranes of small vesicles. These vesicles are transported through the cytoplasm of the inner segment until they reach its apical plasma membrane. At that site, opsin-laden vesicles appear to fuse near the base of the connecting cilium that joins the inner and outer segments. This fusion inserts opsin into the plasma membrane of the photoreceptor. Opsin becomes incorporated into the disk membrane by a process of membrane expansion and fusion to form the flattened disks of the outer segment. Within the disks, opsin is highly mobile, and rapidly rotates and traverses the disk surface. Despite its mobility in the outer segment, quantitative electron microscopic, immunocytochemical, and autoradiographic studies of opsin distribution demonstrate that little opsin is detectable in the inner segment plasma membrane, although its bilayer is in continuity with the plasma membrane of the outer segment. The photoreceptor successfully establishes the polarized distribution of its membrane proteins by restricting the redistribution of opsin after vectorially transporting it to one end of the cell on post-Golgi vesicles. </jats:p

Immunocytochemical localization of opsin in outer segments and Golgi zones of frog photoreceptor cells. An electron microscope analysis of cross-linked albumin-embedded retinas

Adult vertebrate retinal cells (rod and cones) continuously synthesize membrane proteins and transport them to the organelle specialized for photon capture, the outer segment. The cell structures involved in the synthesis of opsin have been identified by means of immunocytochemistry at the electron microscope level. Two indirect detection systems were used: (a) rabbit antibodies to frog opsin were localized with ferritin conjugated F(ab')2 of sheep antibodies to rabbit F(ab')2 and (b) sheep antibodies to cattle opsin were coupled to biotin and visualized by means of avidin-ferritin conjugates (AvF). The reagents were applied directly to the surface of thin sections of frog retinal tissues embedded in glutaraldehyde cross-linked bovine serum albumin (BSA). Specific binding of anti-opsin antibodies indicates that opsin is localized in the disks of rod outer segments (ROS), as expected, and in the Golgi zone of the rod cell inner segments. In addition, we observed quantitatively different labeling patterns of outer segments of rods and cones with each of the sera employed. These reactions may indicate immunological homology of rod and cone photopigments. Because these quantitiative variations of labeling density extend along the entire length of the outer segment, they also serve to identify the cell which has shed its disks into adjacent pigment ipithelial cell phagosomes