David S. Papermaster

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 cells 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.

Cone lamellae and red and green rod outer segment disks contain a large intrinsic membrane protein on their margins: an ultrastructural immunocytochemical study of frog retinas.

In addition to rhodopsin, the disk membranes of rod outer segments (ROS) contain a large integral membrane protein (mol. wt 290,000). This protein was previously localized by immunocytochemistry to the margins and incisures of disks in frog red ROS by specific antibody applied to thin sections of bovine serum albumin embedded retinas (Papermaster et al., 1978b, J. Cell Biol. 78, 415-425). Upon further study of the reactions of this antibody with outer segments of other photoreceptor classes in frog retina, labeling of the short incisures and margins of green ROS and margins of cone outer segment lamellae is also observed. Thus the large protein participates in the structure of the edges of disks and lamellae of all photoreceptors in the frog. In addition, labeling of the inter-incisure surface of all photoreceptor classes was observed at high antibody concentration. In order to interpret this labeling, the effect of dilution on labeling density was determined and double reciprocal plots (Markham and Benton, 1931, J. Am. Chem. Soc. 53, 497) were employed to evaluate the relative affinity and heterogeneity. There was considerable deviation from linearity in the plots of labeling disk interiors compared to the relatively linear plots of disk incisure labeling which suggests that the interior sites contain a weakly cross-reacting antigen or that the serum contains a lower concentration of antibody weakly reactive with another antigen.

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.

[38] Immunocytochemistry of retinal membrane protein biosynthesis at the electron microscopic level by the albumin embedding technique

Publisher Summary Pathways of membrane protein biosynthesis and transport may be investigated at the electron microscopic level using antibodies as highly specific probes. This chapter describes the application of antibodies against opsin and other membrane proteins to thin sections of retinas embedded in bovine serum albumin to study membrane biosynthesis in photoreceptor cells. The chapter presents and discusses embedding protocol, sectioning, preparation of reagents, labeling protocol, quantitation, and application in analysis of opsin biosynthesis in the retina. One of the major advantages of this technique is that sections and blocks appear to be stable indefinitely, while disadvantages of the BSA embedding technique include the time required for processing, and fragility of sections, especially from fresh blocks. The vertebrate photoreceptor cell is a highly compartmentalized sensory neuron; light capture is a function of the opsin-laden membranes of the rod outer segment (ROS), while the biosynthetic apparatus is confined to the adjacent compartment, the inner segment. Labeling of BSA-embedded retinas with affinity purified anti-opsin antibodies demonstrates the high density of opsin in the ROS.

OPSIN mRNA ISOLATION FROM BOVINE RETINA AND PARTIAL SEQUENCE OF THE IN VITRO TRANSLATION PRODUCT

Rod photoreceptor cells of adult vertebrate retinas synthesize membrane proteins in one compartment of the cell, the inner segment, and transport them to an adjacent compartment, the outer segment. The rod outer segment (ROS) is composed of an enclosed stack of closely packed membranous disks, which are continuously renewed in adult vertebrates. Each day new disk formation at the base of the ROS displaces older disks apically until they are shed from the ROS tip.’ Light exposure accelerates disk renewal in Xenopus tadpole previously dark adapted at 20”. Subsequent disk renewal is slower until the next cycle of light following a period of dark adaptation.’ The mechanism by which light stimulates membrane biosynthesis in these amphibia is unclear. Rhodopsin is the light-sensitive protein of the ROS membrane disks and makes up approximately 90% of the total protein in these membranes.’ Recent studies‘.’ of rhodopsin’s NH,-terminal sequence have established that oligosaccharides are attached to two sites at Asn’ and Asn” and are composed primarily of short chains whose structure is

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.

Ultrastructural localization of retinal photoreceptor proteins.

Publisher Summary The development of streptavidin-gold (SAG) has greatly simplified the techniques of immunocytochemical localization at high resolution. The AvF and SaG conjugates can be used in several ways. Either one is suitable for direct visualization of bound biotinyl antibodies. The amplification of the signal from the binding of an antibody or detection of an antibody whose reactivity is destroyed by biotinylation is readily achieved by using a series of multiplier stages. A small aliquot is tested for stability by addition of an equal volume of 1 M NaC1. Unstable particles form a dark blue precipitate. Preembedding labeling is primarily used for the detection of antigens exposed on the external surfaces of cells. Careful observation and quantification of labeling density along the length of the cell often reveal a gradient of labeling from distal to proximal regions of the photoreceptor when such washing is incomplete. The most important change is the introduction of a variety of methacrylate-derived hydrophilic plastics, which provide easily sectioned tissue blocks that are suitable for labeling with antibodies to numerous antigens.

[49] Subcellular fractionation and immunochemical analysis of membrane biosynthesis of photoreceptor proteins

Publisher Summary This chapter discusses the procedure for retinal subcellular fractionation, presents the full details and rationale for the experimental conditions, and explains the results. Rhodopsin biosynthesis and disk formation can be divided into four stages: (a) synthesis in rough endoplasmic reticulum (RER) and Golgi; (b) vectorial transport of opsin on vesicles (cisternae) from the Golgi to the base of the connecting cilium and insertion into the apical plasma membrane of the inner segment; (c) transport out the plasma membrane of the connecting cilium; and (d) disk morphogenesis at the base of the rod outer segment (ROS). The procedure involves subcellular fractionation, electrophoretic analysis, and immunochemical analysis of membrane and cytosol fractions. Using the two-dimensional electrophoretic technique, the chapter reveals the presence of immunoprecipitable, newly synthesized opsin and large ROS protein only in membrane fractions. However, this approach to retinal subcellular fractionation must still be considered an early stage in the development of the study of retinal membrane biosynthesis. Until procedures for subfractionation of smooth membrane vesicles with similar biophysical properties become available, it at least ensures a rapid recovery of the inner segment membranes in a reasonable yield.

[19] Two-dimensional immunoelectrophoresis of membrane antigens

Publisher Summary This chapter provides complete directions for the preparation of the first- and second-dimension gels and their recording by photography. The procedure consists of a first-dimension separation of membrane proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, followed by electrophoretic elution of the proteins from this gel, in the second dimension, through a two-layer agarose gel containing Lubrol PX in the first layer and specific antibodies in the second layer, where precipitin arcs form. An outline of the procedure is also presented in the chapter including first dimensional electrophoresis, modifications of first-dimension conditions, preparation of second-dimension gel, modifications to second-dimension gel, second-dimension electrophoresis, and photography and staining. The chapter describes that the two-dimensional immunoelectrophoresis method discussed is quick, and gives useful, relatively unambiguous information about relationships among proteins that can be separated on SDS-polyacrylamide gels.