Interphotoreceptor Retinoid-Binding Protein in the Golgi Apparatus of Monkey Foveal Cones

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1 Investigative Ophthalmology & Visual Science, Vol. 33, No. 5, April 1992 Copyright Association for Research in Vision and Ophthalmology Interphotoreceptor Retinoid-Binding Protein in the Golgi Apparatus of Monkey Foveal Cones Electron Microscopic Immunocyrochemicol Locolizorion Louvenia Carrer-Dawson and Michael Burroughs The presence of interphotoreceptor retinoid-binding protein (IRBP) in the Golgi apparatus of monkey cone photoreceptors was examined by electron microscopic immunocytochemistry. Intracellular labeling for IRBP was light to moderate over the Golgi complex of foveal and peripheral cones in the retinas of all four monkeys examined. Composite drawings of two to seven serial sections of inner segments indicated that the highest density of intracellular labeling occurred over the Golgi apparatus. The labeling density over the Golgi complex was fold higher than that over a comparable area of inner segment cytoplasm. The presence of immunolabel for IRBP in the Golgi apparatus supported the findings of in situ hybridization studies that cone photoreceptors can synthesize IRBP. Invest Ophthalmol Vis Sci 33: ,1992 Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular protein found largely in the interphotoreceptor matrix (IPM). 1 " 6 This protein may be involved in the transfer of retinoids between the photoreceptors and the retinal pigment epithelium (RPE) 34 or may serve as a buffer protein. 7 Rod photoreceptors are thought to be responsible for the synthesis and secretion of IRBP. Studies using complementary DNA (cdna) probes and in situ hybridization showed IRBP messenger RNA localized mainly to rod photoreceptors. 8 Electron microscopic immunocytochemical studies also have shown IRBP localized intracellularly to rod photoreceptors. 910 Although intracellular labeling for IRBP has not been reported for cones, predominately cone retinas contain IRBP in the IPM. 11 It also has been detected in the all-cone fovea of the monkey. 6 Either the neighboring rods or both rods and cones synthesize the IRBP seen in the IPM of the fovea and cone-dominant retinas. A recent in situ hybridization study 12 showed the From the Sensory Sciences Center, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, Texas. Supported by National Institutes of Health (Bethesda, Maryland) grant EY05596 (LC-D), and National Science Foundation (Washington DC) grant BSN (LC-D). Presented at the Association for Research in Vision and Ophthalmology, Sarasota, Florida, May 1-6, Submitted for publication: August 27, 1990; accepted November 8, Reprint requests: Louvenia Carter-Dawson, PhD, 6420 Lamar Fleming Avenue, Room 316, Sensory Sciences Center, The University of Texas Health Science, Center at Houston, Houston, TX presence of IRBP messenger RNA in the cones of humans and cone-dominant squirrel retinas. The protein IRBP should pass through the Golgi apparatus for glycosylation and packaging because it is a glycoprotein. We examined the Golgi complex of foveal and peripheral cones in monkey retinas for IRBP immunoreactivity using a postembedded immunogold-labeling procedure. The presence of immunoreactivity for this glycoprotein in the Golgi apparatus would provide supporting evidence that cones synthesize IRBP. Materials and Methods One eye from each of four monkeys (Macacafascicularis) was provided by Dr. M. L. J. Crawford (University of Texas Health Science Center at Houston) after vascular perfusion with a mixture of paraformaldehyde 2% and glutaraldehyde 0.5% (ph 7.3, 0.1 M phosphate buffer). The animals were handled in conformance with the ARVO Resolution on the Use of Animals in Research. The anterior segments were removed, and the eyecups were fixed further, and processed for embedding in LR white (E. F. Fullam, Latham, NY). 10 Ultrathin sections of foveal and peripheral retina were mounted on Butvar-coated (BioRad, Cambridge, MA) grids and incubated with goat serum 5% to block nonspecific binding of primary antisera, followed by incubation in 30-/*l drops of rabbit anti-monkey IRBP immunoglobulin G (0.009 ng/ix\ or 30 n\ of affinity-purified rabbit antimonkey IRBP serum. Polyclonal antibodies to purified IRBP were gifts from Drs. B. Wiggert and G. 1589

2 1590 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 Vol. 33 Chader, National Eye Institute, Bethesda, MD) using the two-stage labeling method. 10 Control specimens consisted of sections incubated in rabbit immunoglobulin G instead of rabbit anti-monkey immunoglobulin G. All sections were washed in Tris buffer, incubated in goat anti-rabbit immunoglobulin G conjugated to 5-nm gold. The grids were washed, and the sections were counterstained with uranyl acetate and exposed to osmium vapor as previously described. 10 The sections were examined and photographed with a JEOL (Peabody, MA) 100B electron microscope. Foveal and peripheral cones, rods, and RPE cells with prominent Golgi regions were photographed in two to seven consecutive sections and printed to a final magnification of 40,000. Four to eight foveal cones, three peripheral cones, three rods, and four RPE cells were examined in each of four, two, and three monkeys, respectively. Composite drawings were made of the inner segments or RPE cell bodies, including outlines of the Golgi apparatus and membranous vesicles and all gold particles. The area of the inner segments or RPE cell bodies and the Golgi apparatus was determined using the Hewlett Packard (Fort Collins, CO) area program and digital tablet (model 9111 A). The area of the inner segments or RPE cytoplasm included only the region surrounding the Golgi complex. The number of gold particles were counted for each area, and the number per 0.25 /xm 2 area was determined. (Vesicles heavily labeled for IRBP were excluded from the analysis because it was not clear whether they were secretory or endocytotic.) Results Examination of the Golgi regions of foveal cones in a single section showed unlabeled, lightly labeled, or moderately labeled areas. Composite drawings (two M2 FOVEAL CONE IPM QLM Fig. 1. A composite drawing and representative section of a cone from monkey 2. (A) The distribution of labeling for IRBP in two consecutive sections is seen in this composite drawing. The highest density of label is present over the Golgi region (arrows). (B) Labeling over the Golgi region (arrows) is seen in a portion of one section used in the drawing (sections incubated with nonaffinity purified antiserum). OLM, outer limiting membrane; IPM, interphotoreceptor matrix. Original magnification X 40,000; bar = 0.5 Mm.

3 No. 5 IRBP LOCALIZATION IN MONKEY CONE5 / Correr-Dowson and Durroughs 1591 to four sections) of representative cells that were unlabeled to lightly labeled in one section showed most of the label localized to the Golgi apparatus and membranous vesicles. This pattern of labeling was observed in sections that reacted with nonaffinity (Figs. 1A-1B) or affinity-purified antiserum (Figs. 2A-B). The Golgi apparatus of peripheral cones and rods also were unlabeled to lightly labeled in single sections, but they were heavily labeled in composite drawings (Fig. 3, seven serial sections; Fig. 4, two serial sections). This pattern of labeling was seen in the cones of all monkeys examined. The Golgi region of RPE cells were labeled at background levels. Quantitation of the number of gold particles, representing IRBP labeling of inner segments, showed the highest labeling over the Golgi complex. The density of label over the Golgi and inner segment in representative foveal cones was fold higher than in the inner segment cytoplasmic region (range, 0.93/ /0.1 per 0.25 ^m 2 ). Over peripheral cones, the labeling density for Golgi and inner segment cytoplasm was fold higher (range, 2.51/ /0.18 per 0.25 /urn 2 ). The Golgi regions of peripheral rod inner segments were labeled fold higher than other cytoplasmic regions (rod range, 2.5/ /0.06 per 0.25 jim 2, excluding membranous vesicles and rough endoplasmic reticulum). Similar quantitation of IRBP labeling over the Golgi region in RPE cells showed the labeling density over the Golgi region to be only fold higher than that over other cytoplasmic regions (range, 0.09/ /0.06 per 0.25 ftm 2 ). The labeling for IRBP over the Golgi region in control samples was similar to that over cytoplasmic regions (range, 0.16/ /0.24 per fim 2 ). Discussion Our findings provide supporting evidence that cones may synthesize IRBP. We found IRBP immunoreactivity associated with the Golgi complex and M2 FOVEAL CONE (affinity purified serum) V IPM 2A Fig. 2. Localization of IRBP in foveal cones with affinity purified serum. (A) Similar to Figure 1A, the composite drawing shows labeling concentrated in the region of the Golgi (arrows), except for a heavily labeled vesicle (arrow head). (B) The Golgi region is moderately labeled in one of the sections used for the composite drawing (arrows). The heavily labeled membranous vesicle seen in the composite drawing is present in one of the sections used in the drawing (arrow head). M2, monkey 2; IPM, interphotoreceptor matrix. Original magnification X4O,OOO; bar = 0.5 Mm.

4 1592 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / April 1992 Vol. 30 M3 PERIPHERAL CONE 1PM iv QLM \ : 3B NUC Fig. 3. Composite drawing and a representative section of a peripheral cone from monkey 3. (A) The composite drawing shows the distribution of label for IRBP in seven serial sections. Label is associated with the Golgi region (arrows) and other membranous vesicles. (B) A portion of the Golgi region from one of seven sections is lightly labeled for IRBP in this micrograph (arrows; nonaffinity purified antiserum was used). NUC, nucleus; OLM, outer limiting membrane; IPM, interphotoreceptor matrix. Original magnification X4O,OOO; bar = 0.5 ^m. membranous vesicles in cone and rod inner segments. The labeling in rods was similar to that described earlier. 9 In addition, the Golgi apparatus of foveal and peripheral cones were labeled for IRBP. Single sections through the Golgi apparatus of cones appeared unlabeled, lightly labeled, or moderately labeled. Most often the Golgi region appeared unlabeled to lightly labeled. Therefore, if only a single section is examined and background labeling is high, the Golgi region will appear unlabeled. This may explain why labeling of the Golgi region was not detected previously. The labeling of the Golgi complex in cones and rods appeared to be specific for IRBP. The IRBP labeling of the Golgi region was observed with affinity- and nonaffinity-purified antisera. However, labeling for IRBP in the Golgi region of RPE cells by affinity- or nonaffinity-purified antiserum was only one to threefold higher than background. This slightly elevated labeling density in the RPE may represent an error related to the identification of the Golgi apparatus. The Golgi in the RPE is not recognized as easily as it is in the photoreceptor inner segment. Some non-golgi apparatus membranous structures may have been included in the analysis. The absence of a high density of labeling in the Golgi region of RPE cells and cone inner segments of control cells for IRBP suggested that the antibody was not recognizing a protein component common to all Golgi apparatus, but rather was recognizing IRBP in the Golgi. We used a secondary antibody conjugated to 5-nm gold particles to increase the labeling density and examined two to seven serial sections of cones through the Golgi region. Composite drawings of the labeling in serial sections also were made. The combination of high density and specificity of labeling, low background, the use of serial sections, composite drawings, and quantitative analysis of labeling densities made it clear that the Golgi apparatus of monkey cones showed labeling for IRBP. Does the label for IRBP in the Golgi apparatus represent synthesis or recycling of protein that had undergone endocytosis? Recent studies have shown cellsurface proteins that have undergone endocytosis to be translocated to the trans-golgi network for resialylation. 13 In this study, label for IRBP was not restricted to one or two cisternae, as might be expected for labeling in the trans-golgi area. Because label was

5 No. 5 IRBP LOCALIZATION IN MONKEY CONES / Correr-Dawson and Burroughs 1593 M3 PERIPHERAL ROD IPM 4B S OLM 4A Fig. 4. IRBP labeling in rod photoreceptors of monkey 3 (M3). (A) The composite drawing of two serial sections shows most of the label associated with the Golgi region (arrows) and membranous vesicles. (B) A micrograph of one serial section shows IRBP labeling associated with the Golgi region (arrows) and membranous vesicles (nonaffinity purified IRBP antiserum). OLM, outer limiting membrane; 1PM, interphotoreceptor matrix. Original magnification X 40,000; bar = 0.5 pm. present in most cisternae, some of it probably was related to IRBP synthesis. Our findings of IRBP immunoreactivity in the Golgi region of monkey cone photoreceptors supported cdna hybridization studies 12 that cone photoreceptors can synthesize IRBP. Key words: interphotoreceptor retinoid-binding protein, retina, immunocytochemistry, monkey, cones, rods, Golgi apparatus Acknowledgments The authors thank Drs. B. Wiggert and G. Chader for their generous supply of IRBP antibodies and purified protein and Dr. M. L. J. Crawford for the monkey eyes. References 1. Adler AJ and Martin KJ: Retinol-binding proteins in bovine interphotoreceptor matrix. Biochem Biophys Res Commun 108:1601, Bunt-Milam AH and Saari JC: Immunocytochemical localization of two retinoid-binding proteins in vertebrate retina. J Cell Biol 97:703, Chader GJ, Wiggert B, Lai Y-L, Lee L, and Fletcher R: Interphotoreceptor retinoid-binding protein: A possible role in retinoid transport to the retina. In Progress in Retinal Research, Osborne N and Chader GJ, editors. Oxford, Pergamon Press, 1983, pp Okajima T-I, Pepperberg DR, Ripps H t Wiggert B, and Chader GJ: Interphotoreceptors retinoid-binding protein promotes rhodopsin regeneration in toad photoreceptors. Proc Natl Acad Sci USA 87:6907, 1990.

6 1594 INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / April 1992 Vol Fong S-L, Liou GI, Landers RA, Alvarez RA, Gonzalez-Fernandez F, Glazebrook PA, Lam DMK, and Bridges CDB: The characterization, localization, and biosynthesis of an interstitial retinol-binding glycoprotein in the human eye. J Neurochem 42:1667, Wiggert B, Lee L, Rodrigues M, Hess H, Redmond TM, and Chader GJ: Immunochemical distribution of interphotoreceptor retinoid-binding protein in selected species. Invest Ophthalmol Vis Sci 27:1041, Ho M-T, Massey H, Pownall RE, Anderson RE, and Hollyfield JG: Mechanism of vitamin A movement between rod outer segments, interphotoreceptor retinoid-binding protein, and liposomes. J Biol Chem 26:928, van Veen T, Katial A, Shinohara T, Barrett D, Wiggert B, Chader GJ, and Nickerson J: Retinal photoreceptor neurons and pinealocytes accumulate mrna for interphotoreceptor retinoid-binding protein (IRBP). FEBS Lett 208:133, Rodrigues M, Hackett J, Wiggert B, Gery I, Spiegel A, Krishna G, Stein P, and Chader G: Immunoelectron microscopic localization of photoreceptor-specific markers in the monkey retina. Curr Eye Res 6:369, Carter-Dawson L and Burroughs M: Interphotoreceptor retinoid-binding protein (IRBP) in the postnatal developing rds mutant retina: EM immunocytochemical localization. Exp Eye Res 49:829, Anderson DH, Neitz JC, Saari JC, Kaska DD, Fenwick J, Jacobs GH, and Fisher SK: Retinoid-binding proteins in conedominant retinas. Invest Ophthalmol Vis Sci 27:1015, Porrello K, Bhat SP, and Bok D: Detection of Interphotoreceptor retinoid binding protein (IRBP) mrna in human and cone-dominant squirrel retinas by in situ hybridization. J Histochem Cytochem 29:171, Gruenberg J and Howell KE: Membrane traffic in endocytosis: Insights from cell-free assays. Annu Rev Cell Biol 5:453, 1989.