Garlipp MA and Gonzalez-Fernandez F (2013), Cone outer segment and Müller microvilli perice...

XB-ART-46782

Exp Eye Res 2013 Aug 01;113:192-202. doi: 10.1016/j.exer.2013.02.003.

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Cone outer segment and Müller microvilli pericellular matrices provide binding domains for interphotoreceptor retinoid-binding protein (IRBP).

Garlipp MA , Gonzalez-Fernandez F .

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The close packing of vertebrate photoreceptors presents a challenge to the exchange of molecules between the outer segments, retinal pigmented epithelium (RPE), and Müller glia. An extracellular hyaluronan scaffold separates these cells while soluble interphotoreceptor matrix (IPM) proteins traffic visual cycle retinoids, fatty acids, and other molecules between them. In the IPM, retinoids and fatty acids are carried by interphotoreceptor retinoid-binding protein (IRBP). The fact that much of the retina's IRBP can be extracted by saline wash has led to the notion that IRBP does not bind to the retina, but freely distributes itself within the subretinal space. In this study, we challenge this idea by asking if there are specialized IPM domains that bind IRBP, perhaps facilitating its ability to target delivery/uptake of its ligands. Xenopus is an ideal animal model to study the role of the IPM in RPE-photoreceptor interactions. Here, we took advantage of the large size of its photoreceptors, ability to detach the retina in light, sustainability of the retina in short term organ culture, and the availability of recombinant full-length Xenopus IRBP and antisera directed against Xenopus IRBP. We compared the distribution of wash resistant native IRBP, and that of IRBP-Alexa 647 binding in Xenopus retina. IRBP and cone opsin were localized using anti-Xenopus IRBP serum, and monoclonal COS-1 respectively. Cone matrix sheath proteoglycans were localized with wheat germ agglutinin (WGA), and diffuse IPM proteoglycans with peanut agglutinin (PNA). Wholemounts and frozen sections were compared by immunofluorescence from retinas detached under Ringer's followed by additional washes, or detached directly under 4% paraformaldehyde without Ringer's wash. Undetached Lowicryl embedded retinas were subjected to IRBP immunogold electron microscopy (EM). Immunogold labeled a diffuse network of filamentous structures, and a separate distinct flocculant material directly coating the outer segments, filling the rod periciliary ridge, and associated with Müller microvilli. By immunofluorescence, Ringer's wash removed most of the diffuse IRBP, but not that coating the outer segments. IRBP-Alexa 647 bound to the cone outer segments and Müller villi region, and comparably less to rod outer segments. Co-incubation with unlabeled IRBP markedly reduced this binding; ovalbumin-Alexa 647 and Alexa 647 dye alone showed no binding. Our data suggest that the pericellular matrix of the cone outer segments and Müller microvilli provide specialized domains that facilitate IRBP's functions.

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Species referenced: Xenopus laevis
Genes referenced: dnai1 grb10 rbp3 rpe txn

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	Fig. 1. Experiment overview. Using light adapted Xenopus laevis, the spatial distribution of native IRBP in undetached retina frozen and Lowicryl sections, and isolated retinas (paradigm 1) were compared to the location of IRBP-Alexa 647 binding sites in detached retina (paradigm 2). Native IRBP was localized using a rabbit anti-serum directed against Xenopus IRBP. To localize tissue-binding sites, full-length Xenopus IRBP was expressed in E. coli as a soluble thioredoxin (trx) fusion protein (expression construct, Supplementary Fig. 1). A representative purification of full-length Xenopus IRBP free of the trx is provided in Supplementary Fig. 2. Alexa-647 was coupled to Xenopus IRBP in a 1:1 ratio. Co-labeling with cone opsin (mAb COS-1), and wheat germ agglutinin (WGA), which bind to the cone outer segment and its matrix sheath respectively. Controls listed are described in the text.
	Fig. 2. Adherence of IRBP to washed rod outer segments. Retinas were either detached directly under 4% paraformaldehyde, or washed in Ringer's prior to fixation, and then probed with rabbit anti-Xenopus IRBP serum followed by goat anti-rabbit IgG-Alexa 647, and viewed as flatmounts. A) DIC shows numerous rod outer segments in a retinal flat mount. B) IRBP is diffusely distributed throughout the IPM of unwashed retinas. Arrows point to same rods as in panel A. Inset shows no fluorescence with IgG-Alexa 647 alone. C,D) Ringer's washed outer segments in cross-section, and longitudinal orientations respectively. Insets are higher magnifications of bracketed areas. Although Ringer's wash removes most of the IRBP in the IPM, a wash-resistant component remains coating the rod outer segments (arrows). Scale bar = 10 μm in A–D; 3 μm in inset panels.
	Fig. 3. Sequestration of IRBP to the pericellular cone outer segment matrix. Diagram of relative photoreceptor position and IPM associated IRBP. Dotted lines indicate relative Z-plane of confocal section through the outer segments matched with corresponding immunofluorescence. Two IRBP distributions were observed in IPM: a diffuse IRBP that associated with filamentous network of IPM scaffold (pink); and IRBP that coating the outer segments (red). CMS, cone matrix sheath; PCM, pericellular matrix; ELM, external limiting membrane. A–C) IRBP was abundant throughout the IPM in unwashed retinas. However, IRBP was more concentrated around the cone outer segments (arrows) compared to elsewhere in the same Z-plane. D–F) Ringer's wash removed most of the IRBP between the outer segments, but not that coating individual rod outer segments (arrowhead in D), and that surrounding the cone outer segments (arrows). Retinas were detached directly under 4% paraformaldehyde (“unwashed” condition), or under Ringer's followed by 3 washes with gentle agitation (“washed” condition). The retinas were then probed with rabbit anti-Xenopus IRBP serum followed by goat anti-rabbit IgG-Alexa 647, and viewed by confocal microscopy at three planes through the outer segments (diagram on left). Omission of the primary antibody showed no fluorescence (data not illustrated). Scale bar = 10 μm.
	Fig. 4. IRBP accumulates within the rod periciliary ridge complex (PCR). Panels A–C: Confocal 3 μm Z-stacks from a Xenopus retina wholemount (A, IRBP indirect immunofluorescence; B, WGA binding; C, merged fluorescence). IRBP was associated with WGA binding glycoconjugates in the cone outer segment associated matrix (asterisk), and PCR (arrowheads). Panel D: Immuno-EM of the PCR. Lowicryl sections were probed with rabbit anti-Xenopus IRBP serum followed by protein-A 10 nm colloidal gold. Immunogold labeling was associated with amorphous material (arrowheads) in the periciliary ridge complex. Calibration bar = 10 μm in A–C; OS, outer segment; C, cilium; V, vacuoles; IPM, interphotoreceptor matrix.
	Fig. 5. Wash resistant IRBP associates with the extracellular matrix surrounding the outer segments in semi-isolated cone cells. Gentle trituration was used to deplete rod outer segments allowing exposure of the cones prior to wholemount IRBP immunofluorescence. A,B) DIC of cone rich area. Tapering cone outer segments are marked with arrows; oil droplet (asterisk); cone matrix sheath (arrowhead), and inner segment (IS). C,D) Immunofluorescence localizes IRBP to the matrix surrounding the outer segments (arrowheads). Immunofluorescence did not extend beyond the outer segment ending abruptly above the oil droplet (asterisk). E,F) Merged DIC and fluorescence. Scale in panel F is 10 μm in A,C,E; 5 μm in B,D,F.
	Fig. 6. IRBP localization to diffuse filamentous and amorphous pericellular matrix of rod- and cone-associated IPM. Immuno-EM photomicrographs of longitudinally oriented sections through the cone matrix sheath. Lowicryl sections from undetached retinas were probed with rabbit anti-Xenopus IRBP serum followed by protein-A 10 nm colloidal gold. A) Immunogold labeling was associated with amorphous material on the rod outer segment surface (brackets), and matrix strands crisscrossing through the IPM (arrowheads, inset). B) Paracentral cone matrix sheath section showing immunogold labeling throughout the matrix extending between two rod outer segments. C) Parasagittal central cone matrix sheath section showing more of the cone outer segment. Brackets indicate IRBP associated with amorphous extracellular material coating the cone outer segment. IRBP was also associated with fine matrix strands sometimes appearing to extend between the rod and cone outer segments (arrowheads). Sections treated with pre-immune serum showed no immune-specific labeling (data not illustrated). Asterisk, IPM (panel A), cone oil droplet (panel B).
	Fig. 7. IRBP accumulates in the Müller cell associated IPM. Panels A,B: Confocal fluorescence of frozen sections probed with WGA-Alexa 555 (A), or anti-Xenopus IRBP serum followed by goat anti-rabbit IgG-647 (B). IRBP was detected at the cone outer segment (arrowhead), and along the proximal IPM in a punctate distribution (arrows). Panels C–E: Immuno-EM photomicrographs of sections probed with rabbit anti-Xenopus IRBP serum followed by protein-A 10 nm colloidal gold. Immunogold labeling was most dense in the IPM associated with the Muller cell villi region (within boxed area in C). The boxed area is shown as panel D. The IPM is indicated by an asterisk. The area of high IRBP concentration is between the arrowheads. E, Cross-section at the level of the Muller cell villi shows heavy labeling among processes represented in cross (arrows), and longitudinal orientations (arrowheads). M, Muller cell; IS, inner segment; N, nucleus.
	Fig. 8. Xenopus IRBP-Alexa 647 binding is restricted to a domain surrounding cone outer segments. Washed retinas incubated with 1 μM Xenopus IRBP-Alexa 647, rewashed, and viewed in wholemount. A,C) 7.5 μm thick confocal Z-stacks showing inner and outer segments in obliquely oriented photoreceptors. B,D) Higher magnification of bracketed region from A,C; IRBP-Alexa 647 binding was restricted to cone outer segment regions (arrow), and did not extend beyond oil droplet (arrowhead). Scale bar = 10 μm in A,C; 3 μm in B,D.
	Fig. 9. Xenopus IRBP-Alexa 647 binds the cone outer segment matrix, and Müller cell villi region. Washed retinas were incubated with IRBP-Alexa 647, washed, fixed in 4% paraformaldehyde, and embedded in OCT. A) Merged DIC and fluorescence of retina frozen cross-sections. Rod outer segments (ROS); external limiting membrane (ELM); cone outer segment matrix (arrows). B) Magnified region from panel A (asterisk). Xenopus IRBP-Alexa 647 bound to the cone outer segment matrix (COSM). Faint labeling was also appreciated at the rod pericellular ridge complex (slanted arrows) and ELM (horizontal arrowheads). Ellipsoid (Ellip); myoid (Myd); oil droplet (OD). Scale bar = 10 μm in A; 4.0 μm in B.
	Fig. 10. IRBP-Alexa 647 binds to pericellular cone-associated IPM. Washed retinas were probed with Xenopus IRBP-Alexa 647 (red, arrowhead), fixed, and incubated with WGA-Alexa 555 (green, arrow). A) Merged fluorescence show both markers labeling cones. B–E) Z-series of boxed cells from panel A taken in 4 μm steps shown with DIC. Asterisk, oil droplet. Scale bar in E = 5 μm for panels B–E, 2.5 μm in panel A.