Soluble Matrix Proteins Research Articles

Expression of green fluorescent protein from a recoded gene inserted into Saccharomyces cerevisiae mitochondrial DNA

To generate a visible reporter of mitochondrial gene expression, we have synthesized a DNA fragment that specifies an enhanced variant of the green fluorescent protein (GFP) in the Saccharomyces cerevisiae mitochondrial genetic code. This reporter gene, GFP m - 3, was inserted into mtDNA at the eighth codon of the COX3 gene. Mitochondria containing this mtDNA could be detected by fluorescence microscopy. Mitochondrially encoded GFP accumulated as soluble matrix protein, whose level could be measured both immunologically and fluorometrically. Quantitation of relative fluorescence by flow cytometry confirmed that cox3∷ GFP m - 3 expression was affected by carbon source and dependent upon COX3 mRNA-specific translational activation. GFP m - 3 will be a valuable tool for studying mitochondrial gene regulation and the intracellular fates of mitochondrially synthesized proteins.

An Electrophoretic Analysis of the Seed Protein Body Proteins from Pinus Nigra

Protein bodies (PBs) of European black pine (Pinus nigra Arn.) were isolated from mature seeds. Extracted soluble matrix proteins and crystalloid proteins PBs proteins were investigated by SDS-PAGE electrophoresis in presence and absence of 2-mercaptoethanol. The proteins of molecular masses 16, 17, 18, 61 and 65 kDa were presented only in crystalloid protein samples. Only 15 kDa protein was present in soluble matrix proteins and not in crystalloid proteins. Another protein bands were present in both soluble matrix and crystalloid proteins. 20, 37, 38, 39 and 48 kDa proteins were strongly visible among crystalloid proteins. Bands of 23 and 32 kDa were more visible in soluble matrix protein samples. Different composition in crystalloid proteins was found in absence of 2-mercaptoethanol: no proteins with molecular mass 71 kDa and more proteins in soluble matrix. In case of crystalloid proteins we detected 7 protein bands in interval from 71 to 212 kDa.

The influence of curli, a MHC-I-binding bacterial surface structure, on macrophage-T cell interactions.

Escherichia coli express thin surface fimbriae called curli which bind soluble matrix proteins and major histocompatibility complex (MHC)-I molecules. The present study addressed the ability of purified curli or curliated E. coli to influence peptide presentation on MHC-I, T cell proliferation and bacterial uptake by macrophages. In vitro studies with curli-proficient E. coli YMel and the isogenic curli-deficient strain YMel-1, both expressing the model antigen Crl-OVA, showed that curli expression by E. coli does not appear to influence the efficiency by which the bacteria are processed by murine macrophages for OVA(257-264) presentation on K(b). Furthermore, curli expression by E. coli did not influence the binding of exogenously added OVA(257-264) peptide to K(b) on the surface of prefixed macrophages. In addition, neither curliated nor non-curliated heat-killed bacteria influenced proliferation of either murine or human T cells stimulated with anti-CD3. Finally, curliated E. coli adhered to and were internalized by macrophages from C57BL/6 and MHC-I-deficient TAP1(-/-) mice equally well. Together these studies show that curli expression by E. coli does not appear to influence phagocytic processing of bacteria expressing Crl-OVA for OVA(257-264)/K(b) presentation, the binding of exogenously added OVA(257-264) to K(b) or T cell proliferation. In addition, although curli expression by E. coli enhances bacterial interaction with macrophages, curli interaction with MHC-I does not significantly contribute to this adherence.

The influence of curli, a MHC-I-binding bacterial surface structure, on macrophage–T cell interactions

Escherichia coli express thin surface fimbriae called curli which bind soluble matrix proteins and major histocompatibility complex (MHC)-I molecules. The present study addressed the ability of purified curli or curliated E. coli to influence peptide presentation on MHC-I, T cell proliferation and bacterial uptake by macrophages. In vitro studies with curli-proficient E. coli YMel and the isogenic curli-deficient strain YMel-1, both expressing the model antigen Crl–OVA, showed that curli expression by E. coli does not appear to influence the efficiency by which the bacteria are processed by murine macrophages for OVA(257–264) presentation on K b. Furthermore, curli expression by E. coli did not influence the binding of exogenously added OVA(257–264) peptide to K b on the surface of prefixed macrophages. In addition, neither curliated nor non-curliated heat-killed bacteria influenced proliferation of either murine or human T cells stimulated with anti-CD3. Finally, curliated E. coli adhered to and were internalized by macrophages from C57BL/6 and MHC-I-deficient TAP1 −/− mice equally well. Together these studies show that curli expression by E. coli does not appear to influence phagocytic processing of bacteria expressing Crl–OVA for OVA(257–264)/K b presentation, the binding of exogenously added OVA(257–264) to K b or T cell proliferation. In addition, although curli expression by E. coli enhances bacterial interaction with macrophages, curli interaction with MHC-I does not significantly contribute to this adherence.

Sequestration of the Active Site by Interdomain Shifting: CRYSTALLOGRAPHIC AND SPECTROSCOPIC EVIDENCE FOR DISTINCT CONFORMATIONS OF l-3-HYDROXYACYL-CoA DEHYDROGENASE

Sequestration of the Active Site by Interdomain Shifting: CRYSTALLOGRAPHIC AND SPECTROSCOPIC EVIDENCE FOR DISTINCT CONFORMATIONS OF l-3-HYDROXYACYL-CoA DEHYDROGENASE

Mesenchymal regulation of alveolar repair in pulmonary fibrosis.

Induction of alveolar epithelial cell phenotypes via mesenchymal signaling regulates pneumocyte differentiation not only during lung development (1, 2) but also during the response to a myriad of injuries inciting fibrogenesis (3). Signals generated in the epithelium and the mesenchyme regulate critical functions of cells in both compartments reciprocally, with transduction mediated by soluble growth factors, membrane-anchored receptors, and extracellular matrix proteins (4). Such epithelial‐mesenchymal interactions characterize remodeling in a variety of tissues, including airways and lung parenchyma (3, 5, 6). Transition from injury to the repair phase in alveolar remodeling requires epithelialization to regenerate pneumocyte populations and the epithelial component of the alveolar barrier. Ordered layering of type I and type II pneumocytes, basement membranes, and capillary endothelial cells establishes compartmentalization that normalizes communication among cells and extracellular matrix components in distinct epithelial and mesenchymal layers. Restoration of alveolar quiescence reverses ventilation‐perfusion defects to equilibrate gas exchange. By contrast, unrelenting signaling between the compartments leads to continued invasion of fibroblasts and excessive deposition of matrix proteins, causing intra-alveolar fibrosis and the loss of surface area, which defines the pathophysiology of pulmonary fibrosis (3). Epithelial cells differentiate into subpopulations, which reconstruct a protective barrier and regulate clearance of alveolar exudates. Type II cells differentiate from cuboidal progenitor cells at septal intersections into thin type I cells that line approximately 90% of the alveolar unit. Subpopulations of type II pneumocytes synthesize surfactants needed to reduce alveolar surface tension and also produce matrix constituents such as fibronectin, collagen IV, and proteoglycan, essential for remodeling. Other type II cells help resorb proteinaceous exudates by releasing fibrinolytic proteases or by upregulating expression of water and ion channels. Some type II pneumocytes also serve as stem cells to replenish the type II epithelial cell population itself (3). Failure of the alveolar epithelium to perform these functions results both in the unchecked migration of fibroblasts onto the provisional matrix and the continued matrix protein deposition, which propagate fibrogenesis. Premature demise of the epithelial cell population may result from apoptosis due to incessant injury or from apoptotic signals arising from the mesenchyme (7). Thus, epithelial dysfunction permits sequential flooding and increased surface tension in the alveolar space, apposition and fusion of exposed basement membranes, and irreversible collapse and fibrosis—all of which diminish effective alveolar surface area (3).

Identification of nuclear matrix and associated proteins that bind the haptoglobin gene cis-element.

To identify the major nuclear matrix proteins that bind to the rat haptoglobin gene cis-element, we isolated a soluble nuclear matrix protein fraction and analysed it by gel retardation. Two major DNA-binding proteins exhibiting different types of protein-DNA interactions were detected: a DNA sequence-specific 32-kDa isoform of transcription factor C/EBP beta, and a nuclear matrix protein p55 that bound to the DNA nonspecifically. During increased transcription of the haptoglobin gene in the course of the acute-phase reaction, the DNA-binding affinities and concentrations of these proteins in the soluble nuclear matrix fraction were increased. These data lend further evidence that the nuclear matrix is an active support structure that localizes gene regulatory proteins and participates in transcriptional regulation.

Paradoxical Decrease of an Adipose-Specific Protein, Adiponectin, in Obesity

We isolated the human adipose-specific and most abundant gene transcript, apM1 (Maeda, K., et al.,Biochem. Biophys. Res. Commun.221, 286–289, 1996). The apM1 gene product was a kind of soluble matrix protein, which we named adiponectin. To quantitate the plasma adiponectin concentration, we have produced monoclonal and polyclonal antibodies for human adiponectin and developed an enzyme-linked immunosorbent assay (ELISA) system. Adiponectin was abundantly present in the plasma of healthy volunteers in the range from 1.9 to 17.0 mg/ml. Plasma concentrations of adiponectin in obese subjects were significantly lower than those in non-obese subjects, although adiponectin is secreted only from adipose tissue. The ELISA system developed in this study will be useful for elucidating the physiological and pathophysiological role of adiponectin in humans.

Primary structure of a soluble matrix protein of scallop shell; implications for calcium carbonate biomineralization

Soluble proteins in the scallop (Patinopecten yessoensis) foliated calcite shell layer were characterized using biochemical and molecular biological techniques. SDS PAGE of these molecules revealed three major protein bands, 97 kD, 72 kD, and 49 kD in molecular weight, when stained with Coomassie Brilliant Blue. Periodic Acid Schiff staining and Stains-All staining indicated that these proteins are slightly glycosylated and may have cation-binding potential. N-terminal sequencing of the three proteins revealed that all three share the same amino acid sequence at least for the first 20 residues. A partial amino acid sequence of 436 amino acids of one of these proteins (MSP-1) was deduced by characterization of the complementary DNA encoding the protein. The deduced sequence is composed of a high proporton of Ser (31%), Gly (25%), and Asp (20%), typifying an acidic glycoprotein of mineralized tissues. The protein has a basic domain near the Nterminus and two highly conserved Asp-rich domains interspersed in three Ser and Glyrich regions. In contrast with prevalent expectations, (Asp-Gly)n-, (Asp-Ser)n-, and (AspGly-X-Gly-X-Gly)n-type sequence motifs do not exist in the Asp-rich domains, demanding revision of previous theories of protein-mineral interactions.

Primary structure of a soluble matrix protein of scallop shell; implications for calcium carbonate biomineralization

Primary structure of a soluble matrix protein of scallop shell; implications for calcium carbonate biomineralization

Modulation of calcium current in arteriolar smooth muscle by alphav beta3 and alpha5 beta1 integrin ligands.

Vasoactive effects of soluble matrix proteins and integrin-binding peptides on arterioles are mediated by alphav beta3 and alpha5 beta1 integrins. To examine the underlying mechanisms, we measured L-type Ca2+ channel current in arteriolar smooth muscle cells in response to integrin ligands. Whole-cell, inward Ba2+ currents were inhibited after application of soluble cyclic RGD peptide, vitronectin (VN), fibronectin (FN), either of two anti-beta3 integrin antibodies, or monovalent beta3 antibody. With VN or beta3 antibody coated onto microbeads and presented as an insoluble ligand, current was also inhibited. In contrast, beads coated with FN or alpha5 antibody produced significant enhancement of current after bead attachment. Soluble alpha5 antibody had no effect on current but blocked the increase in current evoked by FN-coated beads and enhanced current when applied in combination with an appropriate IgG. The data suggest that alphavbeta3 and alpha5 beta1 integrins are differentially linked through intracellular signaling pathways to the L-type Ca2+ channel and thereby alter control of Ca2+ influx in vascular smooth muscle. This would account for the vasoactive effects of integrin ligands on arterioles and provide a potential mechanism for wound recognition during tissue injury.

Intrauterine Growth Retardation (IUGR) Alters Muscle Gene Expression of L-3-Hydroxy-CoA Dehydrogenase (L3HD): A Pivotal Enzyme of β Oxidation • 398

Uteroplacental insufficiency leading to IUGR is associated with an increased risk for the development of insulin resistance and NIDDM. Because increased lipid oxidation is associated with insulin resistance, we hypothesized that gene expression of mitochondrial enzymes involved in fatty acid oxidation would be altered in muscle of IUGR juvenile rats. We performed bilateral uterine artery ligation to induce IUGR and sham surgery (C) in pregnant rats on day 19 of gestation. Muscle was harvested on day 14(J14) and day 21(J21) of neonatal life. Using RT-PCR with an internal control, we measured mRNA levels of carnitine palmitoyl-transferase II (CPTII)(transports fatty acids across the inner mitochondrial membrane), L3HD (a component of the beta-oxidation complex), electron transfer flavoprotein (ETF)(a soluble matrix protein which participates in transfer of electrons to the respiratory chain at complex II), and succinate dehydrogenase (SD) (a component of complex II). Gene expression of all four enzymes were not significantly different in IUGR J14 muscle (C=1.0, CPTII=1.03, L3HD=0.95, ETF=0.74, SD=1.1). In J21 muscle, gene expression of CPTII, ETF, and SD were not significantly altered in IUGR muscle; however, in contrast, gene expression of L3HD was increased almost two fold (C=1.0, CPTII=1.24, L3AD=1.94*, ETF-1.08, SD=1.12; *p<0.05). L3HD is a unique component of the beta oxidation enzyme complex because it alone competes with three NAD-dependent dehydrogenases of the Krebs cycle. As a result, L3HD may play an important role in determining the dynamic balance between glucose and lipid oxidation. Our findings are consistent with our previous observation of an increased mitochondrial NADH/NAD ratio in J21 IUGR muscle which also favors beta oxidation. We conclude that the altered metabolic milieu associated with IUGR leads to increased gene expression of L3HD. We speculate that the phenomena of metabolic imprinting may eventually lead to the development of insulin resistance in IUGR offspring.

A carbonic anhydrase from the nacreous layer in oyster pearls.

It is believed that the polymorphism observed in calcium carbonate crystals, such as aragonite and calcite in mollusk shells, is controlled by organic matrix proteins secreted from the mantle epithelia. However, the fine structures of these proteins are still unknown, and to understand the molecular mechanisms of mineralization process, detailed structural analyses of the organic matrix proteins are essential. For this, we have carried out purification, characterization, and cDNA cloning of nacrein, which is a soluble organic matrix protein in the nacreous layer of oyster pearls. Northern blot analysis showed that the nacrein transcript was specifically expressed in mantle pallial. Analysis of the deduced amino acid sequence revealed that the protein contained two functional domains: one was a carbonic anhydrase and another was a Gly-Xaa-Asn (Xaa = Asp, Asn, or Glu) repeat domain; however, the carbonic anhydrase domain was split into two subdomains with insertion of the Gly-Xaa-Asn repeat domain between them. Our findings suggest that nacrein actually functions as a matrix protein whose repeated Gly-Xaa-Asn domain possibly binds calcium and as a carbonic anhydrase that catalyzes the HCO3- formation, thus participating in calcium carbonate crystal formation of the nacreous layer.

Production of Chemokines, Interleukin-8 and Monocyte Chemoattractant Protein-1, During Monocyte:Endothelial Cell Interactions

The extravasation of leukocytes from the lumen of the vessel to a site of inflammation requires specific binding events. The interaction of leukocytes with endothelium, via specific receptors, may provide intracellular signals that activate extravasating cells. In the present study, we have investigated the production of chemokines, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) during monocyte: endothelial cell interactions. Both unstimulated and interferon-gamma (IFN-gamma)-prestimulated human umbilical vein endothelial cells (HUVEC) produced low constitutive levels of IL-8 and MCP-1. The addition of enriched monocytes with unstimulated HUVEC resulted in synergistic increases in production of both IL-8 and MCP-1. Monocytes cultured with IFN-gamma-preactivated HUVECs demonstrated little additional increase in IL-8 and MCP-1 production in coculture assays compared with unstimulated HUVEC. Northern blot analysis paralleled the protein data, demonstrating upregulated expression of IL-8 and MCP-1 mRNA in stimulated and unstimulated coculture assays. Culture of enriched monocytes and endothelial cells in transwells demonstrated no increases in IL-8 or MCP-1, indicating the necessity for cellular contact for chemokine production. In previous investigations, we have demonstrated that increased monocyte-derived MIP-1 alpha production was induced by intracellular adhesion molecule-1 (ICAM-1) interactions on activated HUVECs. In contrast, addition of anti-ICAM-1 monoclonal antibodies (MoAbs) did not diminish the production of IL-8 and MCP-1 in the present study. Furthermore, neither antibodies to IL-1 nor tumor necrosis factor (TNF) diminished the production of either IL-8 or MCP-1. However, when soluble matrix proteins were added to the coculture to block cellular interactions, the chemokine protein and mRNA levels were significantly decreased. IL-8 production was decreased by both soluble collagen and fibronectin, whereas MCP-1 was decreased by only soluble collagen, suggesting differential activation pathways. These results indicate that IL-8 and MCP-1 production are increased during monocyte and endothelial cell interactions in part due to matrix protein binding mechanisms. This mechanism may serve a role in cell activation, production of chemokines, as well as extravasation and recruitment of additional leukocytes during inflammatory responses.

Production of chemokines, interleukin-8 and monocyte chemoattractant protein-1, during monocyte: endothelial cell interactions

The extravasation of leukocytes from the lumen of the vessel to a site of inflammation requires specific binding events. The interaction of leukocytes with endothelium, via specific receptors, may provide intracellular signals that activate extravasating cells. In the present study, we have investigated the production of chemokines, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) during monocyte: endothelial cell interactions. Both unstimulated and interferon-gamma (IFN-gamma)-prestimulated human umbilical vein endothelial cells (HUVEC) produced low constitutive levels of IL-8 and MCP-1. The addition of enriched monocytes with unstimulated HUVEC resulted in synergistic increases in production of both IL-8 and MCP-1. Monocytes cultured with IFN-gamma-preactivated HUVECs demonstrated little additional increase in IL-8 and MCP-1 production in coculture assays compared with unstimulated HUVEC. Northern blot analysis paralleled the protein data, demonstrating upregulated expression of IL-8 and MCP-1 mRNA in stimulated and unstimulated coculture assays. Culture of enriched monocytes and endothelial cells in transwells demonstrated no increases in IL-8 or MCP-1, indicating the necessity for cellular contact for chemokine production. In previous investigations, we have demonstrated that increased monocyte-derived MIP-1 alpha production was induced by intracellular adhesion molecule-1 (ICAM-1) interactions on activated HUVECs. In contrast, addition of anti-ICAM-1 monoclonal antibodies (MoAbs) did not diminish the production of IL-8 and MCP-1 in the present study. Furthermore, neither antibodies to IL-1 nor tumor necrosis factor (TNF) diminished the production of either IL-8 or MCP- 1. However, when soluble matrix proteins were added to the coculture to block cellular interactions, the chemokine protein and mRNA levels were significantly decreased. IL-8 production was decreased by both soluble collagen and fibronectin, whereas MCP-1 was decreased by only soluble collagen, suggesting differential activation pathways. These results indicate that IL-8 and MCP-1 production are increased during monocyte and endothelial cell interactions in part due to matrix protein binding mechanisms. This mechanism may serve a role in cell activation, production of chemokines, as well as extravasation and recruitment of additional leukocytes during inflammatory responses.

Purification and immunochemistry of a soluble matrix protein of the chicken eggshell (ovocleidin 17)

The protein components of biomineralized structures (matrix proteins) are believed to modulate crystal nucleation and growth, and thereby influence the shape and strength of the final structure. The chicken eggshell contains a complex array of distinct matrix proteins. The most abundant of these was purified to homogeneity by a combination of anionic exchange and hydroxyapatite chromatographies. Antibodies to this protein were raised in rabbit, and utilized for Western blotting and immunohistochemistry. These studies indicated that the 17 kDa antigen (ovocleidin 17, OC-17) is found in the shell gland mucosa, and that only the tubular gland cells were positive. Immunohistochemistry with decalcified shell indicated that OC-17 is uniformly distributed throughout the shell matrix, but concentrated in the mammillary bodies. Our results indicate that this protein is secreted during shell formation and becomes incorporated into this structure. It may therefore play a role in the crystallization process and influence the properties of the resulting eggshell.

Chapter 14 Isolation of Respiratory Cilia

The membrane that surrounds the ciliary axoneme of mammals is active in interaction with the external environment. This activity may result in the alteration of ciliary function. Zwitterionic 3-[(3-cholamidopropyI) dimethylammonio]-1-propanesulfonate (CHAPS) detergent at 0.1% is effective combined with brushing the epithelium. This injury multiplies the yield of cilia approximately 10-fold compared with nontraumatic extraction, but also increases cellular debris. For CHAPS at 0.5% without brushing, there is release ciliary embranes into solution during axoneme isolation. A different approach homogenizes epithelial cells scraped off the trachea, followed by MgCl 2 aggregation and ouabain affinity chromatography to remove intracellular and basolateral membranes from the apical membrane preparation; however, this method results in a mixture of epithelial cells other than just ciliated ones. The method given in this chapter specifically yields isolated intact cilia, derives membranes from the isolated cilia, and identifies apical membrane surface components by prior labeling of the epithelium with biotin, as similarly applied to Chlamydomonas. Solubilizing ciliary membranes releases the dense matrix proteins from within the axoneme. Reconstitution of membrane vesicles after detergent removal separates integral membrane components from soluble matrix proteins. Use of Triton X-114 in a study of Tetrahymena ciliary membrane proteins facilitates further partitioning into aqueous and detergent phases. The chapter discusses the materials and their sourcing, and the procedure involved. Standard transmission electron microscopy (TEM) techniques are employed in embedment, sectioning, staining, and examination. Presence of membranes is ascertained by TEM that also detects a well-camouflaged Gram-negative bacterium, the cilia-associated respiratory bacillus. This microbe is morphologically indistinguishable from cilia by light microscopy unless immunostained with specific antisera.

T cell epitope analysis with peptides simultaneously synthesized on cellulose membranes: Fine mapping of two DQ dependent epitopes

Several MHC class II restricted, CD4 + human T cell clones from three donors were induced with soluble matrix protein of influenza virus. The epitopes recognized by these clones were mapped using a complete set of overlapping 15-mer peptides synthesized with the newly developed SPOT method which allows the simple simultaneous synthesis of numerous peptides on cellulose membranes. Fine analysis of two clones by truncation of the stimulatory peptide by single subsequent amino acids from either the NH 2- or the COOH-terminus revealed the minimal stimulatory determinants of two DQ dependent T cell epitopes.

Mutagenesis of the amino targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals conserved amino acids required for import into peroxisomes in vivo.

Saccharomyces cerevisiae peroxisomal 3-ketoacyl-CoA thiolase is a soluble matrix protein that does not end in a consensus peroxisomal targeting signal-1. The amino terminus of S. cerevisiae peroxisomal thiolase is conserved in 6 of 11 residues with the amino terminus of rat thiolase B, shown to act as a peroxisomal targeting signal-2 (Swinkels, B.W., Gould, S.J., Bodnar, A.G., Rachubinski, R.A., and Subramani, S. (1991) EMBO J. 10, 3255-3262). Unlike mammalian peroxisomal thiolases, there is no extensive cleavage of S. cerevisiae thiolase upon import into peroxisomes. We demonstrate by in vivo expression that the amino-terminal 16 amino acids of S. cerevisiae thiolase are necessary and sufficient for targeting to peroxisomes. This result implies that yeast, like mammalian cells, can target proteins to the peroxisomal matrix by at least two different routes. We also demonstrate by targeted mutagenesis and in vivo expression of mutated thiolase genes that three amino acids conserved in the amino termini of all known thiolases are critical for efficient targeting of S. cerevisiae thiolase to peroxisomes.

Subject index

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.