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• W2098078159 abstract "Polarized epithelial cells are characterized by displaying compartmentalized functions associated with differential distribution of transporters, structural proteins, and signaling molecules on their apical and basolateral surfaces. Their apical surfaces frequently elaborate microvilli, which vary in structure according to the specific type and function of each epithelium. The molecular basis of this heterogeneity is poorly understood. However, differences in function will undoubtedly be reflected in the specific molecular composition of the apical surface in each epithelial subtype. We have exploited a method for isolating microvilli from the mouse eye using wheat germ agglutinin (WGA)-agarose beads to begin to understand the specific molecular composition of apical microvilli of the retinal pigment epithelium (RPE) and expand our knowledge of the potential function of this interface. Initially, apical RPE plasma membranes bound to WGA beads were processed for morphological analysis using known apical and basolateral surface markers. The protein composition of the apical microvilli was then established using proteomic analysis. Over 200 proteins were identified, including a number of proteins previously known to be localized to RPE microvilli, as well as others not known to be present at this surface. Localization of novel proteins identified with proteomics was confirmed by immunohistochemistry in both mouse and rat eye tissue. The data generated provides new information on the protein composition of the RPE apical microvilli. The isolation technique used should be amenable for isolating microvilli in other epithelia as well, allowing new insights into additional functions of this important epithelial compartment. Polarized epithelial cells are characterized by displaying compartmentalized functions associated with differential distribution of transporters, structural proteins, and signaling molecules on their apical and basolateral surfaces. Their apical surfaces frequently elaborate microvilli, which vary in structure according to the specific type and function of each epithelium. The molecular basis of this heterogeneity is poorly understood. However, differences in function will undoubtedly be reflected in the specific molecular composition of the apical surface in each epithelial subtype. We have exploited a method for isolating microvilli from the mouse eye using wheat germ agglutinin (WGA)-agarose beads to begin to understand the specific molecular composition of apical microvilli of the retinal pigment epithelium (RPE) and expand our knowledge of the potential function of this interface. Initially, apical RPE plasma membranes bound to WGA beads were processed for morphological analysis using known apical and basolateral surface markers. The protein composition of the apical microvilli was then established using proteomic analysis. Over 200 proteins were identified, including a number of proteins previously known to be localized to RPE microvilli, as well as others not known to be present at this surface. Localization of novel proteins identified with proteomics was confirmed by immunohistochemistry in both mouse and rat eye tissue. The data generated provides new information on the protein composition of the RPE apical microvilli. The isolation technique used should be amenable for isolating microvilli in other epithelia as well, allowing new insights into additional functions of this important epithelial compartment. Epithelial cells are characterized by the asymmetric distribution of proteins and lipids in their plasma membrane: a basic feature referred to as polarity. The functional polarity of epithelial cells is dependent on the asymmetric distribution of specific enzymes, signaling molecules, and transport proteins between their apical and basolateral surface membranes (1Rodriguez-Boulan E. Nelson W.J. Morphogenesis of the polarized epithelial cell phenotype..Science. 1989; 245: 718-725Crossref PubMed Scopus (816) Google Scholar). The apical surface of cuboidal and columnar epithelial cells commonly faces a luminal cavity and is characterized by the presence of numerous surface membrane elaborations referred to as microvilli. Microvilli greatly increase the apical surface area and, consequently, the number of transport and signaling proteins it contains, thereby enhancing the epithelial functional capacity. Absorptive epithelia such as kidney and intestine have their apical surface decorated with highly organized apical microvilli of uniform length and width. More dynamic and less organized structures are present in the epithelial cells of the placenta and the retinal pigment epithelium (RPE), 1The abbreviations used are: RPE, retinal pigment epithelium; WGA, wheat germ agglutinin (Triticum vulgaris) lectin; Glut-1, glucose transporter type 1; integrin αv, vitronectin receptor α subunit; EBP50, ERM-binding phosphoprotein 50. which perform endocytosis and phagocytosis, respectively. The basis of this morphological heterogeneity is poorly understood and is likely to be related to the specialized function and specific molecular composition of the microvilli in each epithelial cell type. A first step toward the understanding of the relationship between the structure and the functions of specialized epithelial surfaces is through the identification of their constituent molecules (2Arpin M. Crepaldi T. Louvard D. Cross-talk between apical and basolateral domains of epithelial cells regulates microvillus assembly.in: Epithelial Morphogenesis in Development and Disease. Harwood Academic, Amsterdam, The Netherlands1999: 95-116Google Scholar). The RPE is a low-cuboidal epithelium containing very long sheet-like apical microvilli that project into a complex extracellular matrix, referred to as the interphotoreceptor matrix. At this surface, the microvilli interact with the tips of cylindrical photoreceptor outer segments extending from the outer retinal surface. The RPE basal surface is highly infolded and interacts with the underlying Bruch’s membrane (3Zinn K.M. Benjamin-Henkind J.V. Anatomy of the human retinal pigment epithelium.in: The Retinal Pigment Epithelium. Harvard University Press, Cambridge, MA1979: 3-31Google Scholar), an acellular layer separating the RPE from the choriocapillaris. The polarized organization of RPE cells is essential for the vectorial transport of the different molecules between the choriocapillaris and the neural retina and vice versa. The RPE performs highly specialized, unique functions essential for homeostasis of the neural retina. These include phagocytosis of photoreceptors’ shed outer segments, directional transport of nutrients into and removal of waste products from photoreceptor cells, and visual pigment transport and regeneration. The apical microvilli of the RPE play a key role in mediating these activities (3Zinn K.M. Benjamin-Henkind J.V. Anatomy of the human retinal pigment epithelium.in: The Retinal Pigment Epithelium. Harvard University Press, Cambridge, MA1979: 3-31Google Scholar, 4Bok D. The retinal pigment epithelium: A versatile partner in vision..J. Cell Sci. Suppl. 1993; 17: 189-195Crossref PubMed Google Scholar, 5Bonilha V.L. Bhattacharya S.K. West K.A. Crabb J.S. Sun J. Rayborn M.E. Nawrot M. Saari J.C. Crabb J.W. Support for a proposed retinoid-processing protein complex in apical retinal pigment epithelium..Exp. Eye Res. 2004; 79: 419-422Crossref PubMed Scopus (30) Google Scholar). Another unique characteristic of the RPE is the “reversed polarity” of proteins such as the Na,K-ATPase pump, EMMPRIN, and the adhesion molecule N-CAM at the apical surface, rather than at the basolateral surface where these proteins are found in other epithelia (6Clark V.M. The retina: A model for cell biology, Part II.in: The Cell Biology of the Retinal Pigment Epithelium. Academic Press, New York1986: 129-167Google Scholar, 7Gundersen D. Orlowski J. Rodriguez-Boulan E. Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton..J. Cell Biol. 1991; 112: 863-872Crossref PubMed Scopus (156) Google Scholar, 8Gundersen D. Powell S.K. Rodriguez-Boulan E. Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina..J. Cell Biol. 1993; 121: 335-343Crossref PubMed Scopus (67) Google Scholar, 9Marmorstein A.D. Finnemann S.C. Bonilha V.L. Rodriguez-Boulan E. Morphogenesis of the retinal pigment epithelium: Toward understanding retinal degenerative diseases..Ann. N. Y. Acad. Sci. 1998; 857: 1-12Crossref PubMed Scopus (73) Google Scholar). However, the RPE shares many of the common characteristics of other transporting epithelia such as the presence of antioxidative enzymes, amino peptidase (7Gundersen D. Orlowski J. Rodriguez-Boulan E. Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton..J. Cell Biol. 1991; 112: 863-872Crossref PubMed Scopus (156) Google Scholar), and the glucose transporter (10Almers W. Stirling C. Distribution of transport proteins over animal cell membranes..J. Membr. Biol. 1984; 77: 169-186Crossref PubMed Scopus (76) Google Scholar, 11Bergersen L. Johannsson E. Veruki M.L. Nagelhus E.A. Halestrap A. Sejersted O.M. Ottersen O.P. Cellular and subcellular expression of monocarboxylate transporters in the pigment epithelium and retina of the rat..Neuroscience. 1999; 90: 319-331Crossref PubMed Scopus (110) Google Scholar) in their apical microvilli. A more complete definition of the protein composition of the RPE apical microvilli should provide additional insight into other biochemical processes that occur at this interface that are important for the support and maintenance of vision. To this end, we isolated RPE microvilli using wheat germ agglutinin (WGA)-agarose beads (12Cooper N.G. Tarnowski B.I. McLaughlin B.J. Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina..Curr. Eye Res. 1987; 6: 969-979Crossref PubMed Scopus (13) Google Scholar) and defined the protein composition using of MS. Several new proteins identified in this compartment were confirmed with immunocytochemistry. RPE microvilli were isolated using the protocol initially described by Cooper (12Cooper N.G. Tarnowski B.I. McLaughlin B.J. Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina..Curr. Eye Res. 1987; 6: 969-979Crossref PubMed Scopus (13) Google Scholar) with the addition of the mechanical removal of the retina after enzymatic digestion. This procedure was recently described in detail (5Bonilha V.L. Bhattacharya S.K. West K.A. Crabb J.S. Sun J. Rayborn M.E. Nawrot M. Saari J.C. Crabb J.W. Support for a proposed retinoid-processing protein complex in apical retinal pigment epithelium..Exp. Eye Res. 2004; 79: 419-422Crossref PubMed Scopus (30) Google Scholar) and is only briefly summarized here. C57Bl6 mice were sacrificed by CO2 asphyxiation, and the eyes were enucleated. The anterior segments were removed and the eyecups with the exposed neural retina were incubated in 320 U/ml bovine testes hyaluronidase (Sigma, St. Louis, MO) in Hank’s buffered solution for 1h at 37 °C. The neural retina was peeled off from the RPE. Eyecups were extensively washed with TBS plus 1 mm CaCl2 for 1 h at 4 °C followed by incubation with WGA-agarose beads (Sigma) in TBS for 2–3 h at 4 °C. WGA beads were gently scrapped from the eyecups, collected into eppendorf tubes, washed extensively with TBS, and processed for biochemical, morphological, or immunohistochemical analysis. For proteomics analyses, the beads were dissolved in 2× Laemmli buffer, boiled, and resolved by SDS-PAGE on 4–15% gradient gels (Bio-Rad, Hercules, CA). The gel lanes were cut from top to bottom into ∼2-mm slices. Gel slices were washed, reduced, alkylated, digested with trypsin, extracted, and resultant peptides subjected to LC MS/MS analysis using a QTOF2 mass spectrometer equipped with a CapLC System (Waters Corp., Milford, MA). Protein identifications from MS/MS data utilized the Swiss-Prot, and the NCBI sequence databases and the search engines Protein Lynx TM Global server and Mascot (Matrix Science, London, United Kingdom) (5Bonilha V.L. Bhattacharya S.K. West K.A. Crabb J.S. Sun J. Rayborn M.E. Nawrot M. Saari J.C. Crabb J.W. Support for a proposed retinoid-processing protein complex in apical retinal pigment epithelium..Exp. Eye Res. 2004; 79: 419-422Crossref PubMed Scopus (30) Google Scholar, 13Crabb J.W. Miyagi M. Gu X. Shadrach K. West K.A. Sakaguchi H. Kamei M. Hasan A. Yan L. Rayborn M.E. Salomon R.G. Hollyfield J.G. Drusen proteome analysis: An approach to the etiology of age-related macular degeneration..Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14682-14687Crossref PubMed Scopus (993) Google Scholar, 14West K.A. Yan L. Shadrach K. Sun J. Hasan A. Miyagi M. Crabb J.S. Hollyfield J.G. Marmorstein A.D. Crabb J.W. Protein database, human retinal pigment epithelium..Mol. Cell. Proteomics. 2003; 2: 37-49Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). For quality control of the isolation methods, samples of the WGA-agarose beads with attached microvilli and bead-treated eyecups were fixed in 2.5% glutaraldehyde in 0.1 m cacodylate buffer, sequentially dehydrated in ethanol and embedded in Epon as previously reported (15Bonilha V.L. Finnemann S.C. Rodriguez-Boulan E. Ezrin promotes morphogenesis of apical microvilli and basal infoldings in retinal pigment epithelium..J. Cell Biol. 1999; 147: 1533-1548Crossref PubMed Scopus (128) Google Scholar). Thin sections were prepared and electron micrographs were taken on a Tecnai 20 200-kV digital electron microscope (Philips, Hillsboro, OR) using a Gatan image filter and digital camera at 3,600 diameters and are printed at identical magnifications. Additional WGA beads with attached intact microvilli were fixed in 4% paraformaldehyde made in PBS, permeabilized in 0.2% Triton X-100 made in PBS for 10 min at room temperature, and reacted with antibodies to specific apical and basolateral domain markers. Samples were observed under an epifluorescence microscope, and images were collected with a cooled CCD camera (Hamamatsu C5810). Image panels were composed using AdobePhotoshop 5.5 (Adobe, San Jose, CA). To confirm the localization of some of the proteins identified by LC MS/MS analysis, immunohistochemical assays were performed using both paraffin and cryosections of rats and mice eyes. Eyes were enucleated and fixed by immersion in 4% paraformaldehyde made in PBS for 3 h at 4 °C, subsequently the anterior segments were removed. For paraffin processing, fixed eyecups were dehydrated and embedded in paraffin. Immunostaining was carried out on 12-μm sections. After deparaffinization and rehydration to PBS, sections were subjected to heat-mediated antigen retrieval by pressure cooking in 10 mm citric acid buffer, pH 6.0. For cryosectioning, eyecups were fixed as described above, quenched with 50 mm NH4Cl made in PBS for 1 h at 4 °C, infused successively with 15 and 30% sucrose made in the same buffer and with Tissue-Tek “4583” (Miles Inc., Elkhart, IN). For labeling, sections were blocked in PBS supplemented with 0.3 mm CaCl2, 1 mm MgCl2, and 1% BSA (PBS/CM/BSA) for 30 min, and incubated with the monoclonal anti-neuroglycan C antibody in PBS/CM/BSA overnight at 4 °C. The sections were washed in PBS/CM/BSA and incubated with secondary antibodies coupled to Alexa 488 and Alexa 594 (Molecular Probes, Eugene, OR) for 1 h at room temperature. Cell nuclei were labeled with 1 μm TO-PRO-3 (Molecular Probes) in PBS for 15 min. A series of 1-μm xy (en face) sections were collected using a laser scanning confocal microscope (Leica TCS-SP, Exton, PA). Each individual xy image of the retinas stained represents a three-dimensional projection of the entire cryosection (sum of all images in the stack). Polyclonal antibody to Lumican was a generous gift from Chia-Yang Liu (Bascom Palmer Eye Institute, Miami, FL) and used at 1:500. Monoclonal antibody to rat neuroglycan C (BD Biosciences Pharmingen, San Diego, CA) was used at 2.5 μg/ml. Phalloidin-FITC (Sigma) was used at 0.5 μg/ml. A schematic overview of the microvilli isolation method is presented in Fig. 1. The procedure relies on the interaction of N-acetylglucosamine and sialic acid-containing glycoconjugates present in abundance on the free surface of epithelial microvilli (12Cooper N.G. Tarnowski B.I. McLaughlin B.J. Lectin-affinity isolation of microvillous membranes from the pigmented epithelium of rat retina..Curr. Eye Res. 1987; 6: 969-979Crossref PubMed Scopus (13) Google Scholar, 16Roper K. Corbeil D. Huttner W.B. Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane..Nat. Cell Biol. 2000; 2: 582-592Crossref PubMed Scopus (483) Google Scholar) with the WGA lectin conjugated to the agarose beads. Mass interactions of the surface glycoconjugates with the immobilized lectin on the bead allow for the detachment of the RPE microvilli upon physical removal of the WGA beads. Analyzing the residual eyecups by transmission electron microscopy, we observed that the RPE layer remains attached to the Bruch’s membrane following the loss of its apical membranes (Fig. 2). The RPE cells that remained following the loss of their apical microvilli displayed normal ultrastructure without any significant damage to the cell body (Fig. 2, A and B). Some cells, however, retained their apical microvilli, demonstrating that not all the microvilli were extracted with this procedure (Fig. 2B). Subsequent analysis of the WGA beads showed extensive surface areas covered with microvilli (Fig. 2, C and D), indicating that this procedure allows for selective removal of apical microvilli from RPE layer.Fig. 2Transmission electron microscopy of mouse eyecups reacted with WGA-agarose beads (A and B) and the WGA beads scraped off the eyecups (C and D).A, residual RPE cell deprived of microvilli but with an intact cytoplasm and basal infoldings still attached to the Bruch’s membrane. B, most of the RPE cells had their microvilli removed by the WGA beads but some cells in the eyecup still have intact microvilli (MV). C and D, RPE Microvilli attached to WGA beads scraped from mouse eyecups (arrows). p, pigment; BI, basal infoldings; M, mitochondria; N, nucleus; BM, Bruch’s membrane; W, WGA agarose beads. Electron micrographs were taken on a Tecnai 20, 200-kV digital electron microscope using a Gatan image filter and digital camera at 3,600 diameters and are printed at identical magnifications. (Bars: A, B, and C, 1 μm; D, 0.5 μm.)View Large Image Figure ViewerDownload Hi-res image Download (PPT) The microvillar fraction while still attached to the WGA beads was also tested for RPE markers using antibodies directed to apical and basolateral proteins. The fixed beads were probed with specific antibodies and observed under an epifluorescence microscope. Fig. 3 shows that beads with microvilli are labeled with antibodies directed to the apical protein Na,K-ATPase (Fig. 3A) and with proteins present both in the apical and basolateral surfaces like ezrin (Fig. 3B) and glucose transporter type 1 (Glut-1) (Fig. 3C) but not with the basolateral marker, laminin (Fig. 3D). A punctate pattern characteristic of microvillar staining was observed. These data suggests that the WGA bead preparations provide a highly purified RPE microvilli fraction (Figs. 2 and 3), lacking significant cytoplasmic and basal protein contaminants. When the isolated microvilli were subjected to fractionation on a gradient SDS-PAGE, they showed a distinctly different banding pattern compared with total RPE lysates (Fig. 4). The RPE microvilli fraction showed several prominent protein bands (ca. 280, 250, 210, 150, 88, and 49 kDa) that were not as prominent in the total RPE lysate (compare Fig. 4, lanes 5 and 6). In contrast, several major bands in the molecular mass range 33–85 kDa that were prominent in the total RPE lysate were missing in the microvilli fraction. None of these enriched proteins in the microvillar fraction were recovered under control conditions, including WGA bead alone (Fig. 4, lane 4) or protein A-agarose before (Fig. 4, lane 2) and after (Fig. 4, lane 3) exposure to eyecups. MS of peptides present in SDS-PAGE following in-gel trypsin digestion resulted in identification of 283 proteins (Supplemental Table I). A summary of selected proteins identified is shown in Table I. The identified protein profile of the RPE microvilli can be divided into different functional categories: retinoid-metabolizing, cytoskeletal, enzymes, extracellular matrix components, membrane proteins and transporters, unknown, and others (Fig. 5). Several proteins identified in the RPE microvilli fraction were previously identified in the microvilli of other epithelial cells. A number of identified RPE microvilli proteins has been shown independently to localize to this compartment, including basigin, Glut-1, vitronectin receptor α subunit (integrin αv), Na,K-ATPase, ezrin, and ERM-binding phosphoprotein 50 (EBP50). In contrast, proteins resident to the basolateral domain like laminin, ZO-1, and SAP-97 were not found in these samples, substantiating the validity that this isolation procedure allows preferential enrichment of RPE apical microvilli. These results therefore provide an unbiased account of proteins present in the apical microvilli.Table ISelected proteins identified on WGA beads after incubation with apical RPEProteinsAccession no.aSwiss-Prot database and NCBI (in italics) accession numbers are shown; for links use the EXPASY server at us.expasy.org/sprot/ and www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein&cmd=search&term=.Peptides matchedFunctionbFunctions: CYT, cytoskeleton and cytoskeleton-associated; E, enzyme; ECM, extracellular matrix; MT, plasma membrane and transporter; RM, retinoid metabolizing.ExperimentcWGA-coated beads were incubated on the apical RPE surface in mouse eyecups, recovered, washed, and bound proteins identified as described in the text. Selected results from three experiments are shown from 16 mouse eyes each.Actin, cytoplasmic 1 (β-actin)P6071011CYT++Actin, γQ9QZ8319CYT+++α enolaseP171825E+++Annexin A2P073562MT+Annexin A5P480363MT+β enolaseP139293E++BasiginP185726MT+++Carbonic anhydrase XIVQ9WVT62E+Chloride intracellular channel 6Q96NY72MT+++Cellular retinaldehyde-binding protein (CRALBP)Q9Z2756RM+++Cytokeratin 15I495955CYT+++DecorinP286544ECM++DermcidinP816053ECM+EBP50Q9JJ191dThe identified peptide sequences: EBP50, AVDPDSPAEASGLR; peroxiredoxin 1, GLFIIDDKGILR; neuroglycan C, ETGSAIEAEELVR.CYT+EzrinP260404CYT++FibromodulinP506084ECM++Fructose-bisphosphate aldolase AP050644E+++Glut-1P178093MT+++GST PP049062E+Glyceraldehyde 3-phosphate dehydrogenaseP168588E+++Glycogen phosphorylaseQ9WUB35E++Interphotoreceptor retinoid-binding protein (IRBP)P491945RM+++l-lactate dehydrogenase A chain (LDH)P061513+LumicanP518859ECM+++Malate dehydrogenaseP141522++Membrane-associated adenylate kinaseQ9R0Y42MT+MoesinP260413CYT++Monocarboxylate transporter 1AAC137203MT+++Monoglyceride lipaseO356782E++Na,K-transporting ATPase α1 chainP066856MT+++Neuroglycan CQ9QY321cWGA-coated beads were incubated on the apical RPE surface in mouse eyecups, recovered, washed, and bound proteins identified as described in the text. Selected results from three experiments are shown from 16 mouse eyes each.ECM+Neuronal membrane glycoprotein M6-aP516741MT+Peroxiredoxin 1Q637161cWGA-coated beads were incubated on the apical RPE surface in mouse eyecups, recovered, washed, and bound proteins identified as described in the text. Selected results from three experiments are shown from 16 mouse eyes each.E+Peroxiredoxin 2Q9CWJ42E+Phosphoglycerate kinaseP094112E++Profilin IP109242CYT+Pyruvate kinase, M1 isozymeP146184E+Pyruvate kinase, M2 isozymeP524802E+Retinol-binding protein, cellular (CRBP)Q009155RM++Retinol dehydrogenase, 11-cis (RDH5)Q279793RM+++Sodium/potassium-transporting ATPase α-1P066856MT+++Spectrin β chainQ00963CYT++Tubulin α-2 chainP052136CYT++Undulin 1A409707ECM++Vitronectin receptor α subunit (integrin αv)P434063MT++TotaleTotal of proteins identified by LC MS/MS and bioinformatics in each preparation.8697100a Swiss-Prot database and NCBI (in italics) accession numbers are shown; for links use the EXPASY server at us.expasy.org/sprot/ and www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=protein&cmd=search&term=.b Functions: CYT, cytoskeleton and cytoskeleton-associated; E, enzyme; ECM, extracellular matrix; MT, plasma membrane and transporter; RM, retinoid metabolizing.c WGA-coated beads were incubated on the apical RPE surface in mouse eyecups, recovered, washed, and bound proteins identified as described in the text. Selected results from three experiments are shown from 16 mouse eyes each.d The identified peptide sequences: EBP50, AVDPDSPAEASGLR; peroxiredoxin 1, GLFIIDDKGILR; neuroglycan C, ETGSAIEAEELVR.e Total of proteins identified by LC MS/MS and bioinformatics in each preparation. Open table in a new tab Many extracellular molecules were identified in this preparation, which were not characterized as being present in the RPE microvilli-interphotoreceptor matrix. Localization of lumican and neuroglycan C in adult mouse and rat eye sections was investigated to gain further understanding of the distribution of these proteins. Lumican was detected by immunofluorescence of cryosections of mouse eyecups at the RPE apical and basal surfaces and around the photoreceptor outer segments (Fig. 6A). Lumican localization greatly overlapped with the phalloidin-FITC staining of actin filaments (Fig. 6B), as demonstrated by the yellow color in merged images (Fig. 6C). Similarly, rat paraffin sections probed with neuroglycan C antibody revealed localization in association with both the RPE apical and basal surfaces and the photoreceptor outer segment layers (Fig. 6, D–F). Our data suggests that some of the new extracellular components identified with this method are indeed localized to the interphotoreceptor matrix, which is isolated along with the apical microvilli. This report describes a simple and efficient method that produces a highly enriched RPE microvilli fraction. This conclusion is supported by several observations. First, we provide morphological evidence for specific removal of microvilli from cells that remain otherwise intact and for association of microvilli with the isolated beads. Second, we show the presence of known apical markers in the microvilli-enriched fraction, whereas there was absence of basolateral. Third, MS analysis allowed the identification of novel proteins confirmed to be present on the apical surface through subsequent morphological analysis of the tissue. This method produces a fraction of intact microvilli characterized by the presence of proteins such as annexin A2 (17Hansen G.H. Pedersen J. Niels-Christiansen L.L. Immerdal L. Danielsen E.M. Deep-apical tubules: Dynamic lipid-raft microdomains in the brush-border region of enterocytes..Biochem. J. 2003; 373: 125-132Crossref PubMed Scopus (46) Google Scholar, 18Massey-Harroche D. Mayran N. Maroux S. Polarized localizations of annexins I, II, VI and XIII in epithelial cells of intestinal, hepatic and pancreatic tissues..J. Cell Sci. 1998; 111: 3007-3015Crossref PubMed Google Scholar), annexin A5 (19Turnay J. Olmo N. Lizarbe M.A. von der Mark K. Changes in the expression of annexin A5 gene during in vitro chondrocyte differentiation: Influence of cell attachment..J. Cell. 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• W2098078159 created "2016-06-24" @default.
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• W2098078159 date "2004-11-01" @default.
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• W2098078159 title "Proteomic Characterization of Isolated Retinal Pigment Epithelium Microvilli" @default.
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