scholarly journals Apical Polarity of N-CAM and EMMPRIN in Retinal Pigment Epithelium Resulting from Suppression of Basolateral Signal Recognition

1998 ◽  
Vol 142 (3) ◽  
pp. 697-710 ◽  
Author(s):  
Alan D. Marmorstein ◽  
Yunbo C. Gan ◽  
Vera L. Bonilha ◽  
Silvia C. Finnemann ◽  
Karl G. Csaky ◽  
...  
Keyword(s):  
Mdck Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Neurotrophin Receptor ◽  
P75 Neurotrophin Receptor ◽  
Apical Surface ◽  
Rpe Cells ◽  
Apical Polarity ◽  
P75 Ntr

Retinal pigment epithelial (RPE) cells apically polarize proteins that are basolateral in other epithelia. This reversal may be generated by the association of RPE with photoreceptors and the interphotoreceptor matrix, postnatal expansion of the RPE apical surface, and/or changes in RPE sorting machinery. We compared two proteins exhibiting reversed, apical polarities in RPE cells, neural cell adhesion molecule (N-CAM; 140-kD isoform) and extracellular matrix metalloproteinase inducer (EMMPRIN), with the cognate apical marker, p75-neurotrophin receptor (p75-NTR). N-CAM and p75-NTR were apically localized from birth to adulthood, contrasting with a basolateral to apical switch of EMMPRIN in developing postnatal rat RPE. Morphometric analysis demonstrated that this switch cannot be attributed to expansion of the apical surface of maturing RPE because the basolateral membrane expanded proportionally, maintaining a 3:1 apical/basolateral ratio. Kinetic analysis of polarized surface delivery in MDCK and RPE-J cells showed that EMMPRIN has a basolateral signal in its cytoplasmic tail recognized by both cell lines. In contrast, the basolateral signal of N-CAM is recognized by MDCK cells but not RPE-J cells. Deletion of N-CAM's basolateral signal did not prevent its apical localization in vivo. The data demonstrate that the apical polarity of EMMPRIN and N-CAM in mature RPE results from suppressed decoding of specific basolateral signals resulting in randomized delivery to the cell surface.

scholarly journals Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton.

1991 ◽  
Vol 112 (5) ◽  
pp. 863-872 ◽  
Author(s):  
D Gundersen ◽  
J Orlowski ◽  
E Rodriguez-Boulan
Keyword(s):  
Retinal Pigment Epithelium ◽  
Epithelial Cells ◽  
Current Knowledge ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Opposite Polarity ◽  
Apical Surface ◽  
Rpe Cells ◽  
Membrane Cytoskeleton

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.

scholarly journals SLC26A7 constitutes the thiocyanate-selective anion conductance of the basolateral membrane of the retinal pigment epithelium

2020 ◽  
Vol 319 (4) ◽  
pp. C641-C656
Author(s):  
Xu Cao ◽  
Manoocher Soleimani ◽  
Bret A. Hughes
Keyword(s):  
Retinal Pigment Epithelium ◽  
Ph Regulation ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Subretinal Fluid ◽  
Wild Type Mouse ◽  
Biologically Active ◽  
Rpe Cells ◽  
Anion Conductance

Anion channels in the retinal pigment epithelium (RPE) play an essential role in the transport of Cl− between the outer retina and the choroidal blood to regulate the ionic composition and volume of the subretinal fluid that surrounds the photoreceptor outer segments. Recently, we reported that the anion conductance of the mouse RPE basolateral membrane is highly selective for the biologically active anion thiocyanate (SCN−), a property that does not correspond with any of the Cl− channels that have been found to be expressed in the RPE to date. The purpose of this study was to determine the extent to which SLC26A7, a SCN− permeable-anion exchanger/channel that was reported to be expressed in human RPE, contributes to the RPE basolateral anion conductance. We show by quantitative RT-PCR that Slc26a7 is highly expressed in mouse RPE compared with other members of the Slc26 gene family and Cl− channel genes known to be expressed in the RPE. By applying immunofluorescence microscopy to mouse retinal sections and isolated cells, we localized SLC26A7 to the RPE basolateral membrane. Finally, we performed whole cell and excised patch recordings from RPE cells acutely isolated from Slc26a7 knockout mice to show that the SCN− conductance and permeability of its basolateral membrane are dramatically smaller relative to wild-type mouse RPE cells. These findings establish SLC26A7 as the SCN−-selective conductance of the RPE basolateral membrane and provide new insight into the physiology of an anion channel that may participate in anion transport and pH regulation by the RPE.

scholarly journals The Basolateral Targeting Signal of CD147 (EMMPRIN) Consists of a Single Leucine and Is Not Recognized by Retinal Pigment Epithelium

2004 ◽  
Vol 15 (9) ◽  
pp. 4148-4165 ◽  
Author(s):  
Ami A. Deora ◽  
Diego Gravotta ◽  
Geri Kreitzer ◽  
Jane Hu ◽  
Dean Bok ◽  
...  
Keyword(s):  
Amino Acids ◽  
Retinal Pigment Epithelium ◽  
Mdck Cells ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Site Directed Mutagenesis ◽  
Immunoglobulin Superfamily ◽  
Type I ◽  
Rpe Cells ◽  
Basolateral Targeting

CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells.

Monocarboxylate transporter MCT1 is located in the apical membrane and MCT3 in the basal membrane of rat RPE

1998 ◽  
Vol 274 (6) ◽  
pp. R1824-R1828 ◽  
Author(s):  
Nancy J. Philp ◽  
Heeyong Yoon ◽  
Evelyn F. Grollman
Keyword(s):  
Apical Membrane ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Basolateral Membrane ◽  
Basal Membrane ◽  
Monocarboxylate Transporter ◽  
Apical Surface ◽  
Membrane Domains ◽  
Blood Retinal Barrier ◽  
Specific Proteins

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier and regulates the movement of nutrients, water, and ions between the choroidal blood supply and the retina. The transport properties of the RPE maintain retinal adhesion and regulate the pH and osmolarity in the space surrounding the photoreceptor cell outer segments. In this report we identify two monocarboxylate transporters, MCT1 and MCT3, expressed in rat RPE. On the basis of Northern and Western blot analyses, MCT1 is expressed in both the neural retina and the RPE, whereas the expression of MCT3 is restricted to the RPE. Using indirect immunolocalization we show that the two transporters are polarized to distinct membrane domains. MCT1 antibody labels the apical surface and the apical processes of the RPE. A polyclonal antibody produced against the carboxy terminus of rat MCT3 labels only the basolateral membrane of the RPE. The demonstration of MCT1 on the apical membrane and MCT3 on the basal membrane identifies specific proteins involved in the discriminate and critical regulation of water and lactate transport from the retina to the choroid.

Apical sorting of influenza hemagglutinin by transcytosis in retinal pigment epithelium

1997 ◽  
Vol 110 (15) ◽  
pp. 1717-1727 ◽  
Author(s):  
V.L. Bonilha ◽  
A.D. Marmorstein ◽  
L. Cohen-Gould ◽  
E. Rodriguez-Boulan
Keyword(s):  
Plasma Membrane ◽  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Subretinal Space ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Apical Surface ◽  
Rpe Cells

The retinal pigment epithelium is endowed with a unique distribution of certain plasma membrane proteins. Na+,K+-ATPase, for instance, is polarized to the apical surface of RPE, rather than to the basolateral surface as in most other epithelia. To study the sorting pathways of RPE cells, we used temperature sensitive mutants of influenza and vesicular stomatitis virus (VSV) to synchronize the transport of hemagglutinin (HA) and VSV G protein (VSV G) along the biosynthetic pathway of the RPE cell line RPE-J. After HA and VSV G accumulated in the trans-Golgi network of RPE-J cells kept at 20 degrees C, transfer to the permissive temperature (32 degrees C) resulted in the transport of both HA and VSV G to the basolateral plasma membrane. Later, while VSV G remained basolateral, HA progressively reversed its polarity, eventually becoming apical. Further analysis demonstrated that the reversal of HA polarity was due to transcytosis of HA from the basolateral to the apical surface of RPE-J cells. To determine whether HA followed a transcytotic route in RPE in vivo, influenza and VSV were injected into the subretinal space of rat eyes. Again, both HA and VSV G were initially observed at the basolateral surface of RPE cells. However, whereas VSV G remained there, HA progressively redistributed to the apical surface. These findings demonstrated that RPE cells use a transcytotic pathway for the targeting of at least some apical proteins to their destination.

scholarly journals Basolateral Targeting of ERBB2 Is Dependent on a Novel Bipartite Juxtamembrane Sorting Signal but Independent of the C-Terminal ERBIN-Binding Domain

2002 ◽  
Vol 22 (18) ◽  
pp. 6553-6563 ◽  
Author(s):  
Christian Dillon ◽  
Anna Creer ◽  
Karen Kerr ◽  
Angelika Kümin ◽  
Clive Dickson
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Binding Domain ◽  
Neurotrophin Receptor ◽  
P75 Neurotrophin Receptor ◽  
Endosomal Trafficking ◽  
Membrane Domain ◽  
Sorting Signal ◽  
Necessary And Sufficient ◽  
Basolateral Targeting

ABSTRACT ERBB2 is a receptor tyrosine kinase present on the basolateral membrane of polarized epithelia and has important functions in organ development and tumorigenesis. Using mutagenic analyses and Madin-Darby canine kidney (MDCK) cells, we have investigated the signals that regulate basolateral targeting of ERBB2. We show that basolateral delivery of ERBB2 is dependent on a novel bipartite juxtamembrane sorting signal residing between Gln-692 and Thr-701. The signal shows only limited sequence homology to known basolateral targeting signals and is both necessary and sufficient for correct sorting of ERBB2. In addition we demonstrate that this motif can function as a dominant basolateral targeting signal by its ability to redirect the apically localized P75 neurotrophin receptor to the basolateral membrane domain of polarized epithelial cells. Interestingly, LLC-PK1 cells, which are deficient for the μ1B subunit of the AP1B adaptor complex, missort a large proportion of ERBB2 to the apical membrane domain. This missorting can be partially corrected by the introduction of μ1B, suggesting a possible role for AP1B in ERBB2 endosomal trafficking. Furthermore, we find that the C-terminal ERBIN binding domain of ERBB2 is not necessary for its basolateral targeting in MDCK cells.

scholarly journals Functional and morphological analysis of the subretinal injection of retinal pigment epithelium cells

2012 ◽  
Vol 29 (2) ◽  
pp. 83-93 ◽  
Author(s):  
MAREN ENGELHARDT ◽  
CHINATSU TOSHA ◽  
VANDA S. LOPES ◽  
BRYAN CHEN ◽  
LISA NGUYEN ◽  
...  
Keyword(s):  
Retinal Pigment Epithelium ◽  
Host Cell ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Primary Cultures ◽  
Host Cells ◽  
Apical Surface ◽  
Noninvasive Methods ◽  
Rpe Cells ◽  
Subretinal Injection

AbstractReplacement of retinal pigment epithelium (RPE) cells by transplantation is a potential treatment for some retinal degenerations. Here, we used a combination of invasive and noninvasive methods to characterize the structural and functional consequences of subretinal injection of RPE cells. Pigmented cells from primary cultures were injected into albino mice. Recovery was monitored over 8 weeks by fundus imaging, spectral domain optical coherence tomography (sdOCT), histology, and electroretinography (ERG). sdOCT showed that retinal reattachment was nearly complete by 1 week. ERG response amplitudes were reduced after injection, with cone-mediated function then recovering better than rod function. Photoreceptor cell loss was evident by sdOCT and histology, near the site of injection, and is likely to have been the main cause of incomplete recovery. With microscopy, injected cells were identified by the presence of apical melanosomes. They either established contact with Bruch’s membrane, and thus became part of the RPE monolayer, or were located on the apical surface of the host’s cells, resulting in apposition of the basal surface of the injected cell with the apical surface of the host cell and the formation of a series of desmosomal junctions. RPE cell density was not increased, indicating that the incorporation of an injected cell into the RPE monolayer was concomitant with the loss of a host cell. The transplanted and remaining host cells contained large vacuoles of ingested debris as well as lipofuscin-like granules, suggesting that they had scavenged the excess injected and host cells, and were stressed by the high digestive load. Therefore, although significant functional and structural recovery was observed, the consequences of this digestive stress may be a concern for longer-term health, especially where RPE cell transplantation is used to treat diseases that include lipofuscin accumulation as part of their pathology.

scholarly journals A human model of Batten disease shows role of CLN3 in phagocytosis at the photoreceptor–RPE interface

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Cynthia Tang ◽  
Jimin Han ◽  
Sonal Dalvi ◽  
Kannan Manian ◽  
Lauren Winschel ◽  
...  
Keyword(s):  
Photoreceptor Cell ◽  
Vision Loss ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cell Loss ◽  
Human Model ◽  
Lysosomal Storage ◽  
Rpe Cells ◽  
Cln3 Disease

AbstractMutations in CLN3 lead to photoreceptor cell loss in CLN3 disease, a lysosomal storage disorder characterized by childhood-onset vision loss, neurological impairment, and premature death. However, how CLN3 mutations cause photoreceptor cell death is not known. Here, we show that CLN3 is required for phagocytosis of photoreceptor outer segment (POS) by retinal pigment epithelium (RPE) cells, a cellular process essential for photoreceptor survival. Specifically, a proportion of CLN3 in human, mouse, and iPSC-RPE cells localized to RPE microvilli, the site of POS phagocytosis. Furthermore, patient-derived CLN3 disease iPSC-RPE cells showed decreased RPE microvilli density and reduced POS binding and ingestion. Notably, POS phagocytosis defect in CLN3 disease iPSC-RPE cells could be rescued by wild-type CLN3 gene supplementation. Altogether, these results illustrate a novel role of CLN3 in regulating POS phagocytosis and suggest a contribution of primary RPE dysfunction for photoreceptor cell loss in CLN3 disease that can be targeted by gene therapy.

The cytoskeletal elements of human retinal pigment epithelium: in vitro and in vivo

1988 ◽  
Vol 91 (2) ◽  
pp. 303-312
Author(s):  
N.M. McKechnie ◽  
M. Boulton ◽  
H.L. Robey ◽  
F.J. Savage ◽  
I. Grierson
Keyword(s):  
Retinal Pigment Epithelium ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Cytokeratin 18 ◽  
Rpe Cells ◽  
Human Retinal Pigment Epithelium ◽  
Cytoskeletal Elements

The cytoskeletal elements of normal (in situ) and cultured human retinal pigment epithelium (RPE) were studied by a variety of immunocytochemical techniques. Primary antibodies to vimentin and cytokeratins were used. Positive immunoreactivity for vimentin was obtained with in situ and cultured material. The pattern of reactivity obtained with antisera and monoclonals to cytokeratins was more complex. Cytokeratin immunoreactivity could be demonstrated in situ and in cultured cells. The pattern of cytokeratin expression was similar to that of simple or glandular epithelia. A monoclonal antibody that specifically recognizes cytokeratin 18 identified a population of cultured RPE cells that had particularly well-defined filamentous networks within their cytoplasm. Freshly isolated RPE was cytokeratin 18 negative by immunofluorescence, but upon culture cytokeratin 18 positive cells were identifiable. Cytokeratin 18 positive cells were identified in all RPE cultures (other than early primaries), regardless of passage number, age or sex of the donor. In post-confluent cultures cytokeratin 18 cells were identified growing over cytokeratin 18 negative cells, suggesting an association of cytokeratin 18 immunoreactivity with cell proliferation. Immunofluorescence studies of retinal scar tissue from two individuals revealed the presence of numerous cytokeratin 18 positive cells. These findings indicate that RPE cells can be identified by their cytokeratin immunoreactivity and that the overt expression of cytokeratin 18 may be associated with proliferation of human RPE both in vitro and in vivo.

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