Differential expression of N- and B-cadherin during lens development

PURPOSE: To analyze the dynamics of N- and B-cadherin cell adhesion molecule expression and cytoskeletal interaction during embryonic chick lens development. METHODS: Localization of N- and B-cadherin, F-actin, and connexin 56 were determined by immunohistochemistry of developing lenses or immunocytochemistry of differentiating primary lens cultures. Biochemical analysis of cytoskeletal linkage of N- or B-cadherin was assessed by differential detergent extraction, electrophoresis, and immunoblotting. RESULTS: The results indicate that although both cadherins are expressed throughout lens development, N-cadherin expression detected was similar in both lens epithelial and fiber cells, whereas B-cadherin was preferentially localized to the lens fiber cells. During differentiation, both cadherins become increasingly associated with the lens cytoskeleton, as indicated biochemically by a transition from largely Triton X-100-soluble to Triton X-100-insoluble pools and immunocytologically by cadherin localization to cell-cell borders and colocalization with the actin cytoskeleton. Although a significant fraction of N-cadherin remains Triton X-100-soluble as the lens cells differentiate, B-cadherin becomes resistant to extraction by both Triton X-100 as well as RIPA buffers. As detected immunocytochemically in lens cell cultures, the temporal localization of N-cadherin to cell-cell interfaces precedes that of B-cadherin. Furthermore, temporal localization of B-cadherin, as opposed to N-cadherin, to cell-cell borders more closely parallels that of connexin 56 in vitro as well as in vivo. CONCLUSIONS; These results suggest that while both N- and B-cadherin are expressed during lens cell differentiation, both their patterns of expression as well as their cytoskeletal association differ between epithelial and fiber cells.     

High levels of E-/P-cadherin: correlation with decreased apical polarity of Na/K ATPase in bovine RPE cells in situ

PURPOSE: The adherens junction protein E-cadherin induces a basolateral polarity of Na/K ATPase in most epithelial cells that express it, whereas in retinal pigment epithelium (RPE) cells, Na/K ATPase is largely apical. The purpose of this study was to determine whether the distribution of Na/K ATPase differs in RPE cells in situ, that differ in levels of junctional E-cadherin. METHODS: Bovine RPE cells in situ were immunostained with an E-cadherin antibody (which has some cross-reactivity with the closely related epithelial cadherin P-cadherin), and RPE cells with different levels of junctional stain were identified. RPE cells with low and high E-/P-cadherin were costained in various combinations with Na/K ATPase and interacting proteins of the membrane cytoskeleton (ankyrin, fodrin, and actin) and analyzed by confocal imaging. RESULTS: Individual RPE cells within the same monolayer differed in amount of Na/K ATPase, with a lower frequency of high expressing cells in the area centralis. High expressing Na/K ATPase cells were found among cells with both low and high E-/P-cadherin levels. In cells with low E/P-cadherin, Na/K ATPase localized to apical microvilli, whereas in high E-/P-cadherin cells, Na/K ATPase was on basolateral surfaces in addition to microvilli. Actin staining showed that microvillar domains were smaller and that lateral membrane domains were taller in high E-/P-cadherin cells. In high but not low E-/P-cadherin cells, ankyrin and fodrin levels varied among cells, with a subset of cells showing distinctly higher expression. Both ankyrin and fodrin had complex subcellular distribution patterns, although they tended to be enriched basal to rather than apical to the adherens junction. Cells with high Na/K ATPase did not necessarily have commensurately higher levels of ankyrin or fodrin. Where both Na/K ATPase and ankyrin were high, they codistributed weakly in apical microvilli but more prominently on the basal cell surface. CONCLUSIONS: Within the same RPE monolayer, the polarity of Na/K ATPase differs among cells, with a more basal polarity found in cells with high levels of junctional E-/P-cadherin. The increased basal Na/K ATPase was due to a combination of a smaller microvillar domain, a taller lateral domain, and more basolateral staining for Na/K ATPase, perhaps because of an enrichment of a basal ankyrinfodrin membrane cytoskeleton with which Na/K ATPase is known to associate.     

Expression of motility-related protein MRP1/CD9, N-cadherin, E-cadherin, alpha-catenin and beta-catenin in retinoblastoma

In our earlier study we showed that invasive retinoblastoma (RB) had down regulated tetraspanin protein KAI1/CD82, a family of cell surface glycoprotein. KAI1 may link to the cell surface molecules, such as integrins, E-cadherin, and other TM4SF members, and loss of KAI1 function may have a significant role in the progression of retinoblastoma. We also showed that epithelial cell adhesion molecule (EpCAM) is overexpressed in invasive RB. EpCAM expression decreases adhesion mediated by cadherins. Thus, we were further interested in studying the role of other adhesion molecules like cadherins and catenins in RB. We studied the expression of Motility-Related Protein 1 (MRP-1)/CD9, E-cadherin, N-cadherin, alpha-catenin and beta-catenin in RB and correlated clinicopathologically in 62 archival paraffin-embedded tumors by immunohistochemistry. There were 29 tumors with no invasion of choroids/optic nerve and 33 tumors with invasion of choroid/optic nerve/orbit. Western blotting was performed on 20 tumors using the same antibodies. We observed higher expression of CD9 (P<0.001), E-cadherin (P<0.001) and alpha-catenin (P<0.001) in the non-invasive RB and higher expression of N-cadherin (P<0.001) in invasive RB. The expression of beta-catenin was not significantly different between two groups of tumors. In Western blotting, we were able to see CD9 and E-cadherin expression in a minority of tumors while N-cadherin, alpha-catenin and beta-catenin were expressed with differing intensities in a majority of tumors. Thus, invasive tumors expressed increased N-cadherin, alpha-catenin and decreased E-cadherin and CD9. Thus, it appears that loss of E-cadherin and gain of N-cadherin expression are features of invasiveness. Further functional studies are required to evaluate the role of beta-catenin in RB.     

过氧化物酶体增生物激活受体γ在鼠眼球中的分布

背景过氧化物酶体增生物激活受体γ（PPAR-γ）是一类由配体激活的核转录因子,是潜在的抗炎、抗纤维增生、抗新生血管形成及神经保护因子,其在动物和人体组织中的生理病理功能是目前的研究热点之一,PPARγ与眼科疾病的研究受到关注。目的研究PPARγ在眼部不同组织细胞中的表达,为PPARγ激动剂在眼科疾病治疗中的应用提供参考依据。方法取SPF级C57BL／6J小鼠6只及SD大鼠1只,用质量分数3％水合氯醛麻醉处死后立即摘除眼球,采用Western blot法检测小鼠角膜、晶状体和视网膜组织中PPARγ蛋白的表达;采用免疫组织化学和免疫荧光化学法检测PPARγ在小鼠角膜、晶状体、视网膜、睫状体及视神经组织中的表达及定位。结果Western blot法检测表明,PPARγ在小鼠角膜、晶状体、视网膜中均呈阳性表达。免疫组织化学和免疫荧光化学法检测显示,PPARγ在角膜组织中主要表达于上皮层,以基底细胞染色最强,而角膜内皮及基质细胞上仅有弱表达。PPARγ在晶状体中主要表达于上皮细胞和浅皮质层;在视网膜组织中,PPARγ主要表达于视网膜节细胞层、内丛状层、外丛状层和内核层,此外PPARγ在SD大鼠睫状体组织中主要表达于无色素上皮。免疫荧光化学法检测显示,其在视网膜中与Muller细胞标志物谷氨酰胺合成酶（GS）共定位表达明显;PPARγ在视神经组织中的表达与星形胶质细胞标志物胶质纤维酸性蛋白（GFAP）共定位表达明显。结论PPARγ广泛分布于眼不同组织中并呈特异性表达,该结果为相关眼科疾病的靶向治疗提供了依据。     

Patterned expression of BDNF and NT-3 in the retina and anterior segment of the developing mammalian eye.

PURPOSE: The neurotrophins brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are hypothesized to play an important role in vertebrate eye development because of their patterned expression in the developing and adult neuroretina, their regulated response to retinal and optic nerve injury, and the effects of altered neurotrophin signaling on retinal development. To further characterize the role of these neurotrophins in mammalian eye development and maintenance, the pattern of expression of BDNF and NT-3 was analyzed in the developing and mature mouse eye. METHODS: Using mouse strains in which the reporter gene lacZ, encoding the enzyme beta-galactosidase, was targeted to either the BDNF or NT-3 locus, the expression of BDNF and NT-3 in the eyes of mice heterozygous for these mutations was analyzed by enzyme histochemistry during embryogenesis, postnatal development, and adulthood. RESULTS: BDNF and NT-3 expression were first observed in the inner and outer segments of the developing optic cup at embryonic days 10.5 to 11.5. As the retina matured, BDNF expression was restricted to retinal ganglion cells and a subset of cells in the inner nuclear layer (INL), whereas NT-3 expression was confined to a small subset of cells in the INL and ganglion cell layer. Both neurotrophins were expressed within the developing retinal pigment epithelium. In the anterior segment, BDNF and NT-3 were expressed at high levels in the developing and mature ciliary epithelium. In the lens and cornea, however, these neurotrophins displayed distinct patterns of expression during development and adulthood. BDNF expression was found in the lens epithelium, immature trabecular meshwork, corneal endothelium, and corneal epithelium, whereas NT-3 expression was confined to the corneal epithelium. CONCLUSIONS: BDNF and NT-3 exhibit different, yet overlapping, patterns of expression during the development and differentiation of the mouse eye. In addition to the neuroretina, the spatiotemporal expression of BDNF and NT-3 may play an important role in the development and maintenance of the lens, ciliary body, trabecular meshwork, and cornea.     

N-cadherin expression in a rat model of retinal detachment and reattachment

PURPOSE: To observe the changes in N-cadherin expression in the retina after experimental retinal detachment (RD) and reattachment in the rat and to explore the role N-cadherin might play after RD. METHODS: Forty rat retinas were detached by transscleral injection of 1.4% sodium hyaluronate into the subretinal space. The eyes were enucleated at different time intervals (n = 5), followed by fixation, embedding, and sectioning. The differences in N-cadherin expression in the normal retina, detached retina, and spontaneously reattached retina were determined. Furthermore, an N-cadherin antagonist was injected in combination with 1.4% sodium hyaluronate into the subretinal space in another 10 eyes, in an attempt to demonstrate the role N-cadherin plays after RD. RESULTS: N-cadherin was not expressed in the RPE layer of the normal rat retina. After RD, intense immunolabeling of N-cadherin was seen in the RPE cells, the photoreceptors, and the outer limiting membrane (OLM). An increasing number of cytokeratin (CK)-positive cells likely to be RPE cells was found attached to the outer surface of the detached neural retina. Where the retina was reattached, the N-cadherin immunolabeling rapidly decreased. In eyes treated with an N-cadherin antagonist, the retinas appeared thinner than that in eyes without treatment, and the photoreceptor nuclei showed significantly loss. Moreover, CK-positive cells attached to the outer surface of the detached retina were markedly fewer in number. CONCLUSIONS: Increased expression of N-cadherin in the RPE cells, the photoreceptor cells, and the OLM of the retina after RD may contribute to RPE cell migration and photoreceptor survival. These changes could be reversed by retinal reattachment.     

Differential expression of cadherin adhesion receptors in neural retina of the postnatal mouse

PURPOSE: To determine the expression pattern of multiple subtypes of cadherin adhesion receptor in postnatal mouse neural retina. METHODS: The expression of N-cadherin, R-cadherin, cadherin-6, cadherin-8, and cadherin-11 in retinas at postnatal days 0 to 42 was analyzed by in situ hybridization of mRNA as well as by immunohistochemistry. RESULTS: Each cadherin was expressed by different cell populations of the retina, and the following expression patterns were established by postnatal day 14: in the ganglion cell layer, all these molecules were expressed, but each occurred only in a subset of the cells. Likewise, in the inner nuclear layer, R-cadherin and cadherin-6 and -8 were expressed by a restricted population of amacrine cells, and cadherin-8 also by a subpopulation of bipolar cells. All horizontal cells expressed R-cadherin, and Muller cells expressed N-cadherin and cadherin-11. Proteins of R-cadherin and cadherin-6 were concentrated in neuropil layers. CONCLUSIONS: The pattern of differential expression of the five cadherins supports the idea that these molecules may play a role in selective cell interactions within the heterogeneous cell pool of the neural retina.     

Expression and localisation of apical junctional complex proteins in lens epithelial cells

The lens epithelium possesses an apical junctional complex (AJC) comprising adherens and tight junctions (AJs and TJs) and yet several key structural components and associated regulatory proteins have not been identified or localised in these cells. Here we determine the subcellular distribution of the archetypal TJ markers (ZO-1, claudin-1, and occludin) and TJ-associated cell polarity proteins (aPKC, Par3 and Par6beta) with AJ markers, E- and N-cadherin. As seen in other polarised epithelia, all these markers were located by confocal immunofluorescence microscopy to the apical ends of the lateral plasma membranes of bovine lens epithelial cells at sites of cell-cell interaction. Using immunoelectron microscopy, we show that ZO-1 concentrated at "kissing points" between neighbouring cells and these data, when taken in the context of our confocal immunofluorescence microscopy and blotting data, suggest the presence of TJs within the AJC. Likewise, immunogold labelling for E-cadherin identified AJs within these AJCs. We also report aPKC immunogold labelling localised to the AJC. These data show that the AJC of lens epithelial cells are a composite of TJs and AJs.     

年龄相关性黄斑病变易感因子2蛋白在正常人眼组织中的表达和定位

背景研究表明,年龄相关性黄斑病变易感因子2（ARMS2）基因A69S位点的变异与年龄相关性黄斑变性（AMD）的发病及病变的进展高度相关,但ARMS2蛋白在正常成人眼组织中的定位仍存在争议,相关功能尚未完全明确。目的研究ARMS2蛋白在正常成人视网膜和脉络膜各层的表达定位及其在视网膜色素上皮（RPE）细胞的分布,为进一步从蛋白水平研究ARMS2基因的功能提供实验基础。方法收集死亡后立即摘除的正常供体眼球10只,来自年龄为28～42岁的男性,将其中3只眼球的视网膜和脉络膜组织制作冰冻切片,采用组织免疫荧光法在激光共焦显微镜下检测ARMS2蛋白在视网膜和脉络膜各层的分布。另7只眼球用于分离视网膜组织,并用组织块培养法进行RPE细胞的原代培养,培养的细胞用CK32抗体进行免疫荧光染色鉴定。应用荧光显微镜检测ARMS2蛋白在RPE细胞内的表达及定位情况。结果激光共焦显微镜下可见ARMS2蛋白在正常人视网膜血管、RPE层、Bruch膜、脉络膜血管内呈强阳性表达,而在视网膜神经节细胞层、内核层、外丛状层、外核层、内丛状层ARMS2蛋白表达较弱。组织块法培养的RPE细胞呈多角形,细胞质中可见大量的棕褐色色素颗粒,CK32染色表明原代培养RPE细胞的细胞质中可见大量的绿色荧光颗粒。在RPE细胞内ARMS2阳性蛋白呈簇状红色荧光,主要分布于细胞质中,细胞核为蓝色荧光。结论成年人正常眼视网膜血管中、RPE层、Bruch膜、脉络膜血管内均有ARMS2蛋白的强表达,此外,RPE细胞的细胞质中也可见呈簇状分布的ARMS2蛋白。     

二肽基肽酶II在正常大鼠眼球各组织中的免疫表达

目的:探讨二肽基肽酶II(dipeptidyl peptidaseII,DPPII)在正常大鼠眼球各组织中的免疫表达。方法:应用抗生物素蛋白-生物素-过氧化物酶复合体法,检测DPPII在正常大鼠眼球各组织中的免疫表达。结果:发现DPPII在角膜上皮细胞、角膜基质层、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维、视网膜神经纤维层、神经节细胞、视网膜色素上皮细胞中免疫染色均呈阳性。结论:DPPII存在于角膜上皮细胞、角膜基质层、角膜内皮细胞、睫状体无色素上皮细胞、虹膜色素上皮细胞、晶状体上皮细胞、晶状体纤维、视网膜神经纤维层、神经节细胞、视网膜色素上皮细胞。     

Expression patterns of beta1-related alpha integrin subunits in murine lens during embryonic development and wound healing

PURPOSE: To study the expression patterns of b1-related alpha integrin subunits in murine lens epithelial cells, comparing embryonic fiber differentiation with injury-induced epithelial mesenchymal transition (EMT). METHODS: Adult mice type C57BL/6, pregnant as well as with an eye injured, were sacrificed at different time-course intervals.The embryonic and the injured eyes were obtained and deparaffinized sections of these eyes were processed for immunohistochemistry staining for detection of integrin a subunits. RESULTS: Embryonic lens epithelial cells expressed primarily a3 and a5 subunits, whereas embryonic fiber cells expressed a2, a5, and a6 subunits.Adult lens epithelial cells expressed a3, and a6 subunits,whereas injured lens cells expressed a2, a3, and a6 integrin subunits. CONCLUSIONS: The phenotypic changes of lens epithelial cells during embryonic fiber differentiation and EMT are characterized by different expression of integrin subunits as a result both of the altered extracellular matrix conditions and of the altered cell signaling pathways recruited in each process.     

Expression of tissue plasminogen activator during eye development

Tissue plasminogen activator (tPA) is a serine protease responsible for the activation of plasminogen to plasmin as well as extracellular matrix remodeling. While tPA is used clinically to treat some retinal disorders and it is expressed at low levels in the adult eye, its expression pattern during eye development had never been determined. tPA protein is broadly dispersed in the lens placode and optic vesicle of the mouse eye and it becomes highly localized to the apical surfaces of both the lens pit and the optic cup as they invaginate. In the lens, tPA remains at the apical tips of both lens epithelial and fiber cells from the lens vesicle stage until birth in the mouse, when it begins to downregulate to barely detectable levels in adults. In humans, tPA is found in a similar pattern in the lens vesicle and early lens, however, appreciable protein is also detected in the cytoplasm of lens epithelial cells until adulthood. In the retina, tPA is found at the apical interface between the developing retinal pigmented epithelium and neural retina, then begins to downregulate once the photoreceptors have differentiated. In conclusion, tPA protein is found in a different pattern in embryonic versus adult eyes and may be involved in remodeling of the extracellular environment during eye development.     

羊毛甾醇合酶及羊毛甾醇在大鼠角膜、晶状体和视网膜组织中的分布

背景 已有研究证实与羊毛甾醇结构相似的三萜类化合物对于全身多种疾病具有治疗作用,近年来发现羊毛甾醇合酶(LSS)及羊毛甾醇对白内障具有治疗作用,但羊毛甾醇及其抑制剂与其他眼病的关系尚不清楚.了解羊毛甾醇在眼组织中的分布有助于阐明其与眼病的关系. 目的 研究正常大鼠角膜、晶状体和视网膜中LSS及羊毛甾醇的表达及分布,为相关眼科疾病的靶向治疗提供依据.方法 取SPF级雄性SD大鼠15只,采用戊巴比妥钠过量麻醉法处死实验大鼠,立即摘取眼球,分别采用Western blot和逆转录(RT)-PCR法检测大鼠角膜、晶状体和视网膜组织中LSS蛋白及其mRNA的表达情况;采用免疫荧光化学法对LSS在角膜、晶状体和视网膜组织中的表达进行定位;采用液相色谱-质谱联用仪(LC-MS)检测羊毛甾醇在正常大鼠角膜、晶状体和视网膜组织中的含量. 结果 Western blot法检测显示,大鼠视网膜中无LSS蛋白表达,LSS蛋白在晶状体组织的相对表达量为0.43±0.05,明显高于角膜组织中的0.25±0.03,差异有统计学意义(t =-5.35,P＜0.01).RT-PCR结果显示,正常大鼠视网膜中无LSS mRNA表达,大鼠角膜和晶状体组织中LSS mRNA的相对表达量为0.51±0.04,明显高于角膜组织中的0.29±0.02,差异有统计学意义(t=-8.34,P＜0.01).免疫荧光化学法结果表明,LSS主要表达于角膜上皮、基质和内皮层角膜细胞的细胞质以及晶状体上皮细胞和浅皮质层细胞,而视网膜组织中未检测到LSS表达,且视网膜中LSS与NeuN标记的神经元及谷氨酰胺合成酶(GS)标记的Müller细胞无共表达.LC-MS检测显示,正常大鼠视网膜中未检测到羊毛甾醇,大鼠晶状体中含羊毛甾醇为(24.37±2.91) ng/mg,明显高于角膜组织中的(5.31±0.58) ng/mg,差异有统计学意义(t=-11.13,P＜0.01).结论 LSS及羊毛甾醇分布于大鼠角膜各层组织以及晶状体组织中,在视网膜各层组织中均无LSS表达.     

错配修复基因MSH3在人晶状体中的表达

目的　研究错配修复基因MSH3在人晶状体上皮细胞系SRA01/04、年龄相关性白内障（age-related cataract,ARC）患者晶状体组织以及24周人胎眼晶状体组织中的表达情况，探讨其与ARC形成的关系。方法　采用RT-PCR检测人晶状体上皮细胞系SRA01/04、ARC患者晶状体组织和健康人脐带（阳性对照）中MSH3基因的表达水平，免疫荧光检测24周人胎眼晶状体中MSH3蛋白的表达。结果　MSH3在晶状体上皮细胞系SRA01/04中高表达，在ARC患者晶状体组织中表达下调。MSH3在24周人胎眼晶状体纤维细胞、晶状体上皮细胞以及晶状体前囊组织中均有表达，且在晶状体上皮细胞中高表达。结论　MSH3在人胎眼晶状体组织和SRA01/04中高表达，而在ARC患者晶状体组织中表达下调，这种表达差异可能会影响DNA的错配修复过程，进而影响晶状体发育导致ARC的发生。     

Structure and function of the N-cadherin/catenin complex in retinoblastoma

PURPOSE: To identify in human retinoblastoma and normal retinal tissue the type of cadherin, its relationship with cytoplasmic catenins, and its participation in invasion. METHODS: The cadherin/catenin complex was characterized in surgical retinoblastoma specimens from five patients and human retinas from four donor eyes by immunocytochemistry, flow cytometry, and coimmunoprecipitation with antibodies against N-cadherin, alpha-catenin, and beta-catenin, followed by Western blot analysis or autoradiography. Y79 and WERI-Rb-1 retinoblastoma cell lines serve the evaluation of the cadherin/catenin complex in aggregation and collagen type I invasion in vitro. The association of the cadherin/catenin complex with the cytoskeleton was examined by an antibody-capping assay. RESULTS: In retinoblastoma and normal retina N-cadherin associated with alpha-catenin and beta-catenin but not E- or P-cadherin. The N-cadherin/catenin complex formed a regular, linear, and continuous honeycomb pattern in normal retina that was irregular, clustered, and interrupted in retinoblastoma. The N-cadherin/catenin complex was found also in the retinoblastoma cell lines WERI-Rb and Y79, in which it also showed an irregular pattern. Both cell lines were invasive in collagen type I, and invasion was inhibited by the GC-4 antibody, which functionally neutralizes N-cadherin. Less GC-4 antibody was needed to inhibit invasion of Y79 cells, which expressed N-cadherin at a lower level, than to inhibit invasion of WERI-Rb-1 cells. In both cell lines, antibody capping of the N-cadherin/catenin complex indicated that its linkage with the cytoskeleton were weak or absent. CONCLUSIONS: Retinoblastoma cells, in contrast with normal retina, express an N-cadherin/catenin complex that is irregularly distributed and weakly linked to the cytoskeleton. In retinoblastoma, this complex acts as an invasion promoter.     

Cell phenotype in normal epithelial cell lines with high endogenous N-cadherin: comparison of RPE to an MDCK subclone

PURPOSE: Unlike most monolayer epithelial cells, cultured RPE are competent to form a zonular adhesion of N- rather than E-cadherin. To determine whether other normal epithelial cells do likewise, cells with high endogenous N-cadherin were cloned from the typically E-cadherin dominant epithelial line Madin-Darby canine kidney cells (MDCK) to analyze cell and junction phenotype in the presence of N-cadherin. METHODS: A MDCK subclonal line, clone-YH, was selected for high endogenous N-cadherin and was compared with the RPE line hTERT-RPE1 with regard to cell phenotype, cadherin gene expression and cadherin protein distribution, glycosylation state, and catenin complex composition. RESULTS: In early cultures, hTERT-RPE1 cells are moderately epithelioid with junctional N-cadherin, but clone-YH cells are initially highly fusiform with N-cadherin in multiple sites. With time, N-cadherin in clone-YH becomes deglycosylated, resistant to detergent extraction, and zonular, and cells become epithelioid. Treatment with the N-glycosylation inhibitor tunicamycin induces an epithelioid phenotype in clone-YH, like time in culture but disrupts the hTERT-RPE1 phenotype. N-cadherin traffics to surface membranes and complexes with catenins regardless of cell type or glycosylation state, although catenin complex composition varied, showing enriched alpha-catenin under the cell-type-specific conditions in which N-cadherin was junctional. Clone-YH continued to express E-cadherin as a very minor cadherin, which trafficked to membranes but did not accumulate at junctions. CONCLUSIONS: RPE cells are not unique in localizing N-cadherin to a zonular adhesion typical of a monolayer epithelium, because even epithelial cells derived from a typically E-cadherin dominant line (clone-YH) form a zonular N-cadherin junction if the protein is abundant. However, there are cell and cadherin differences in mechanisms of cadherin accumulation in a zonular pattern, and a previously unrecognized cell-type-specific role for protein glycosylation in epithelial phenotype development.     

Retinal cadherins and the retinal cadherinopathies: Current concepts and future directions

Cadherins are a superfamily of calcium-dependent intercellular adhesion molecules that are widely expressed in living tissues. Within the retina and retinal pigment epithelium (RPE), cadherins contribute to tissue morphogenesis, neural circuit formation, adherens junctions of the outer blood-retinal barrier, photoreceptor disc morphogenesis, maintenance and survival. Four monogenic disorders involving genes which encode cadherins have been identified as causes of inherited retinal degeneration: the retinal cadherinopathies (CDHR1, CDH23, PCDH15, CDH3). Biallelic variants in CDHR1 result in cone-rod dystrophy, rod-cone dystrophy or late-onset macular dystrophy which may be misclassified as dry age-related macular degeneration. Biallelic variants in CDH23 and PCDH15 underlie Usher Syndrome type 1D and 1F. Hypotrichosis with juvenile macular dystrophy results from biallelic variants in CDH3, which contributes to adherens tight junctions between RPE cells. In this review, we summarise the classification of cadherins, and the role of cadherins in the physiology and morphogenesis of the inner and outer retina. Cadherins expressed in primate photoreceptors (CDHR1, CDH23 and PCDH15) have evolved complex roles in outer segment disc morphogenesis and maintenance involving intracellular heterophilic interactions which are as yet incompletely characterised. We highlight what is currently unknown about the molecular function of these cadherins, and review the pathogenesis, clinical phenotype and molecular genetics of each monogenic retinal cadherinopathy. Genes regulating the expression and post-translational modification of retinal cadherins, or those coding for as yet unidentified interacting partners, are candidates for unsolved cases of retinal degeneration. This group of disorders is potentially treatable; we summarise the likely molecular therapeutic approaches and future directions for each retinal cadherinopathy.     

Cultured chicken embryo lens cells resemble differentiating fiber cells in vivo and contain two kinetic pools of connexin56

The lens is an avascular organ in which gap junctions play a pivotal role for cell physiology and transparency. Here we evaluate a lens culture system as a model for studies of lens gap junction dynamics. In culture, chicken embryo lens cells initially form a monolayer of epithelial cells. Subsequently, the epithelial cells differentiate into lentoids, birefringent multicellular structures composed of fiber-like cells. We examined the cultures for the expression of cellular markers and lens fiber specific proteins using immunofluorescence and immunoblot analysis. We also determined the half-life of connexin56 (Cx56), a fiber-specific gap junction protein. All lens cells in culture expressed actin, endoplasmic reticulum proteins and N-cadherin. Only lentoid cells expressed the lens fiber connexins, Cx45.6 and Cx56. Cx56 localized at appositional membranes and did not co-localize with endoplasmic reticulum proteins or N-cadherin. Two pools of Cx56 were detected in these cultures, one with a half-life of a few hours and the other with a half-life of days. The two pools contained phosphorylated forms of Cx56 of different apparent molecular weights. These results suggest that lens cells in culture can be used as a model for the study of lens biology. They also suggest that phosphorylation of Cx56 might be regulating the stability of the protein.