Evidence for beta(1)-integrins on both apical and basal surfaces of Xenopus retinal pigment epithelium
Evidence for beta(1)-integrins on both apical and basal surfaces of Xenopus retinal pigment epithelium
复制标题
DOI：
10.1006/exer.1996.0183
复制
发表时间：
1997-01-01
影响因子：
3.4
通讯作者：
Defoe, DM
中科院分区：
文献类型：
--
作者：
Chen, WH;Joos, TO;Defoe, DM
视网膜色素上皮（RPE）是一种具有偏振膜域的运输上皮。这些细胞的一个独特特征是它们的顶端表面不面向管腔空间，而是直接与一层神经元（光感受器）及其相关的细胞外基质相连。由于该位点发生的相互作用对于视网膜附着和颗粒吞噬作用很重要，因此尝试鉴定可能介导细胞-细胞或细胞-基质粘附的上皮分子。在本报告中，β(1)-整合素（细胞外基质配体的主要受体）的亚细胞定位已在爪蟾 RPE 中进行了检查。本分析中使用了几种先前表征的抗体，包括：两种针对纯化的鸡肌纤连蛋白受体的兔多克隆抗体（pAb 编号 3818 和编号 2999），以及一种针对非洲爪蟾 β(1)-整合素亚基特异的单克隆抗体 (mAb 8C8)。在整个上皮细胞提取物的蛋白质印迹中，每种抗体强烈标记 115 kDa 条带，与 β(1)-整合素反应性一致。其中一种试剂（pAb 编号 3818）也对 50 和 100 kDa 的未识别条带进行了微弱染色。预澄清实验表明，pAb No. 3818 和 mAb 8C8 均识别相同的去污剂可溶性整联蛋白：当通过 mAb 8C8 免疫沉淀去除细胞提取物中的 β(1)-整联蛋白时，未观察到 pAb No. 3818 识别的 115 kDa 抗原。与它们相似的免疫化学反应性一致，每种抗体对许多眼杯组织产生相同的免疫细胞化学染色，包括眼外骨骼肌细胞、巩膜和脉络膜成纤维细胞以及脉络膜和神经视网膜的血管内皮。然而，在天然 RPE 和分离的上皮片中，这些抗体之间的标记的质量差异是明显的。共聚焦显微镜分析表明，虽然所有三种抗体都对上皮基底表面进行染色，但 pAb No. 3818 还强烈标记了顶端微绒毛表面。由于在对照实验中相邻光感受器不与该抗体发生交叉反应，因此顶端 RPE 染色不能解释为分离过程中视网膜组织的污染。此外，当顶端细胞表面被选择性地原位生物素化，并且通过链霉亲和素-琼脂糖沉淀生物素化的蛋白质时，通过使用 mAb 8C8 和 pAb No. 3818 进行免疫印迹来检测 β(1)-整合素。然而，这种域特异性材料仅代表整个细胞表面整合素的一小部分：当分离的上皮片被生物素化时，可以检测到大量的标记分子，最有可能代表基础蛋白。基于这些结果，可以得出结论，β(1)-整合素存在于基底和顶端 RPE 质膜中。顶膜中存在的分子可能代表负责视网膜-上皮相互作用的粘附受体的成分。 (C) 1997 学术出版社有限公司
The retinal pigment epithelium (RPE) is a transporting epithelium with polarized membrane domains. A unique characteristic of these cells is that their apical surface does not face a lumenal space, but is directly apposed to a layer of neurons (photoreceptors) and their associated extracellular matrix. Because the interaction occurring at this site is important for retinal attachment and particle phagocytosis, an attempt was made to identify epithelial molecules which potentially could mediate cell-cell or cell-matrix adhesion. In the present report, the subcellular localization of beta(1)-integrins, the main receptors for extracellular matrix ligands, has been examined within Xenopus RPE. Several previously characterized antibodies were used in this analysis including: two rabbit polyclonal antibodies directed against purified chick muscle fibronectin receptor (pAbs No. 3818 and No. 2999), and a monoclonal antibody specific for Xenopus beta(1)-integrin subunit (mAb 8C8). In Western blots of whole epithelial cell extracts, each of the antibodies intensely labeled a 115 kDa band, consistent with beta(1)-integrin reactivity. One of the reagents (pAb No. 3818) also weakly stained unidentified bands of 50 and 100 kDa. Pre-clearing experiments demonstrated that pAb No. 3818 and mAb 8C8 both recognize the same detergent-soluble integrin: when cell extracts were depleted of beta(1)-integrin by immunoprecipitation with mAb 8C8, the 115 kDa antigen recognized by pAb No. 3818 was not observed. Consistent with their similar immunochemical reactivities, each of the antibodies produced equivalent immunocytochemical staining of many eyecup tissues, including extraocular skeletal muscle cells, scleral and choroidal fibroblasts and vascular endothelium of the choroid and neural retina. In the native RPE, and isolated sheets of epithelium, however, qualitative differences in labeling between these antibodies were evident. Analysis by confocal microscopy showed that, while all three antibodies stained the basal surface of the epithelium, pAb No. 3818 also strongly labeled the apical microvillar surface. As the adjacent photoreceptors did not crossreact with this antibody in control experiments, the apical RPE staining could not be accounted for as contamination with retinal tissues during isolation. Furthermore, when the apical cell surface was selectively biotinylated in situ, and biotinylated proteins precipitated by streptavidin-agarose, beta(1)-integrin was detected by immunoblotting with both mAb 8C8 and pAb No. 3818. This domain-specific material, however, represented only a fraction of the whole cell surface integrin: substantially greater amounts of tagged molecules could be detected when isolated epithelial sheets were biotinylated, most likely representing the basal protein. Based on these results, it can be concluded that beta(1)-integrin is present in both basal and apical RPE plasma membranes. Molecules present in the apical membrane may represent components of adhesion receptors responsible for retina-epithelium interactions. (C) 1997 Academic Press Limited