Bonilha Vera L, Bhattacharya Sanjoy K, West Karen A, Sun Jian, Crabb John W, Rayborn Mary E, Hollyfield Joe G
The Cole Eye Institute, Department of Ophthalmic Research, The Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Mol Cell Proteomics. 2004 Nov;3(11):1119-27. doi: 10.1074/mcp.M400106-MCP200. Epub 2004 Sep 14.
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.
极化上皮细胞的特征是具有分区化功能，这与转运蛋白、结构蛋白和信号分子在其顶端和基底外侧表面的差异分布相关。它们的顶端表面常常有精心构建的微绒毛，其结构根据每种上皮细胞的特定类型和功能而有所不同。这种异质性的分子基础尚不清楚。然而，功能上的差异无疑会反映在每种上皮细胞亚型顶端表面的特定分子组成中。我们利用一种方法，即使用麦胚凝集素（WGA）-琼脂糖珠从小鼠眼睛中分离微绒毛，以开始了解视网膜色素上皮（RPE）顶端微绒毛的特定分子组成，并扩展我们对该界面潜在功能的认识。最初，使用已知的顶端和基底外侧表面标记物，对与WGA珠结合的RPE顶端质膜进行形态学分析。然后使用蛋白质组学分析确定顶端微绒毛的蛋白质组成。鉴定出了200多种蛋白质，包括一些先前已知定位于RPE微绒毛的蛋白质，以及其他未知在此表面存在的蛋白质。通过免疫组织化学在小鼠和大鼠眼组织中证实了蛋白质组学鉴定出的新蛋白质的定位。所产生的数据提供了有关RPE顶端微绒毛蛋白质组成的新信息。所使用的分离技术应该也适用于在其他上皮细胞中分离微绒毛，从而使我们能够对这个重要上皮区室的其他功能有新的认识。
