Proteomic Analysis of the Retina

描述(申请人提供)：尽管经过几十年的研究，调控光感受器外段组装的机制仍然很大程度上是未知的。我的实验室的重点是了解视网膜色素上皮(RPE)和神经视网膜之间的相互作用如何调节外节组装。在我们之前对非洲爪哇胚胎视网膜的研究中，我们已经表明，去除RPE不仅破坏了外节的组装，而且还对光感受器和Muller细胞的蛋白质表达谱进行了差异调节。我们还确定了一些因素，当加入培养时，可以模拟RPE的存在，允许正常的外节组装和正常的视网膜蛋白表达谱。为了促进和加快描绘光感受器外节段组装复杂过程的潜在机制，在请求的赠款期间，我们将把蛋白质的二维(2-D)分离和基质辅助激光解吸电离飞行时间(MALDI-ToF)质谱仪纳入我们的实验策略。我们建议使用这些强大的蛋白质组学工具来比较非洲爪哇眼睛在四种典型实验条件下(差异蛋白质组学)的视网膜蛋白质组的表达谱：对照完整的RPE-神经视网膜蛋白质组；缺乏RPE且外层节段混乱的视网膜蛋白质组；作为阳性对照的在支持正常外节组装的不可代谢的糖聚糖存在下培养的视网膜的蛋白质组；以及暴露于“不允许的”多糖中的视网膜的蛋白质组，以控制假阳性。通过使用2D差示凝胶内电泳(2D-DGE)和ETTAN12多播凝胶系统，将最大限度地减少凝胶间的变异性，提高重复性。在不同条件下上调或下调的蛋白质将被鉴定为MALDI-ToF MS，通过聚类分析进行分类，并随后进行表征。我们预测：(1)大多数差异调节的蛋白质将聚集在三个功能群(细胞黏附分子、细胞骨架蛋白和细胞内信号通路)中；以及(2)大多数涉及的蛋白质将在光感受器和Muller细胞中表达。因此，我们将使用这些标准来优先分析差异调节的蛋白质。然后，我们将通过使用Morpholino基因敲除方法下调它们的表达，然后量化外段组织，来确定选定的符合这些标准的目标蛋白是否必要且足以支持外段组装。我们的方法将使我们能够识别任何足以进行光感受器外段组装的功能基团和/或新的蛋白质。这些研究还将为未来的项目期提供框架，在这些框架中，将确定控制光感受器外段组装的精确分子机制和详细途径。

DESCRIPTION (provided by applicant): Despite several decades of studies, the mechanisms that regulate photoreceptor outer segment assembly remain largely unknown. The focus of my laboratory is to understand how the interactions between the retinal pigment epithelium (RPE) and the neural retina regulate outer segment assembly. In our previous studies of the Xenopus laevis embryonic retina, we have shown that removal of the RPE not only disrupts outer segment assembly, but also differentially regulates the protein expression profiles of both photoreceptors and Muller cells. We have also identified factors that, when added to culture, can mimic the presence of the RPE, allowing for normal outer segment assembly and normal retinal protein expression profiles. To facilitate and expedite the delineation of the mechanisms underlying the complex process of photoreceptor outer segment assembly, during the requested grant period, we will incorporate two-dimensional (2-D) separation of proteins followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-ToF) mass spectrometry into our experimental strategies. We propose to use these powerful proteomic tools to compare the protein expression profiles of the retina, i.e., the retinal proteome, of Xenopus laevis eyes under four well-characterized experimental conditions (differential proteomics): the control whole RPE-neuroretinal proteome; that of RPEdeprived retinas with disorganized outer segments; that of retinas cultured in the presence of a nonmetabolizable glycan that supports normal outer segment assembly ("permissive" glycan) as a positive control; and that of retinas exposed to a "non permissive" glycan, to control for false positives. Inter-gel variability will be minimized and reproducibility enhanced by using 2D Differential In-Gel Electrophoresis (2D-DIGE) and the ETTANtwelve multicasting gel system. Proteins that are up- or downregulated under the different conditions will be identified MALDI-ToF MS, categorized by cluster analysis, and subsequently characterized. We predict that: (1) the majority of the differentially regulated proteins will cluster within three functional groups (cell adhesion molecules, cytoskeletal proteins, and intracellular signaling pathways); and (2) that the majority of the involved proteins will be expressed in photoreceptors and Muller cells. Therefore, we will use these criteria to prioritize our analysis of the differentially regulated proteins. We will then determine if selected target proteins that fulfill these criteria are necessary and sufficient to support outer segment assembly by down regulating their expression using a Morpholino gene knockdown approach followed by quantification of outer segment organization. Our approach will allow us to identify any functional group and/or novel protein that are sufficient for photoreceptor outer segment assembly. These studies will also generate the framework for future project periods in which the precise molecular mechanisms and detailed pathways that control photoreceptor outer segment assembly will be determined.