Proteomic Analysis of the Retina

描述（由申请人提供）：尽管经过几十年的研究，调节光感受器外段组装的机制在很大程度上仍然未知。我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段组装。在我们之前对非洲爪蟾胚胎视网膜的研究中，我们已经表明，去除RPE不仅会破坏外节段的组装，还会对光感受器和Muller细胞的蛋白质表达谱进行差异调节。我们还确定了一些因素，当添加到培养物中时，可以模拟RPE的存在，允许正常的外段组装和正常的视网膜蛋白表达谱。为了促进和加快描述光感受器外段组装复杂过程的机制，在申请的资助期间，我们将把二维（2-D）蛋白质分离以及基质辅助激光解吸电离飞行时间（MALDI-ToF）质谱法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蟾视网膜的蛋白质表达谱，即视网膜蛋白质组，在四种表征良好的实验条件下（差异蛋白质组学）：对照全rpe -神经视网膜蛋白质组；外节紊乱的rped视网膜；在支持正常外段组装的非代谢聚糖（“允许的”聚糖）存在下培养的视网膜作为阳性对照；以及暴露于“非允许”聚糖的视网膜，以控制假阳性。通过使用2D差分凝胶电泳（2D- dige）和ettan12多播凝胶系统，凝胶间的可变性将最小化，可重复性增强。在不同条件下上调或下调的蛋白质将被MALDI-ToF MS识别，通过聚类分析进行分类，并随后进行表征。我们预测：(1)大多数差异调节蛋白将聚集在三个功能基团中（细胞粘附分子、细胞骨架蛋白和细胞内信号通路）；(2)大多数相关蛋白将在光感受器和穆勒细胞中表达。因此，我们将使用这些标准来优先分析差异调节蛋白。然后，我们将确定满足这些标准的选定靶蛋白是否必要和足以支持外段组装，方法是使用Morpholino基因敲低方法下调其表达，然后对外段组织进行量化。我们的方法将使我们能够识别任何功能基团和/或新的蛋白质，足以进行光感受器外段组装。这些研究还将为未来的项目阶段提供框架，其中精确的分子机制和控制光感受器外段组装的详细途径将被确定。

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.