Formation and function of Membrane Contact Sites between the ER and the phagocytic pathway in the Retinal Pigment Epithelium.

视网膜色素上皮(RPE)是一种保护视网膜免受过量光线影响的有色细胞层，对感光细胞(视网膜中探测光线的细胞)的生存起着至关重要的作用。这包括每天吞噬从光感受器外段(POSS)脱落的富含胆固醇的圆盘，以及它们在称为吞噬小体的细胞内间隔内的处理。内质网(ER)和吞噬小体的局部钙水平升高可以促进吞噬作用，对维持光感受器功能可能很重要。人们对POS源脂质的加工过程知之甚少，但为了包装成脂蛋白并从RPE中分泌出来在血流中清除，POS膜上的胆固醇必须经过相当长的加工过程。胆固醇在包装成脂蛋白或以脂滴形式储存在细胞中之前被转化为中性脂类。唯一可以发生酯化的地方是内质网，这表明POS产生的游离胆固醇可能从吞噬小体运输到内质网。细胞也可以通过一种称为内吞作用的过程从血液中吸收与蛋白质(LDL)复合的胆固醇，这一过程允许在内吞体内加工之前内化到细胞内。使用高分辨率电子显微镜，我们以前在不同类型的细胞中确定了膜接触部位(MCSs)，即两种不同的膜非常接近(&lt；30 nm)的内质网和内容体之间的区域，最近发现这些接触点在将低密度脂蛋白(LDL)衍生的胆固醇从内容体运输到内质网进行酯化过程中发挥作用。我们已经确定了这些MCS的一些调节因子，可以通过操纵它们来增加或减少MCS的形成。我们还确定了内质网和RPE中的吞噬体间的接触部位。内体和吞噬体之间有许多相似之处，我们已经证明两者经常融合在一起。因此，似乎在这些ER：吞噬小体接触部位的功能和调控方式上存在相似性。我现在建议利用我们在内质网和内体之间建立的MCSs研究工具，来研究RPE中内质网和吞噬细胞途径之间的MCSs的调节和功能。我将使用电子显微镜来测量RPE中ER：吞噬小体接触的程度，并将调查这些接触是如何调节的，操纵已知的ER：内吞体MCS的调节因子和在其他细胞系统中确定的ER：吞噬小体接触的候选调节因子。我也在为新奇的MCS调节器做筛选。在建立了内质网和吞噬细胞途径之间MCS的关键调节之后，我将利用这些发现来操纵MCS的形成，并测量对胆固醇运输和酯化的影响。此外，还将研究钙在MCSs调节中的潜在作用，以及钙水平、MCSs和有效的吞噬小体成熟之间的关系。这个项目将增加我们对吞噬小体成熟的分子调控的理解，吞噬小体成熟对RPE和光感受器的健康至关重要，因此对维持正常的视觉功能是必不可少的。我们还将解决我们在RPE中胆固醇运输知识中的一个根本差距：胆固醇如何运输到内质网进行酯化反应。在老年性黄斑变性(AMD)中，RPE中含有酯化胆固醇的脂肪沉积在眼睛中积累，AMD是西方世界老年人失明的主要原因。因此，该项目可能确定AMD的新治疗靶点。

The retinal pigment epithelium (RPE), a pigmented cell layer that shields the retina from excess light, plays many important roles essential for the survival of the photoreceptors, the cells of the retina that detect light. This includes the daily engulfment of cholesterol-rich disks shed from photoreceptor outer segments (POSs) and their processing within intracellular compartments called phagosomes. Localised rises in calcium levels at the endoplasmic reticulum (ER) and phagosome can boost phagocytosis and could be important in maintaining photoreceptor function. Little is known about the processing of POS-derived lipids but in order to be packaged into lipoproteins and secreted out of the RPE for clearance in the blood stream, the cholesterol from the membranes of the POSs must undergo considerable processing. The cholesterol is converted to neutral lipid prior to packaging into lipoprotein or storage in the cell in lipid droplets. The only place that esterification can occur is in the ER, suggesting that the POS-derived free cholesterol might be transported from phagosomes to the ER.Cells can also take up cholesterol complexed with protein (LDL) from the blood via a process called endocytosis, which allows internalization into the cell prior to processing within endosomes. Using high resolution electron microscopy, we have previously identified membrane contact sites (MCSs), regions where two different membranes are extremely close together (<30nm), between the ER and endosomes in different types of cells and more recently found that these contact sites play a role in the transport of LDL-derived cholesterol from endosomes to the ER for esterification. We have identified a number of regulators of these MCSs, that can be manipulated to increase or reduce MCS formation. We have also identified contact sites between the ER and phagosomes in the RPE. There are many parallels between the endosomes and phagosomes and we have shown the two often fuse together. It therefore seems likely that similarities exist in the way that these ER:phagosome contact sites both function and are regulated. I now propose to investigate the regulation and function of MCSs between the ER and the phagocytic pathway in the RPE, taking advantage of tools we have established in the study of MCSs between the ER and endosomes. I will use electron microscopy to measure the extent of ER:phagosome contacts in the RPE and will investigate how these contacts are regulated, manipulating known regulators of ER:endosome MCSs and candidate regulators of ER: phagosome contacts identified in other cell systems. I was also screen for novel MCS regulators. Having established the key regulators of MCSs between the ER and the phagocytic pathway, I will exploit these findings to manipulate MCS formation and measure the effects on cholesterol transport and esterification. In addition, a potential role for calcium in the regulation of MCSs and the relationship between calcium levels, MCSs and efficient phagosome maturation, will be investigated. This project will increase our understanding of the molecular regulation of phagosome maturation, which is essential to the health of both the RPE and photoreceptors and therefore to maintaining normal visual function. We will also address a fundamental gap in our knowledge of cholesterol transport in the RPE: how cholesterol is transported to the ER for esterification. Lipid deposits containing esterified cholesterol from the RPE accumulate in the eye in Age-related Macular Degeneration (AMD), a leading cause of blindness among the elderly in the Western world. Thus this project may identify novel therapeutic targets for AMD.