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

视网膜色素上皮中 ER 和吞噬途径之间膜接触位点的形成和功能。

基本信息

批准号：
MR/M010767/1
负责人：
Emily Eden
金额：
$ 55.14万
依托单位：
University College London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=MR%2FM010767%2F1
关键词：
Formation function Membrane Contact Sites

项目摘要

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.

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色素细胞层，它对光感受器（视网膜的细胞）的生存起着许多重要的作用。这包括每天吞噬从光感受器外节（POS）脱落的富含胆固醇的盘，以及它们在称为吞噬体的细胞内隔室中的加工。内质网（ER）和吞噬体钙水平的局部升高可以促进吞噬作用，并可能对维持感光功能很重要。关于POS衍生的脂质的加工知之甚少，但是为了包装成脂蛋白并分泌出RPE以在血流中清除，来自POS膜的胆固醇必须经历相当大的加工。胆固醇在包装成脂蛋白或以脂滴形式储存在细胞中之前转化为中性脂质。酯化作用唯一可以发生的地方是在ER中，这表明POS衍生的游离胆固醇可能从吞噬体转运到ER。细胞也可以通过称为内吞作用的过程从血液中摄取胆固醇与蛋白质（LDL）复合，这允许在内体内加工之前内化到细胞中。使用高分辨率电子显微镜，我们以前已经确定了膜接触位点（MCS），在不同类型的细胞中，ER和内体之间的两个不同的膜非常接近（<30 nm）的区域，最近发现这些接触位点在LDL衍生的胆固醇从内体转运到ER进行酯化中发挥作用。我们已经确定了这些MCS的一些监管机构，可以操纵，以增加或减少MCS的形成。我们还确定了ER和RPE中吞噬体之间的接触部位。核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，这些ER：吞噬体接触位点的功能和调节方式似乎存在相似之处。我现在建议研究ER和RPE中吞噬通路之间的MCSs的调节和功能，利用我们在ER和内体之间的MCSs研究中建立的工具。我将使用电子显微镜来测量的程度ER：吞噬体接触的RPE，并将调查这些接触是如何调节，操纵已知的ER：内体MCS和ER：吞噬体接触在其他细胞系统中确定的候选监管机构的监管机构。我也在筛选新型MCS调节器。在ER和吞噬途径之间建立了MCS的关键调节因子后，我将利用这些发现来操纵MCS的形成并测量对胆固醇转运和酯化的影响。此外，钙在调节MCSs中的潜在作用以及钙水平、MCSs和有效吞噬体成熟之间的关系将被研究。该项目将增加我们对吞噬体成熟的分子调节的理解，这对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至关重要。我们还将解决一个根本的差距，我们的知识胆固醇运输的RPE：胆固醇是如何运输到ER酯化。含有来自RPE的酯化胆固醇的脂质沉积物在眼内积累，导致黄斑变性（AMD），这是西方世界老年人失明的主要原因。因此，该项目可能会发现新的治疗AMD的目标。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes.

DOI：
10.15252/embr.201541382
发表时间：
2016-06
期刊：
EMBO reports
影响因子：
7.7
作者：
Starling GP;Yip YY;Sanger A;Morton PE;Eden ER;Dodding MP
通讯作者：
Dodding MP

Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition.

DOI：
10.1242/jcs.164152
发表时间：
2015-01-15
期刊：
Journal of cell science
影响因子：
4
作者：
Hockey LN;Kilpatrick BS;Eden ER;Lin-Moshier Y;Brailoiu GC;Brailoiu E;Futter CE;Schapira AH;Marchant JS;Patel S
通讯作者：
Patel S

Annexin A1 Tethers Membrane Contact Sites that Mediate ER to Endosome Cholesterol Transport.