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

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.

The retinal pigment epithelium (RPE) phagocytoses daily shed photoreceptor outer segments (POSs). Little is known of how POS-derived membrane lipid is processed; considerable processing is required for secretion to the choroid, including cholesterol esterification in the ER, suggesting the existence of phagosome:ER lipid transport mechanisms. The ER forms membrane contact sites (MCSs), microdomains of close membrane apposition (<30nm), with a diverse range of organelles. We have previously used electron microscopy (EM) to identify membrane contact sites (MCSs) between the ER and endocytic pathway in Hela cells. We have identified regulators of MCS formation and established their role in endocytic pathway:ER transport of LDL-derived cholesterol. We and others have observed ER:phagosome MCSs and I now propose to investigate their regulation and function in the RPE, taking advantage of unique EM tools I have developed. I will first characterise the extent of ER:phagosome MCSs in macrophages and cultured RPE cells fed isolated POSs as well as in mouse eye tissue. I will then examine MCSs in macrophages and RPE cells depleted of regulators of ER MCSs with the endocytic pathway, or of STIM1, a phagosome-specific candidate regulator. I will additionally screen for novel MCS regulators, as well as determining the effect of manipulating cytosolic calcium concentrations on ER:phagosome MCS formation and phagosome maturation. The physiological relevance will be tested in eyes isolated from mouse models where available. Having established key ER:phagocytic pathway MCS regulators, I will measure the effects of MCS manipulation on lipid transport and cholesterol esterification in cultured RPE fed with isolated POSs. This project will elucidate the potential role of MCSs in the fundamental process of the transport and esterification of POS-derived free cholesterol in the RPE for secretion of lipoproteins, with potential significance to the development of AMD.

Academics from multiple fields will benefit from this research. The existence of membrane contact sites that provide a novel means of communication between organelles has only recently been recognized. How many different types exist, how they are formed and their functional significance are comparatively new fields of study that are now attracting great interest and research effort. I have played a major part in this effort and continue to do so, taking advantage of my expertise in electron microscopy, the only way to identify membrane contact sites unequivocally. In collaboration with Clare Futter I demonstrated that membrane contact sites form between EGF receptor-containing endosomes and the ER and play a role in regulating tyrosine kinase activity. That this work was published in Nature Cell Biology reflects interest in and perceived importance of this field of research. I have since identified a role for membrane contact sites in the transport of LDL-derived cholesterol to the ER for esterification (manuscript under submission). The proposed research would further these findings on membrane contact sites by characterising their formation with the phagocytic pathway in the retinal pigment epithelium (RPE) their relationship with calcium signaling and their role in intracellular lipid processing in the RPE. Research into interorganellar lipid exchange and localised calcium flux would benefit researchers working in the fields of calcium dynamics and lipid traffic. The interest in fundamental aspects of membrane contact site formation and function and the contribution that we have made to the field is evidenced by invitations to international meetings at which both myself and Clare Futter have presented data on membrane contact sites.
The proposed research will involve continued development of 3D serial electron microscopy imaging, optimising staining and embedding techniques to improve the achievable resolution using the Gatan 3View SEM and will therefore advance the field of 3D EM imaging. The Institute of Ophthalmology obtained, in 2011, the first Gatan 3View in the UK. I have helped to extend the scope of this new technology and will continue to do so through the current application and these efforts will benefit the many academics and industrial partners that use both the system at the Institute of Ophthalmology and newly acquired systems elsewhere in the UK and abroad.
This research will also be of potential benefit to healthcare professionals working in the fields of ophthalmology and degenerative disorders. AMD is the leading cause of blindness among the elderly and its management currently places a huge financial burden on the healthcare system, set to worsen with the predicted continued increase in life expectancy. Furthering understanding of the pathogenesis of this disease could expose novel therapeutic targets for AMD and other retinal pathologies.
Through my internal and external collaborations as well as training a technician on this project in the technically challenging techniques associated with electron microscopy, this project would also promote the accessibility of this immensely powerful technique to cell biologists in the UK.