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

Lead Research Organisation: University College London
Department Name: Institute of Ophthalmology

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

10 25 50
 
Description The regulation of endocytic sorting and cholesterol transport by PTP1B-mediated ESCRT dephosphorylation at ER-endosome membrane contact sites
Amount £447,993 (GBP)
Funding ID MR/P010091/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 01/2017 
End 01/2020
 
Description AMD cells 
Organisation University College London
Department Institute of Ophthalmology UCL
Country United Kingdom 
Sector Academic/University 
PI Contribution I have identified a defect in cholesterol transport in cells from AMD patients
Collaborator Contribution My collaborator provided the AMD patient cells
Impact We have identified a cholesterol transport and endocytic trafficking defect in skin fibroblasts from AMD patients.
Start Year 2016
 
Description Calcium, CT 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution I have provided the means to manipulate membrane contact sites and have started to examine the subcellular localisation of one of their proteins of interest by electron microscopy.
Collaborator Contribution My partners are developing (and sharing) novel fluorescent probes to examine membrane contact sites and have also agreed to perform calcium measurements for me.
Impact We have exchanged both ideas and reagents and are working together to develop tools for measuring and manipulating membrane contact sites.
Start Year 2016
 
Description NPC1-FP 
Organisation University of Oxford
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution My collaborator is interested in NPC1, with a focus on identifying therapeutic targets. I have identified a function for NPC1 at ER-endosome membrane contact sites and am investigating proteins that may be able to compensate for NPC1 loss. In addition I have helped a student from my collaborator's lab to confirm a mitochondrial defect in cells from NPC1 patients by EM. We have worked together on the role of NPC1 at ER-endosome contact sites and our fundings are currently under revision for Nat. Comms.
Collaborator Contribution I have localised NPC1 to phagosomes in the RPE and to ER-phagosome contact sites. My collaborator has provided eyes from NPC1 deficient mice for me to further examine the role of NPC1 in ER-phagosome contact site formation and the processing of lipid derived from photoreceptor outer segments. In addition she has given me tyrosinase inhibitors to enable me to inhibit pigmentation in the early stages of differentiation of human iPS-RPE cells which will facilitate super-resolution imaging. Fran is also a mentor on a recent funding application to moorfields eye charity. We
Impact I have helped confirmation of a mitochondrial defect in NPC1-deficient cells by electron microscopy I have received tyrosinase inhibitors
Start Year 2014
 
Description RAB34 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution I confirmed, by EM, a role for Rab34 at membrane contact sites between the Golgi and lysosomes
Collaborator Contribution This is entirely my collaborator's project that I provided some electron microscopy for since it is directly relevant to my studies.
Impact The EM data that I provided was part of some corrections to a paper published in EMBO J (Starling et al., EMBO Rep 17:823-41, 2016)
Start Year 2015
 
Description SNARES at contact sites 
Organisation University of Sheffield
Department School of Health and Related Research (ScHARR)
Country United Kingdom 
Sector Academic/University 
PI Contribution My collaborator has identified a role for VAMP4 in the transport of LDL-cholesterol to the ER. I am investigating an interaction between VAMP4 and the ER-localised protein VAPA, and whether this interaction might tether ER-endosome membrane contact sites, as well as and the role of ER-endosome membrane contact sites in VAMP4-mediated sterol transport. So far I have demonstrated a VAMP4-VAP interaction and shown, by EM, that VAMP4 is a regulator of ER-endosome membrane contact sites. We are now co-writing a grant to build on these findings.
Collaborator Contribution In addition to fruitful discussions, my collaborator has provided VAMP4 mutant constructs, siRNA and antibodies, as well as other SNARE-related reagents for both this study and in parallel, for studying ER-phagosome membrane contact sites.
Impact I have identified an interaction between VAPA and VAMP4 I have identified a role for VAMP4 in contact site regulation I have received many useful reagents and cell lines from my collaborator
Start Year 2013
 
Description Sphingolipids at contact sites 
Organisation Virginia Commonwealth University
Country United States 
Sector Academic/University 
PI Contribution My collaborator generated SphK1 knock-out cells in which I have identified a defect in the formation of ER-endosome contact sites. I have manipulated ER-endosome contact sites in other cells and sent cell pellets to my collaborator who has used improved MS techniques to measure sphingolipid metabolites.
Collaborator Contribution Provision of SphK1 CRISPR cell lines and plasmids. MS analysis of sphingolipids in AMD patient cells and in cells with reduced contact sites.
Impact Collaboration formed part of collaborator's successful NIH funding application.
Start Year 2017