Formation and function of membrane contact sites between the ER and the endocytic pathway

Membrane contact sites are sites within cells where the membranes of different organelles come very close together, allowing proteins on different organelles to interact directly with each other and also allowing lipids to transfer directly from one organelle to another. We have identified a novel and previously uncharacterised membrane contact site between the endoplasmic reticulum (ER) and endosomes. We know one function of these contacts, which is the interaction between a growth factor receptor on endosomes and a regulatory protein on the ER. Dysregulation of both the growth factor receptor and the regulatory protein have been implicated in human cancer. A likely additional function of these ER:endosome contacts is exchange of cholesterol, as the endosome is the site of cholesterol accumulation derived from the diet and the ER is the site of new cholesterol synthesis. Low cholesterol from the diet stimulates cholesterol synthesis in the ER and so membrane contact sites between endosomes and the ER could be sites of cholesterol sensing. Defects in cholesterol balance underly many human disorders, and contribute to coronary heart disease. This project will identify factors that regulate the formation of membrane contact sites between the ER and endosomes and will develop tools that will allow us to characterise the role of these contact sites in cholesterol sensing and transport. An understanding of the cellular mechanisms underlying membrane contact site formation and cholesterol sensing is the first step towards understanding how those mechanisms are dysregulated under conditions where cholesterol balance is perturbed and how they might be exploited in the design of new therapies.

We have shown that membrane contact sites form between the ER and multivesicular endosomes/bodies (MVBs) and between the ER and lysosomes. The objective of this study is to analyse the formation and function of those contacts. We will use electron microscopy (EM) to directly visualise membrane contact sites between the ER and the endocytic pathway, but in parallel will explore more rapid biochemical assays and fluorescence-based assays that may reflect membrane contact site formation. Such assays will be validated by EM. We have already shown that the protein tyrosine phosphatase, PTP1B, on the ER and endocytosed EGF receptor interact via direct membrane contacts between the ER and MVBs and we will also focus on an ER-localised protein family, VAP, which has been proposed to play a role in membrane contact site formation between the ER and a number of organelles. By manipulating PTP1B:EGFR interaction and VAP interaction with its binding partners we will determine which of these molecules play a role in contact site formation and will, thereby, develop tools to manipulate their formation. This will then allow the dissection of the function of membrane contact sites between the ER and the endocytic pathway. As such contacts form in unstimulated cells they are likely to have functions in addition to PTP1B:EGFR interaction and here we will focus on one probable function, cholesterol exchange between the ER and endosomes. Non-vesicular transport of cholesterol requires the membranes to come into very close apposition and as the main cellular sources of cholesterol are endosomes/lysosomes (derived from endocytosed LDL) and the ER (derived from new synthesis), membrane contact sites between the ER and the endocytic pathway are clearly candidate sites for sensing and exchange of cholesterol. Perturbations in cholesterol homeostasis occur in many human diseases and understanding how cholesterol transport occurs and is regulated will aid the understanding of how these mechanisms break down in disease and how they can be manipulated for therapy. The studies proposed here, which focus on a small number of known or likely functions for membrane contact sites, will provide a basis for future studies aimed at identifying further components and functions of these sites.