Membrane contact sites between endolysosomes and the ER as novel hubs in Ca2+ signalling.

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One critical feature which distinguishes our cells from those of bacteria is the presence of organelles - tiny membrane bound structures dedicated to performing specific tasks. But this division of labour comes at a price namely the need for the organelles to communicate in order for the cell to function properly as a whole. One emerging means by which organelles "chatter" is through membrane contact sites. These sites are regions where the organelles come together via bridges so as to allow exchange of small molecules such as lipids and calcium. Understanding the composition of these sites and how they are regulated is critical for understanding how cell function is coordinated.In this application, we focus on contact sites between the endoplasmic reticulum (ER) where new cellular material is made and endolysosomes that take up, recycle and degrade material. Notably, both of these organelles are also stores of calcium. Calcium is perhaps most familiar as a mineral ion important for bones and teeth. This is certainly true. However, less familiar is its critical role in signalling. When cells are stimulated, calcium levels within the cell rapidly increase and this increase sets in train a series of events which change the behaviour of the cell in significant ways. This is critical for probably all cellular events from fertilisation through to the beating of the heart. In many cases this calcium comes from stores.What is now clear is that release of calcium from endolysosomes "triggers" further calcium release from the ER - an excellent example of organelles working together. What is not so clear is how this occurs leading us to our hypothesis that this happens at contact sites between the two organelles. To this end, we have now succeeded in defining some of the proteins involved in connecting endolysosomes and the ER and shown that disrupting these contacts impairs calcium signalling between the two organelles. Surprisingly, we also find that contacts are regulated by calcium itself.We will build on these findings by combining the expertise of the applicants in studying calcium (Patel) and contact sites (Futter). First, we will study a protein on the endolysosomes which we think channels the calcium to stabilize contacts. We will also interfere with the contacts and study how this impacts calcium changes when calcium release from endolysosomes is activated in different ways. In the second part of the program, we will study new proteins that we think are additional contact site components. Finally, we will examine the consequences of disrupting contacts on cell behaviour. Specifically, we will study how contacts affect the function of receptor proteins that are involved in receiving messages from outside of the cells. It is not easy to study contact sites because of their small size. So within the program we will strive to develop new methods to facilitate study. This will be of relevance to many in the scientific community.The successful outcome of the project will provide us with key new insight into how cells control and use their calcium. Disturbances in calcium have already been linked to many disorders including Parkinson's disease and cancer. Could defective contact sites be an unrecognized culprit? Understanding how calcium regulates membrane contacts sites and vice versa could provide the basis for formulating drugs to make or break contact.