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

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.

Changes in cytosolic Ca2+ form the basis of a critical signalling pathway. Alongside the ER, it is now clear that the
endolysosomal system is also a key mobilizable Ca2+ store. Importantly, these two physically segregated Ca2+ sources are functionally coupled such that endolysosomal Ca2+ release is often amplified by Ca2+ release from the ER to regulate numerous Ca2+-dependent outputs. But the mechanistic and regulatory basis for this cross-talk is unclear.

Here, we consider a role for membrane contact sites in coupling Ca2+ stores. Contact sites are regions of close membrane apposition between organelles that are fast emerging as an important non-vesicular means of communication. This application builds on our new data which has i) revealed the composition of distinct contact sites between endolysosomes and the ER and ii) shown that these contacts are not only required for inter-organellar Ca2+ signalling but also regulated by Ca2+ itself.

Our aims are to bridge complementary expertise in Ca2+ signalling (Patel) and membrane traffic (Futter) to 1) define the role of endolysosome-ER membrane contact sites in integrating Ca2+ signals 2) identify novel endolysosome-ER membrane contact site regulators and 3) establish the physiological relevance of endolysosome-ER membrane contact sites in receptor-mediated signalling events. We will use a combination of molecular and imaging approaches and develop advanced methods to facilitate study of these challenging physiological junctions.

The successful outcome of this project will establish membrane contact sites between endolysosomes and the ER as novel hubs in the control of Ca2+-dependent function.

We identify the following beneficiaries of this collaborative multi-disciplinary project:

1. International science base.
We will provide a broad range of scientific training through the combination of internationally-recognized expertise bought by the applicants within the infrastructure of a world-class university. This will be disseminated not only to the appointee but to the wider scientific community thus adding major value to this proposal.

2. General public.
This application is a basic science proposal addressing a ubiquitous signalling pathway (calcium) that underlies probably all cellular processes. The layman is likely only aware of the role of calcium as a mineral ion. This proposal will provide a better general understanding of the multiple roles that calcium plays physiologically.

3. Women SET.
Futter has been a champion of women in science, engineering and technology (SET). This proposal will provide an ideal vehicle to continue promoting female scientists as role models.

4. Clinicians, patients and the pharmaceutical industry.
The pathways we target are demonstrably relevant to disease. In particular, TPCs are emerging as potential therapeutic targets and our linking of these ubiquitous proteins to membrane contacts sites identifies this cellular locus as novel therapeutic target. The outcomes of this project are thus of potential major relevance to public health and Pharma.