Calcium exchange between endoplasmic reticulum and lysosomes

Lysosomes are small membrane-bound structures present in all animal cells. They are often described as the cellular 'dustbin', but more aptly as a 'recycling bin', because one of their important tasks is to degrade and then recycle biological materials imported from outside the cell or from cellular structures that have outlived their usefulness. The importance of lysosomes is clear from the devastating effects of lysosomal storage diseases, which are often due to a faulty lysosomal enzyme. These degradative processes are precisely regulated to ensure that hungry cells are provided with the raw materials they need. To accomplish this, lysosomes contain an acidic cocktail of digestive enzymes. Transfer of materials into lysosomes relies on fusion of their membrane with the membranes of other intracellular organelles. This process has been hijacked by man to allow drug delivery and by viruses to allow them to invade cells. Similar membrane-fusion events allow lysosomes to fuse with the plasma membrane that surrounds every cell, facilitating release of materials to the outside world and insertion of new membrane for both repair and membrane extension during cell migration. There is persuasive evidence that the final stages of all these membrane-fusion events require release of calcium from within the lysosome via the pores of calcium-permeable channels. Recent work discovered several of these lysosomal calcium channels, and implicated them in the normal activities of lysosomes and in various pathologies (one is required for infection by Ebola virus, for example). Interest in these channels has also revealed a role for lysosomes in regulating the increases in calcium concentration within the cell that regulate many of its activities. Another, more abundant organelle, the endoplasmic reticulum (ER), and its calcium channels play the major role in generating these regulatory calcium signals, but there is accumulating evidence that lysosomes and ER interact to generate calcium signals. It is, therefore, now clear that many functions of lysosomes are dependent on their ability to accumulate calcium. But we do not know how lysosomes acquire calcium. The issue is challenging because the hostility of the lysosome interior makes it difficult to use the calcium indicators that have been so useful in revealing many other aspects of calcium regulation. Our studies suggest that the ER may be responsible for delivering calcium to lysosomes. The ER has a high-affinity calcium pump that allows it to sequester calcium from the low concentrations that prevail within cells, but it seems likely that the uptake system of lysosomes has much lower affinity. We suggest that having accumulated calcium, the ER releases it into the tiny gaps between ER and lysosome membranes, where the high calcium concentration achieved is sufficient to fuel uptake by lysosomes. Our scheme envisages these ER-lysosome contacts as 'refuelling stations' that lysosomes must visit for a periodic top-up with the calcium they need to sustain their activities.

In this proposal, we seek to develop new optical indicators to measure calcium directly within lysosomes using microscopes that can resolve subcellular architecture. With these and other tools, we will test our hypothesis that ER-lysosome interactions are essential for lysosomes to acquire calcium; identify the proteins that mediate tethering of the organelles; and identify the proteins that transport calcium across lysosomal membranes. We can then disrupt the gene(s) encoding critical proteins in a cell line that has only a single copy of each gene (rather than the usual pair), and so cleanly assess the contributions of lysosomal calcium uptake to many cellular behaviours, notably cell migration. Our work addresses a basic problem in cell biology, with additional potential to unmask unanticipated roles for an important intracellular organelle that is widely implicated in many human diseases.

Lysosomes must sequester Ca to function effectively, but in animal cells the Ca uptake mechanism is unknown. An obstacle to progress has been the hostile luminal environment of lysosomes, which is incompatible with conventional Ca indicators. From our extensive pilot data and a re-interpretation of published work, we suggest that lysosomes sequester Ca delivered to them within close contacts between ER and lysosomes. Within these membrane-contact sites, Ca accumulated from the cytosol by the high-affinity ER Ca pump can be released to generate a high local [Ca] sufficient to drive Ca uptake into lysosomes by a transporter with low-affinity for Ca. Our scheme suggests that regulated interactions between lysosomes and ER (a Ca 'refuelling' stop) may be a necessary precursor to subsequent lysosomal activities that need luminal Ca (eg membrane fusion). The proposal has 5 aims:

1. To develop an effective sensor of free [Ca] within the lysosome lumen. This is demanding, but potentially useful. We have two basic approaches: synthesis of a click-chemistry compatible BAPTA-based indicator to allow coupling to lysosome-targeting structures; development of a protease-resistant genetically encoded Ca indicator likely to survive within lysosomes.

The remaining aims will benefit from our development of these new luminal indicators, but we also have alternative approaches. Using optical measurements of intracellular Ca, including a sensor of near-lysosome [Ca] that we have proven to be effective, we will address the following questions:
2. Is the sole purpose of the ER to deliver Ca to lysosomes at a high local concentration or do ER proteins contribute more directly to lysosomal Ca uptake?
3. What are the tethers that link ER to lysosomes?
4. Which proteins mediate Ca uptake by lysosomes?
5. Using the tools and insights gained from aims 1-4, what is the contribution of Ca transfer at ER-lysosome contacts to cell migration.

Our work will advance understanding of a basic feature of all animal cells, namely how lysosomes sequester the Ca they need to support their many and diverse activities. This fundamental question, allied with the many important roles of lysosomes and their relevance to disease, will ensure that our work delivers impact:

Training
Staff are encouraged to develop the skills and experience required for independence. They engage fully with every aspect of the project from developing proposals, managing budgets, reviewing and developing research programmes, to preparing publications and presenting work. Staff apply state-of-the art techniques equipping them for work in the best labs. Staff gain experience of teaching by supervising project/PhD students, teaching practical classes and in a lecture on advanced techniques to final-year students. All staff contribute fully to weekly lab meetings, where they present and critically evaluate work. In my absence, lab meetings are chaired by postdocs. A major impact is our proven ability to train staff equipped to meet future needs of industry, the public sector and academia. Our contributions to providing the UK with well-prepared scientists comes also from our public engagement activities.

International and interdisciplinary interactions
Our work is sustained by international/cross-disciplinary collaborations, and enhanced by international visitors. Development of a luminal Ca indicator is a collaboration with a chemistry lab in India, and we expect our work on chemotaxis to feed into long-standing stochastic modelling interactions with Falcke (Berlin). As our more lysosome-specific interests mature, we expect to develop additional relevant national and international collaborations. Many benefits, additional to those that feed directly into the project and training of associated staff, flow from these collaborations. They encourage interactions at boundaries between disciplines and by fostering extensive international interactions, they address a BBSRC priority by ensuring the UK remains engaged with a world-wide science community.

Public understanding and schools
My lab organises the Young Pharmas scheme, which seeks to inspire year-12 students, ensure that they appreciate the importance of creativity and critical evaluation in science, and the economic impact of pharmacology. Parents and teachers also gain exposure to these activities through the final poster session/guest lecture evening. Our interests in lysosomes will be assimilated into Young Pharmas' activities. We provide at least one placement for an undergraduate to gain research experience before deciding on postgraduate options. Staff contribute to Cambridge Science Festival with a hands-on demonstration of the actions of common drugs on waterfleas. We provide occasional visits to schools, providing practical experience of, for example, insect biology and microscopy. We work with press offices to maximize the impact of our work by bringing it to more diverse audiences than our primary publications can reach. Two press releases from BBSRC described our recent work. These activities encourage informed interest in science from students who have not yet finalised their career choices, and a more widespread appreciation of the importance of addressing fundamental questions in biology.

Health
Lysosomes are associated with numerous pathologies, including cancer, viral and microbial infections, neurodegeneration and lysosomal storage diseases. We suggest that Ca uptake underpins many of the most basic activities of lysosomes, and that understanding this process is likely to contribute to a deeper understanding of the pathologies. The impacts for clinical medicine are impossible to predict. We will actively engage with clinicians and the pharmaceutical industry to ensure that our findings are presented at an early stage to communities with direct interests in clinical development.