Calcium exchange between endoplasmic reticulum and lysosomes

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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.