Calcium exchange between endoplasmic reticulum and lysosomes

溶酶体是存在于所有动物细胞中的小的膜结合结构。它们通常被描述为细胞的“垃圾桶”，但更恰当的说法是“回收箱”，因为它们的重要任务之一是降解并回收从细胞外或从已经失效的细胞结构中进口的生物材料。溶酶体储存疾病的破坏性影响清楚地表明了溶酶体的重要性，溶酶体储存疾病通常是由于溶酶体酶的故障造成的。这些降解过程受到精确的调控，以确保饥饿的细胞获得它们所需的原材料。要做到这一点，溶酶体含有一种酸性的消化酶混合物。物质向溶酶体的转移依赖于溶酶体的膜与其他细胞内细胞器的膜融合。这一过程被人类劫持以允许药物输送，并被病毒劫持以允许它们入侵细胞。类似的膜融合事件允许溶酶体与包围每个细胞的质膜融合，促进材料向外部世界释放，并在细胞迁移过程中插入新的膜用于修复和膜延伸。有令人信服的证据表明，所有这些膜融合事件的最后阶段都需要通过钙渗透通道的孔从溶酶体内释放钙。最近的工作发现了几个这样的溶酶体钙通道，并将它们与溶酶体的正常活动和各种病理联系在一起(例如，感染埃博拉病毒需要一个)。对这些通道的兴趣也揭示了溶酶体在调节细胞内钙浓度增加方面的作用，而细胞内钙浓度的增加调节了细胞的许多活动。另一个更丰富的细胞器是内质网(ER)，它的钙通道在产生这些调节钙信号方面发挥了主要作用，但越来越多的证据表明，溶酶体和ER相互作用产生钙信号。因此，现在很清楚的是，溶酶体的许多功能依赖于它们积累钙的能力。但我们不知道溶酶体是如何获得钙的。这个问题具有挑战性，因为溶酶体内部的敌意使得很难使用钙指示剂，这些指示剂在揭示钙调节的许多其他方面非常有用。我们的研究表明，内质网可能负责向溶酶体输送钙。内质网有一个高亲和力的钙泵，使它能够从细胞内普遍存在的低浓度钙中隔离钙，但似乎溶酶体的摄取系统的亲和力要低得多。我们认为，在积累了钙之后，内质网将其释放到内质网和溶酶体膜之间的微小缝隙中，在那里获得的高钙浓度足以为溶酶体的吸收提供燃料。我们的方案将这些内质网溶酶体接触设想为“加油站”，溶酶体必须访问这些加油站才能定期补充维持其活动所需的钙。在这项建议中，我们寻求开发新的光学指示剂，使用能够分辨亚细胞结构的显微镜来直接测量溶酶体内的钙。使用这些工具和其他工具，我们将检验我们的假设，即内质网-溶酶体相互作用对于溶酶体获得钙是必不可少的；确定介导细胞器拴系的蛋白质；以及确定跨溶酶体膜运输钙的蛋白质。然后，我们可以干扰编码关键蛋白质的基因(S)，在只有每个基因的一个拷贝(而不是通常的一对)的细胞系中，因此可以清楚地评估溶酶体钙摄取对许多细胞行为的贡献，特别是细胞迁移。我们的工作解决了细胞生物学中的一个基本问题，并有可能揭示一个重要的细胞内细胞器的意想不到的角色，该细胞器广泛涉及许多人类疾病。

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.