Mechanisms and Architecture of Endo-lysosomal Ca2+ Signalling

Ca 2+是每个细胞内的重要信号。细胞内的Ca 2+水平非常低，部分原因是它被隔离在Ca 2+储存室中。响应细胞刺激，Ca 2+通过打开驻留离子通道从这些存储器释放，以激活检测器蛋白（“解码器”）并改变细胞过程。细胞内Ca ~（2+）的增加是几乎所有细胞类型的普遍信号，例如受精、肌肉收缩、神经冲动、基因表达。ER Ca 2+通道，IP 3受体，被第二信使IP 3激活，IP 3是响应细胞刺激（例如激素，神经递质，抗体，细胞接触）而合成的。IP 3合成、IP 3R活化和Ca 2+释放在刺激后迅速发生。然而，ER不是唯一的Ca 2+储存，我们发现小的酸性囊泡（包括内体和溶酶体）是重要的Ca 2+储存，但具有自己独特的第二信使（NAADP）和Ca 2+通道（TPC），类似于IP 3/IP 3Rs。虽然内溶体通常被称为细胞废物箱，但它们正在成为动态信号中心，整合并传递信号以响应环境。主要的内-溶酶体信号是Ca 2+。许多刺激耦合到内-溶酶体Ca 2+释放作为转导途径：取决于刺激，细胞合成信使，烟酸腺嘌呤二核苷酸磷酸，NAADP，其打开内-溶酶体上表达的TPC（双孔通道）。这些是Ca 2+渗透性的，并升高细胞溶质Ca 2+。然而，每个囊泡都很小，所以释放的有限量的Ca 2+会产生局部的Ca 2+“纳米结构域”（局部高浓度，空间受限）。为什么细胞含有不同的Ca 2+储存？答案是Ca 2+在胞质溶胶中并不均匀增加，而是在需要的地方离散地传递。因此，不同的商店将Ca 2+输送到不同的目标和不同的下游生理。我们发现内溶酶体通过高度本地化和特权的对话与自己独特的检测器“配对”，这是ER Ca 2+商店无法替代的。因此，每种细胞刺激都为其下游生理选择合适的Ca ~（2+）来源，然而，这些必需的内-溶酶体Ca ~（2+）信号是如何产生和解码的，这是我们关注的焦点。NAADP/TPC轴在目标方面定义不清。更复杂的是，内溶体是小的、异质的、能动的和精确定位的，在专门的连接处与其他细胞器物理/功能相互作用。考虑到输入/输出的多样性，我们的目标是了解内溶酶体如何建立和调节这些“私人”局部Ca 2+与靶点的对话。（蛋白质，细胞器），从而解决了Ca 2+特异性难题。显然，靶标必须与内溶体紧密结合，以检测从TPC发出的局部“Ca 2+羽流”。我们的目标是了解目标是如何被带到内溶体，或者相反，内溶体是如何动态地被带到目标。我们将确定目标和解码器如何、何时以及在何处与TPC相关联。内溶体的位置和运动对于生理学是至关重要的，我们还将测试是否有一个专门的内溶体亚类用于不同的Ca 2+信号作用。潜在的好处和应用。站在多个过程的十字路口，定义内-溶酶体Ca 2+信号将：（a）阐明基础科学（Ca 2+信号，信号区室化，内-溶酶体生物学，细胞器/膜动力学，Ca 2+解码）;（B）为更广泛的细胞生物学社区提供新的工具（报告，药理学）;（c）加强细胞器离子通道作为新药物靶点的情况。

Ca2+ serves an essential signal within every cell. Ca2+ levels inside cells are very low, partly because it is sequestered inside Ca2+-storing compartments. In response to cell stimuli, Ca2+ is released from these stores by opening resident ion channels to activate detector proteins ('decoders') and change cell processes. The increase in intracellular Ca2+ is a universal signal in virtually all cells types e.g. for fertilization, muscle contraction, nerve impulses, gene expression.The largest and best understood Ca2+ store is the endoplasmic reticulum (ER) which contains millimolar Ca2+. ER Ca2+ channels, IP3 receptors, are activated by a second messenger, IP3, which is synthesised in response to cell stimuli (e.g. hormones, neurotransmitters, antibodies, cell contact). IP3 synthesis, IP3R activation and Ca2+ release occur rapidly upon stimulation. However, the ER is not the only Ca2+ store and we discovered that small acidic vesicles (including endosomes and lysosomes) are important Ca2+ stores, but with their own unique second messenger (NAADP) and Ca2+ channels (TPCs), analogous to IP3/IP3Rs. This axis forms our focus.Although they are better known as cellular waste-bins, endo-lysosomes are emerging as dynamic signalling hubs, integrating and delivering signals in response to the environment. A major endo-lysosomal signal is Ca2+. Many stimuli couple to endo-lysosomal Ca2+ release as a transduction pathway: depending on the stimulus, cells synthesise the messenger, nicotinic acid adenine dinucleotide phosphate, NAADP, which opens TPCs (two-pore channels) expressed on endo-lysosomes. These are Ca2+-permeable and elevate cytosolic Ca2+. However, each vesicle is small so the limited amount of Ca2+ that is released generates local Ca2+ 'nanodomains' (a locally high concentration, restricted in space).Why does the cell contain different Ca2+ stores? The answer is that Ca2+ does not increase uniformly in the cytosol but rather is delivered discretely where it is needed. Different stores therefore deliver Ca2+ to different targets and different downstream physiology. We find endo-lysosomes 'pair-up' with their own unique detectors via highly localized and privileged conversations, for which the ER Ca2+ store cannot substitute. Therefore, each cell stimulus selects the appropriate Ca2+ sources for its downstream physiology.However, it is unclear how these essential endo-lysosomal Ca2+ signals are generated and decoded which therefore forms our focus. The NAADP/TPC axis is poorly defined in terms of targets. A further complexity is that endo-lysosomes are small, heterogeneous, motile and exquisitely positioned, interacting physically/functionally with other organelles in specialized junctions (e.g. with ER, mitochondria).Given the multiplicity of inputs/outputs, our aim is to understand how endo-lysosomes establish and regulate these 'private' local Ca2+ conversations with targets (proteins, organelles), thereby solving the Ca2+-specificity conundrum. Clearly, targets must closely associate with endo-lysosomes in order to detect the local 'Ca2+ plume' that emanates from TPCs. We aim to understand how targets are brought to endo-lysosomes or, conversely, how endo-lysosomes are dynamically brought to targets. We will identify how, when and where targets and decoders associate with TPCs. The placement and motility of endo-lysosomes is crucial for the physiology and we also will test whether there is a specialist sub-class of endo-lysosomes for different Ca2+ signalling roles.Potential Benefits & Applications. Standing at the crossroads of multiple processes, defining endo-lysosomal Ca2+ signals will:(a) illuminate basic science (Ca2+ signals, signal compartmentation, endo-lysosomal biology, organelle/membrane dynamics, Ca2+-decoding);(b) provide new tools to the broader cell biology community (reporters, pharmacology);(c) strengthen the case for organelle ion channels as new drug targets.