Mechanisms and Architecture of Endo-lysosomal Ca2+ Signalling

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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.