| Summary: | <p>Multiple families of Ca<sup>2+</sup>-permeable channels co-exist on lysosomal Ca<sup>2+</sup> stores but how each family couples to its own unique downstream physiology is unclear. We have therefore investigated the Ca<sup>2+</sup>-signalling architecture underpinning different channels on the same vesicle that drive separate pathways, using phagocytosis as a physiological stimulus. Lysosomal Ca<sup>2+</sup>-channels are a major Ca<sup>2+</sup> source driving particle uptake in macrophages, but different channels drive different aspects of Fc-receptor-mediated phagocytosis: TPC2 couples to dynamin activation, whilst TRPML1 couples to lysosomal exocytosis. We hypothesised that they are driven by discrete local plumes of Ca<sup>2+</sup> around open channels (Ca<sup>2+</sup> nanodomains). To test this, we optimized Ca<sup>2+</sup>-nanodomain recordings by screening panels of genetically encoded Ca<sup>2+</sup> indicators (GECIs) fused to TPC2 to monitor the [Ca<sup>2+</sup>] next to the channel. Signal calibration accounting for the distance of the GECI from the channel mouth reveals that, during phagocytosis, TPC2 generates local Ca<sup>2+</sup> nanodomains around itself of up to 42 µM, nearly a hundred-fold greater than the global cytosolic [Ca<sup>2+</sup>] rise. We further show that TPC2 and TRPML1, though on the same lysosomes, generate autonomous Ca<sup>2+</sup> nanodomains of high [Ca<sup>2+</sup>] that are largely insulated from one another, a platform allowing their discrete Ca<sup>2+</sup>-decoding to promote unique respective physiologies.</p>
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