Optical profiling of autonomous Ca²⁺ nanodomains generated by lysosomal TPC2 and TRPML1

Multiple families of Ca²⁺-permeable channels co-exist on lysosomal Ca²⁺ stores but how each family couples to its own unique downstream physiology is unclear. We have therefore investigated the Ca²⁺-signalling architecture underpinning different channels on the same vesicle that drive separate pathways, using phagocytosis as a physiological stimulus. Lysosomal Ca²⁺-channels are a major Ca²⁺ 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 Ca²⁺ around open channels (Ca²⁺ nanodomains). To test this, we optimized Ca²⁺-nanodomain recordings by screening panels of genetically encoded Ca²⁺ indicators (GECIs) fused to TPC2 to monitor the [Ca²⁺] next to the channel. Signal calibration accounting for the distance of the GECI from the channel mouth reveals that, during phagocytosis, TPC2 generates local Ca²⁺ nanodomains around itself of up to 42 µM, nearly a hundred-fold greater than the global cytosolic [Ca²⁺] rise. We further show that TPC2 and TRPML1, though on the same lysosomes, generate autonomous Ca²⁺ nanodomains of high [Ca²⁺] that are largely insulated from one another, a platform allowing their discrete Ca²⁺-decoding to promote unique respective physiologies.