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