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.

NAADP-Mediated Ca2+ Signalling.

The discovery of NAADP-evoked Ca2+ release in sea urchin eggs and then as a ubiquitous Ca2+ mobilizing messenger has introduced several novel paradigms to our understanding of Ca2+ signalling, not least in providing a link between cell stimulation and Ca2+ release from lysosomes and other acidic Ca2+ storage organelles. In addition, the hallmark concentration-response relationship of NAADP-mediated Ca2+ release, shaped by striking activation/desensitization mechanisms, influences its actions as an intracellular messenger. There has been recent progress in our understanding of the molecular mechanisms underlying NAADP-evoked Ca2+ release, such as the identification of the endo-lysosomal two-pore channel family of cation channels (TPCs) as their principal target and the identity of NAADP-binding proteins that complex with them. The NAADP/TPC signalling axis has gained recent prominence in pathophysiology for their roles in such disease processes as neurodegeneration, tumorigenesis and cellular viral entry.

Two-pore channels: going with the flows.

In recent years, our understanding of the structure, mechanisms and functions of the endo-lysosomal TPC (two-pore channel) family have grown apace. Gated by the second messengers, NAADP and PI(3,5)P2, TPCs are an integral part of fundamental signal-transduction pathways, but their array and plasticity of cation conductances (Na+, Ca2+, H+) allow them to variously signal electrically, osmotically or chemically. Their relative tissue- and organelle-selective distribution, together with agonist-selective ion permeabilities provides a rich palette from which extracellular stimuli can choose. TPCs are emerging as mediators of immunity, cancer, metabolism, viral infectivity and neurodegeneration as this short review attests.

Acidic Ca2+ stores and immune-cell function.

Acidic organelles act as intracellular Ca2+ stores; they actively sequester Ca2+ in their lumina and release it to the cytosol upon activation of endo-lysosomal Ca2+ channels. Recent data suggest important roles of endo-lysosomal Ca2+ channels, the Two-Pore Channels (TPCs) and the TRPML channels (mucolipins), in different aspects of immune-cell function, particularly impacting membrane trafficking, vesicle fusion/fission and secretion. Remarkably, different channels on the same acidic vesicles can couple to different downstream physiology. Endo-lysosomal Ca2+ stores can act under different modalities, be they acting alone (via local Ca2+ nanodomains around TPCs/TRPMLs) or in conjunction with the ER Ca2+ store (to either promote or suppress global ER Ca2+ release). These different modalities impinge upon functions as broad as phagocytosis, cell-killing, anaphylaxis, immune memory, thrombostasis, and chemotaxis.

Choreographing endo-lysosomal Ca2+ throughout the life of a phagosome.

The emergence of endo-lysosomes as ubiquitous Ca2+ stores with their unique cohort of channels has resulted in their being implicated in a growing number of processes in an ever-increasing number of cell types. The architectural and regulatory constraints of these acidic Ca2+ stores distinguishes them from other larger Ca2+ sources such as the ER and influx across the plasma membrane. In view of recent advances in the understanding of the modes of operation, we discuss phagocytosis as a template for how endo-lysosomal Ca2+ signals (generated via TPC and TRPML channels) can be integrated in multiple sophisticated ways into biological processes. Phagocytosis illustrates how different endo-lysosomal Ca2+ signals drive different phases of a process, and how these can be altered by disease or infection.

A cellular protection racket: How lysosomal Ca2+ fluxes prevent kidney injury.

LC3-lipidation is activated by lysosomal damage by mechanisms that are unknown and divergent from canonical autophagy. In this study, Nakamura et al, show that lysosomal damage induced by lysosomotropic agents or oxalate in renal proximal tubule cells causes lipidated LC3 to insert into the lysosomal membrane to activate TRPML1 channels and release Ca2+ from lysosomes. This leads to TFEB dephosphorylation and translocation into the nucleus which results in clearance of damaged lysosomes and their contents which may reduce the deleterious effects of crystal nephropathy.

Defective platelet function in Niemann-Pick disease type C1.

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder caused by mutations in either NPC1 (95% of cases) or NPC2. Reduced late endosome/lysosome calcium (Ca2+) levels and the accumulation of unesterified cholesterol and sphingolipids within the late endocytic system characterize this disease. We previously reported impaired lysosome-related organelle (LRO) function in Npc1 -/- Natural Killer cells; however, the potential contribution of impaired acid compartment Ca2+ flux and LRO function in other cell types has not been determined. Here, we investigated LRO function in NPC1 disease platelets. We found elevated numbers of circulating platelets, impaired platelet aggregation and prolonged bleeding times in a murine model of NPC1 disease. Electron microscopy revealed abnormal ultrastructure in murine platelets, consistent with that seen in a U18666A (pharmacological inhibitor of NPC1) treated megakaryocyte cell line (MEG-01) exhibiting lipid storage and acidic compartment Ca2+ flux defects. Furthermore, platelets from NPC1 patients across different ages were found to cluster at the lower end of the normal range when platelet numbers were measured and had platelet volumes that were clustered at the top of the normal range. Taken together, these findings highlight the role of acid compartment Ca2+ flux in the function of platelet LROs.

NAADP-regulated two-pore channels drive phagocytosis through endo-lysosomal Ca2+ nanodomains, calcineurin and dynamin.

Macrophages clear pathogens by phagocytosis and lysosomes that fuse with phagosomes are traditionally regarded as to a source of membranes and luminal degradative enzymes. Here, we reveal that endo-lysosomes act as platforms for a new phagocytic signalling pathway in which FcγR activation recruits the second messenger NAADP and thereby promotes the opening of Ca2+ -permeable two-pore channels (TPCs). Remarkably, phagocytosis is driven by these local endo-lysosomal Ca2+ nanodomains rather than global cytoplasmic or ER Ca2+ signals. Motile endolysosomes contact nascent phagosomes to promote phagocytosis, whereas endo-lysosome immobilization prevents it. We show that TPC-released Ca2+ rapidly activates calcineurin, which in turn dephosphorylates and activates the GTPase dynamin-2. Finally, we find that different endo-lysosomal Ca2+ channels play diverse roles, with TPCs providing a universal phagocytic signal for a wide range of particles and TRPML1 being only required for phagocytosis of large targets.

Mechanistic convergence and shared therapeutic targets in Niemann-Pick disease.

Niemann-Pick disease type C (NPC) and Tangier disease are genetically and clinically distinct rare inborn errors of metabolism. NPC is caused by defects in either NPC1 or NPC2; whereas Tangier disease is caused by a defect in ABCA1. Tangier disease is currently without therapy, whereas NPC can be treated with miglustat, a small molecule inhibitor of glycosphingolipid biosynthesis that slows the neurological course of the disease. When a Tangier disease patient was misdiagnosed with NPC and treated with miglustat, her symptoms improved. This prompted us to consider whether there is mechanistic convergence between these two apparently unrelated rare inherited metabolic diseases. In this study, we found that when ABCA1 is defective (Tangier disease) there is secondary inhibition of the NPC disease pathway, linking these two diseases at the level of cellular pathophysiology. In addition, this study further supports the hypothesis that miglustat, as well as other substrate reduction therapies, may be potential therapeutic agents for treating Tangier disease as fibroblasts from multiple Tangier patients were corrected by miglustat treatment.

Revealing the secrets of secretion.

An intracellular ion channel may have a central role in the release of cytokines by macrophages.

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway.

BACKGROUND: Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with persistent mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC. METHODS: The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed. RESULTS: Macrophages infected with persistent intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that persistent intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells. CONCLUSION: These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies.

TPC: the NAADP discovery channel?

The Ca2+-mobilizing second messenger, NAADP (nicotinic acid adenine dinucleotide phosphate), has been with us for nearly 20 years and yet we still cannot fully agree on the identity of its target Ca2+-release channel. In spite of some recent robust challenges to the idea that two-pore channels (TPCs) represent the elusive "NAADP receptor", evidence continues to accumulate that TPCs are important for NAADP-mediated responses. This article will briefly outline the background and review more recent work pertaining to the TPC story.

Expression of Ca²âº-permeable two-pore channels rescues NAADP signalling in TPC-deficient cells.

The second messenger NAADP triggers Ca(2+) release from endo-lysosomes. Although two-pore channels (TPCs) have been proposed to be regulated by NAADP, recent studies have challenged this. By generating the first mouse line with demonstrable absence of both Tpcn1 and Tpcn2 expression (Tpcn1/2(-/-)), we show that the loss of endogenous TPCs abolished NAADP-dependent Ca(2+) responses as assessed by single-cell Ca(2+) imaging or patch-clamp of single endo-lysosomes. In contrast, currents stimulated by PI(3,5)P2 were only partially dependent on TPCs. In Tpcn1/2(-/-) cells, NAADP sensitivity was restored by re-expressing wild-type TPCs, but not by mutant versions with impaired Ca(2+)-permeability, nor by TRPML1. Another mouse line formerly reported as TPC-null likely expresses truncated TPCs, but we now show that these truncated proteins still support NAADP-induced Ca(2+) release. High-affinity [(32)P]NAADP binding still occurs in Tpcn1/2(-/-) tissue, suggesting that NAADP regulation is conferred by an accessory protein. Altogether, our data establish TPCs as Ca(2+)-permeable channels indispensable for NAADP signalling.

Imaging approaches to measuring lysosomal calcium.

Endolysosomes are emerging as key players that generate as well as respond to intracellular Ca(2+) signals. The role of Ca(2+) in modulating acidic organelle function has long been recognized, but it is now emerging that acidic organelles also act as intracellular Ca(2+) stores; they actively sequester Ca(2+) in their lumina and release it to the cytosol upon activation of endolysosomal Ca(2+) channels. This local Ca(2+) signal is crucial for endolysosomal function and/or global Ca(2+) signaling. Importantly, defects in endolysosomal Ca(2+) are associated with disease. This chapter discusses several complimentary approaches to monitor endolysosomal Ca(2+), with particular emphasis on the inherent pitfalls that can plague the unwary.

Preferential Coupling of the NAADP Pathway to Exocytosis in T-Cells.

A cytotoxic T-lymphocyte (CTL) kills an infected or tumorigenic cell by Ca2+-dependent exocytosis of cytolytic granules at the immunological synapse formed between the two cells. However, these granules are more than reservoirs of secretory cytolytic proteins but may also serve as unique Ca2+ signaling hubs that autonomously generate their own signals for exocytosis. This review discusses a selective role for the Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP) and its molecular targets, two-pore channels (TPCs), in stimulating exocytosis. Given that TPCs reside on the exocytotic granules themselves, these vesicles generate as well as respond to NAADP-dependent Ca2+ signals, which may have wider implications for stimulus-secretion coupling, vesicular fusion, and patho-physiology.

Synthesis of NAADP-AM as a membrane-permeant NAADP analog.

Nicotinic acid adenine dinucleotide phosphate (NAADP), like the other major messengers for Ca²âº mobilization, is passively membrane-impermeant. Instead, a cell-permeant acetoxymethyl ester derivative of NAADP (NAADP-AM) can be synthesized as described here and used to study NAADP-mediated Ca²âº release.

Measurement of luminal pH of acidic stores as a readout for NAADP action.

In addition to mobilizing Ca²âº, NAADP plays a role in modulating the luminal pH (pHL) of acidic stores of the endolysosomal system. The effects of NAADP on pHL have been most extensively studied in the sea urchin egg, both in the intact egg and in egg homogenates. Related observations have also been made in mammalian systems (e.g., guinea pig atrial myocytes and pancreatic acinar cells). Although the connection between Ca²âº mobilization and increase in pHL is not understood, pHL can be a useful parameter to measure when studying NAADP-mediated signaling. This protocol describes the fluorescent measurement of pHL of acidic stores. It relies on the use of acridine orange (AO), a standard dye for pHL. AO selectively accumulates to high concentrations in the lumen of organelles as a function of acidity; at these high concentrations it self-quenches. When pHL increases, some AO is lost from the vesicle. As a result, the lower luminal AO concentration relieves the quenching and fluorescence increases in the lumen.

Synthesis of caged NAADP.

Caged derivatives of Ca²âº-mobilizing messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), are particularly useful for establishing the effects of these messengers on Ca²âº signaling. Caged NAADP is no longer commercially available but can be synthesized in house, as described here. In brief, a stable precursor of the caging reagent is made and converted to an unstable reactive reagent immediately before addition to the compound to be caged.

Preparation and use of sea urchin egg homogenates for studying NAADP-mediated Ca²âº release.

NAADP and other Ca(2+)-mobilizing messengers are membrane impermeant and thus must be added directly to cell-free or broken-cell preparations to effect Ca(2+) release. The sea urchin egg homogenate, where the biological activity of NAADP was first reported, remains the gold standard cell-free system for studying NAADP-mediated Ca(2+) release. Here we describe how to prepare sea urchin egg homogenate and use it to measure NAADP-mediated Ca(2+) release.

Synthesis of [³²P]NAADP for the radioreceptor binding assay.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a major messenger for Ca(2+) mobilization in cells. NAADP-binding proteins are highly selective and have a strong affinity for NAADP. This is the basis of the radioreceptor binding assay, which is used to measure NAADP levels in cells and tissues and to identify cellular stimuli that use NAADP as an intracellular messenger. In the radioreceptor binding assay, radiolabeled NAADP ([(32)P]NAADP) competes with endogenous NAADP present in samples for binding to their receptors. Here, we describe the synthesis of [(32)P]NAADP for use in the radioreceptor binding assay.