Optical profiling of autonomous Ca nanodomains generated by lysosomal TPC2 and TRPML1.

Multiple families of Ca-permeable channels co-exist on lysosomal Ca stores but how each family couples to its own unique downstream physiology is unclear. We have therefore investigated the Ca-signalling architecture underpinning different channels on the same vesicle that drive separate pathways, using phagocytosis as a physiological stimulus. Lysosomal Ca-channels are a major Ca 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.

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.

NAADP-Mediated Ca Signalling.

The discovery of NAADP-evoked Ca release in sea urchin eggs and then as a ubiquitous Ca mobilizing messenger has introduced several novel paradigms to our understanding of Ca signalling, not least in providing a link between cell stimulation and Ca release from lysosomes and other acidic Ca storage organelles. In addition, the hallmark concentration-response relationship of NAADP-mediated Ca 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 Ca 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.

Acidic Ca stores and immune-cell function.

Acidic organelles act as intracellular Ca stores; they actively sequester Ca in their lumina and release it to the cytosol upon activation of endo-lysosomal Ca channels. Recent data suggest important roles of endo-lysosomal Ca 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.

Choreographing endo-lysosomal Ca throughout the life of a phagosome.

The emergence of endo-lysosomes as ubiquitous Ca 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 Ca stores distinguishes them from other larger Ca 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 Ca signals (generated via TPC and TRPML channels) can be integrated in multiple sophisticated ways into biological processes.

A cellular protection racket: How lysosomal Ca 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 Ca 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.

Lysosomal agents inhibit store-operated Ca entry.

Pharmacological manipulation of lysosome membrane integrity or ionic movements is a key strategy for probing lysosomal involvement in cellular processes. However, we have found an unexpected inhibition of store-operated Ca entry (SOCE) by these agents. Dipeptides [glycyl-L-phenylalanine 2-naphthylamide (GPN) and L-leucyl-L-leucine methyl ester] that are inducers of lysosomal membrane permeabilization (LMP) uncoupled endoplasmic reticulum Ca-store depletion from SOCE by interfering with Stim1 oligomerization and/or Stim1 activation of Orai.

NAADP-regulated two-pore channels drive phagocytosis through endo-lysosomal Ca 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 Ca -permeable two-pore channels (TPCs). Remarkably, phagocytosis is driven by these local endo-lysosomal Ca nanodomains rather than global cytoplasmic or ER Ca signals.

Does lysosomal rupture evoke Ca release? A question of pores and stores.

Lysosomotropic agents have been used to permeabilize lysosomes and thereby implicate these organelles in diverse cellular processes. Since lysosomes are Ca stores, this rupturing action, particularly that induced by GPN, has also been used to rapidly release Ca from lysosomes. However, a recent study has questioned the mechanism of action of GPN and concluded that, acutely, it does not permeabilize lysosomes but releases Ca directly from the ER instead.

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 , achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood.

Carvedilol inhibits cADPR- and IP-induced Ca release.

Spontaneous Ca waves, also termed store-overload-induced Ca release (SOICR), in cardiac cells can trigger ventricular arrhythmias especially in failing hearts. SOICR occurs when RyRs are activated by an increase in sarcoplasmic reticulum (SR) luminal Ca. Carvedilol is one of the most effective drugs for preventing arrhythmias in patients with heart failure.

Ca2+ dialogue between acidic vesicles and ER.

Extracellular stimuli evoke the synthesis of intracellular second messengers, several of which couple to the release of Ca(2+)from Ca(2+)-storing organelles via activation of cognate organellar Ca(2+)-channel complexes. The archetype is the inositol 1,4,5-trisphosphate (IP3) and IP3receptor (IP3R) on the endoplasmic reticulum (ER). A less understood, parallel Ca(2+)signalling cascade is that involving the messenger nicotinic acid adenine dinucleotide phosphate (NAADP) that couples to Ca(2+)release from acidic Ca(2+)stores [e.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells.

Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals.

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 Ca²⁺-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.

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.

Synthesis of caged NAADP.

Caged derivatives of Ca²⁺-mobilizing messengers, such as nicotinic acid adenine dinucleotide phosphate (NAADP), are particularly useful for establishing the effects of these messengers on Ca²⁺ 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.

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

In addition to mobilizing Ca²⁺, 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.

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

Nicotinic acid adenine dinucleotide phosphate (NAADP), like the other major messengers for Ca²⁺ 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 Ca²⁺ 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.

Preparation and use of sea urchin egg homogenates for studying NAADP-mediated Ca²⁺ 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.

Preparation and use of sea urchin egg homogenates.

Cell homogenates provide a simple and yet powerful means of investigating the actions of Ca(2+)-mobilizing second messengers and their target Ca(2+) stores. The sea urchin egg homogenate is particularly useful and almost unique in retaining robust Ca(2+) responses to all three major messengers, i.e.

Two-pore channels (TPCs): current controversies.

Much excitement surrounded the proposal that a family of endo-lysosomal channels, the two-pore channels (TPCs) were the long sought after targets of the Ca(2+) -mobilising messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, the role of TPCs in NAADP signalling may be more complex than originally envisaged. First, NAADP may not bind directly to TPCs but via an accessory protein.

Altered distribution and function of natural killer cells in murine and human Niemann-Pick disease type C1.

Niemann-Pick type C (NPC) is a neurodegenerative lysosomal storage disorder caused by defects in the lysosomal proteins NPC1 or NPC2. NPC cells are characterized by reduced lysosomal calcium levels and impaired sphingosine transport from lysosomes. Natural killer (NK) cells kill virally infected/transformed cells via degranulation of lysosome-related organelles.