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Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca2+ signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P[sub 2]] and buffering local Ca2+ increases. NAADP-evoked Ca2+ signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca Ca2+-dependent trafficking in Parkinson disease. [ABSTRACT FROM AUTHOR]

Acidic organelles form an important intracellular Ca2+ pool that can drive global Ca2+ signals through coupling with endoplasmic reticulum (ER) Ca2+ stores. Recently identified lysosome-ER membrane contact sites might allow formation of Ca2+ microdomains, although their size renders observation of Ca2+ dynamics impractical. Here, we generated a computational model of lysosome-ER coupling that incorporated a previous model of the inositol trisphosphate (IP3) receptor as the ER Ca2+ 'amplifier' and lysosomal leaks as the Ca2+ 'trigger'. The model qualitatively described global Ca2+ responses to the lysosomotropic agent GPN, which caused a controlled but substantial depletion of small solutes from the lysosome. Adapting this model to physiological lysosomal leaks induced by the Ca2+ mobilising messenger NAADP demonstrated that lysosome-ER microdomains are capable of driving global Ca2+ oscillations. Interestingly, our simulations suggest that the microdomain Ca2+ need not be higher than that in the cytosol for responses to occur, thus matching the relatively high affinity of IP3 receptors for Ca2+. The relative distribution and overall density of the lysosomal leaks dictated whether microdomains triggered or modulated global signals. Our data provide a computational framework for probing lysosome-ER Ca2+ dynamics. [ABSTRACT FROM AUTHOR]

Accumulating evidence implicates acidic organelles of the endolysosomal system as mobilisable stores of Ca2+ but their relationship to the better-characterised endoplasmic reticulum (ER) Ca2+ store remains unclear. Here we show that rapid osmotic permeabilisation of lysosomes evokes prolonged, spatiotemporally complex Ca2+ signals in primary cultured human fibroblasts. These Ca2+ signals comprised an initial response that correlated with lysosomal disruption and secondary long-lasting spatially heterogeneous Ca2+ oscillations that required ER-localised inositol trisphosphate receptors. Electron microscopy identified extensive membrane contact sites between lysosomes and the ER. Mobilisation of lysosomal Ca2+ stores is thus sufficient to evoke ER-dependent Ca2+ release probably through lysosome-ER membrane contact sites, and akin to the proposed mechanism of action of the Ca2+ mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP). Our data identify functional and physical association of discrete Ca2+ stores important for the genesis of Ca2+ signal complexity. [ABSTRACT FROM AUTHOR]

Nuclear calcium signalling has been a controversial battlefield for many years and the question of how permeable the nuclear pore complexes (NPCs) are to Ca2+ has been the subject of a particularly hot dispute. Recent data from isolated nuclei suggest that the NPCs are open even after depletion of the Ca2+ store in the nuclear envelope. Other research has suggested that a new Ca2+releasing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP), can liberate Ca2+ only from acidic organelles, probably lysosomes, rather than from the traditional Ca2+ store in the endoplasmic reticulum (ER). Recent work indicates that NAADP can release Ca2+ from the nuclear envelope (NE), which has a thapsigargin-sensitive, ER-type Ca2+ store. NAADP acts in a manner similar to inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] or cyclic ADP-ribose (cADPR): all three messengers are equally able to reduce the Ca2+ concentration inside the NE and this is associated with a transient rise in the nucleoplasmic Ca2+ concentration. The NE contains ryanodine receptors (RyRs) and Ins(1,4,5)P3 receptors [Ins(1,4,5)P3Rs], and these can be activated separately and independently: the RyRs by either NAADP or cADPR, and the Ins(1,4,5)P3Rs by Ins(1,4,5)P3. [ABSTRACT FROM AUTHOR]

Lysosomes are acidic Ca2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca2+ stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca2+ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca2+ signals. NH4Cl evoked comparable changes in pH but failed to affect Ca2+. The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca2+ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca2+ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca2+ release. We conclude that Ca2+ signals evoked by GPN most likely derive from acidic organelles. [ABSTRACT FROM AUTHOR]

Lysosomes are acidic Ca 2+ stores often mobilised in conjunction with endoplasmic reticulum (ER) Ca 2+ stores. Glycyl-L-phenylalanine 2-naphthylamide (GPN) is a widely used lysosomotropic agent that evokes cytosolic Ca 2+ signals in many cells. However, whether these signals are the result of a primary action on lysosomes is unclear in light of recent evidence showing that GPN mediates direct ER Ca 2+ release through changes in cytosolic pH. Here, we show that GPN evoked rapid increases in cytosolic pH but slower Ca 2+ signals. NH 4 Cl evoked comparable changes in pH but failed to affect Ca 2+ The V-type ATPase inhibitor, bafilomycin A1, increased lysosomal pH over a period of hours. Acute treatment modestly affected lysosomal pH and potentiated Ca 2+ signals evoked by GPN. In contrast, chronic treatment led to more profound changes in luminal pH and selectively inhibited GPN action. GPN blocked Ca 2+ responses evoked by the novel nicotinic acid adenine dinucleotide phosphate-like agonist, TPC2-A1-N. Therefore, GPN-evoked Ca 2+ signals were better correlated with associated pH changes in the lysosome compared to the cytosol, and were coupled to lysosomal Ca 2+ release. We conclude that Ca 2+ signals evoked by GPN most likely derive from acidic organelles., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Acidic organelles form an important intracellular Ca(2+) pool that can drive global Ca(2+) signals through coupling with endoplasmic reticulum (ER) Ca(2+) stores. Recently identified lysosome-ER membrane contact sites might allow formation of Ca(2+) microdomains, although their size renders observation of Ca(2+) dynamics impractical. Here, we generated a computational model of lysosome-ER coupling that incorporated a previous model of the inositol trisphosphate (IP3) receptor as the ER Ca(2+) 'amplifier' and lysosomal leaks as the Ca(2+) 'trigger'. The model qualitatively described global Ca(2+) responses to the lysosomotropic agent GPN, which caused a controlled but substantial depletion of small solutes from the lysosome. Adapting this model to physiological lysosomal leaks induced by the Ca(2+) mobilising messenger NAADP demonstrated that lysosome-ER microdomains are capable of driving global Ca(2+) oscillations. Interestingly, our simulations suggest that the microdomain [Ca(2+)] need not be higher than that in the cytosol for responses to occur, thus matching the relatively high affinity of IP3 receptors for Ca(2+). The relative distribution and overall density of the lysosomal leaks dictated whether microdomains triggered or modulated global signals. Our data provide a computational framework for probing lysosome-ER Ca(2+) dynamics., (© 2014. Published by The Company of Biologists Ltd.)