Bethan S. Kilpatrick

Direct mobilisation of lysosomal Ca2+ triggers complex Ca2+ signals.

Summary 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.

Dysregulation of lysosomal morphology by pathogenic LRRK2 is corrected by TPC2 inhibition

ABSTRACT 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)P2] 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 Ca2+-dependent trafficking in Parkinson disease.

A computational model of lysosome-ER Ca2+ microdomains.

ABSTRACT 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.

Coupling acidic organelles with the ER through Ca2+ microdomains at membrane contact sites

Acidic organelles such as lysosomes serve as non-canonical Ca(2+) stores. The Ca(2+) mobilising messenger NAADP is thought to trigger local Ca(2+) release from such stores. These events are then amplified by Ca(2+) channels on canonical ER Ca(2+) stores to generate physiologically relevant global Ca(2+) signals. Coupling likely occurs at microdomains formed at membrane contact sites between acidic organelles and the ER. Molecular analyses and computational modelling suggest heterogeneity in the composition of these contacts and predicted Ca(2+) microdomain behaviour. Conversely, acidic organelles might also locally amplify and temper ER-evoked Ca(2+) signals. Ca(2+) microdomains between distinct Ca(2+) stores are thus likely to be integral to the genesis of complex Ca(2+) signals.

An Endosomal NAADP-Sensitive Two-Pore Ca2+ Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling

Summary Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER). Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca2+-dependent hubs for signaling.

Endoplasmic reticulum and lysosomal Ca2+ stores are remodelled in GBA1-linked Parkinson disease patient fibroblasts

Graphical abstract

Endo-lysosomal TRP mucolipin-1 channels trigger global ER Ca2+ release and Ca2+ influx

ABSTRACT Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca2+ release but their role in Ca2+ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca2+ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca2+ release and Ca2+ entry. Ca2+ release required lysosomal and ER Ca2+ stores suggesting that TRPMLs, like other endo-lysosomal Ca2+ channels, are capable of ‘chatter’ with ER Ca2+ channels. Our data identify new modalities for TRPML1 action. Summary: The endolysosomal ion channel TRP mucolipin 1 was thought to mediate local Ca2+ signals. However, as reported here, it can also mediate global elevations in Ca2+.

Methods for monitoring lysosomal morphology.

Lysosomes are abundant organelles best known for their crucial role in macromolecule turnover. Lysosome dysfunction features in several diseases exemplified by the lysosomal storage disorders and is often associated with marked changes in lysosome structure. Lysosomal morphology may therefore serve as a sensitive readout of endocytic well-being. Here we describe methods for monitoring lysosome morphology in fixed and live cells using fluorescent probes and electron microscopy.

A “mix-and-match” approach to designing Ca2+ microdomains at membrane-contact sites

Ca2+ microdomains are critical for regulating cellular activity and often form at membrane contact sites. Such sites between lysosomes and the ER potentially provide a platform for signaling by the Ca2+ mobilizing messenger NAADP. However, at present we know little of how Ca2+ release events are coordinated at these experimentally intractable junctions. We therefore developed a computational model of lysosome-ER microdomains, which suggested that small leaks of Ca2+ from the lysosome couple to Ca2+-sensitive Ins(1,4,5)P3 receptors on the ER to generate global, microdomain-dependent Ca2+ signals. Here we discuss how the “mix-and-match” arrangement of different Ca2+ signaling proteins on the “source” and “target” membranes might generate functionally heterogeneous Ca2+ microdomains.

Two-pore channels and disease

Two-pore channels (TPCs) are Ca2+-permeable endo-lysosomal ion channels subject to multi-modal regulation. They mediate their physiological effects through releasing Ca2+ from acidic organelles in response to cues such as the second messenger, NAADP. Here, we review emerging evidence linking TPCs to disease. We discuss how perturbing both local and global Ca2+ changes mediated by TPCs through chemical and/or molecular manipulations can induce or reverse disease phenotypes. We cover evidence from models of Parkinsons disease, non-alcoholic fatty liver disease, Ebola infection, cancer, cardiac dysfunction and diabetes. A need for more drugs targeting TPCs is identified.