Mechanisms and Architecture of Endo-lysosomal Ca2+ Signalling

Ca2+ serves an essential signal within every cell. Ca2+ levels inside cells are very low, partly because it is sequestered inside Ca2+-storing compartments. In response to cell stimuli, Ca2+ is released from these stores by opening resident ion channels to activate detector proteins ('decoders') and change cell processes. The increase in intracellular Ca2+ is a universal signal in virtually all cells types e.g. for fertilization, muscle contraction, nerve impulses, gene expression.

The largest and best understood Ca2+ store is the endoplasmic reticulum (ER) which contains millimolar Ca2+. ER Ca2+ channels, IP3 receptors, are activated by a second messenger, IP3, which is synthesised in response to cell stimuli (e.g. hormones, neurotransmitters, antibodies, cell contact). IP3 synthesis, IP3R activation and Ca2+ release occur rapidly upon stimulation. However, the ER is not the only Ca2+ store and we discovered that small acidic vesicles (including endosomes and lysosomes) are important Ca2+ stores, but with their own unique second messenger (NAADP) and Ca2+ channels (TPCs), analogous to IP3/IP3Rs. This axis forms our focus.

Although they are better known as cellular waste-bins, endo-lysosomes are emerging as dynamic signalling hubs, integrating and delivering signals in response to the environment. A major endo-lysosomal signal is Ca2+. Many stimuli couple to endo-lysosomal Ca2+ release as a transduction pathway: depending on the stimulus, cells synthesise the messenger, nicotinic acid adenine dinucleotide phosphate, NAADP, which opens TPCs (two-pore channels) expressed on endo-lysosomes. These are Ca2+-permeable and elevate cytosolic Ca2+. However, each vesicle is small so the limited amount of Ca2+ that is released generates local Ca2+ 'nanodomains' (a locally high concentration, restricted in space).

Why does the cell contain different Ca2+ stores? The answer is that Ca2+ does not increase uniformly in the cytosol but rather is delivered discretely where it is needed. Different stores therefore deliver Ca2+ to different targets and different downstream physiology. We find endo-lysosomes 'pair-up' with their own unique detectors via highly localized and privileged conversations, for which the ER Ca2+ store cannot substitute. Therefore, each cell stimulus selects the appropriate Ca2+ sources for its downstream physiology.

However, it is unclear how these essential endo-lysosomal Ca2+ signals are generated and decoded which therefore forms our focus. The NAADP/TPC axis is poorly defined in terms of targets. A further complexity is that endo-lysosomes are small, heterogeneous, motile and exquisitely positioned, interacting physically/functionally with other organelles in specialized junctions (e.g. with ER, mitochondria).

Given the multiplicity of inputs/outputs, our aim is to understand how endo-lysosomes establish and regulate these 'private' local Ca2+ conversations with targets (proteins, organelles), thereby solving the Ca2+-specificity conundrum. Clearly, targets must closely associate with endo-lysosomes in order to detect the local 'Ca2+ plume' that emanates from TPCs. We aim to understand how targets are brought to endo-lysosomes or, conversely, how endo-lysosomes are dynamically brought to targets.
We will identify how, when and where targets and decoders associate with TPCs. The placement and motility of endo-lysosomes is crucial for the physiology and we also will test whether there is a specialist sub-class of endo-lysosomes for different Ca2+ signalling roles.

Potential Benefits & Applications. Standing at the crossroads of multiple processes, defining endo-lysosomal Ca2+ signals will:
(a) illuminate basic science (Ca2+ signals, signal compartmentation, endo-lysosomal biology, organelle/membrane dynamics, Ca2+-decoding);
(b) provide new tools to the broader cell biology community (reporters, pharmacology);
(c) strengthen the case for organelle ion channels as new drug targets.

Endo-lysosomes are emerging as physically small but physiologically crucial Ca2+ stores whose dysfunction contributes to a growing list of pathologies. We find that endo-lysosomal Ca2+ signals couple to their own unique downstream responses for which other Ca2+ sources cannot substitute by virtue of local Ca2+ nanodomains. Our aim is to understand the architectures and pathways that regulate, maintain and decode these privileged Ca2+ conversations.

We focus upon endo-lysosomal Ca2+ release evoked by the NAADP/TPC axis. However, our incomplete understanding requires us to address the following:

(a) Genetically engineer new fluorescent tools to monitor/manipulate previously intractable endo-lysosomal fundamentals that impact TPC action e.g. inter-organelle junction dynamics, Ca2+ nanodomains, endo-lysosomal Ca2+-integrator ('activity mapper'), endo-lysosomal membrane potential.

(b) How/when do TPCs and targets associate to selectively respond to Ca2+ nanodomains? We will compare dynamic/static associations (protein-protein, organelle-organelle). A focus is endo-lysosomal positioning and motility.

(c) Integrate these mechanistic details into physiological contexts. Our main model is phagocytosis since it is a multi-step pathway of major importance, involves processes of universal interest (membrane fusion/fission, vesicle trafficking) and is remarkably driven only by endo-lysosomal Ca2+. Our main focus will be to understand; (i) how Ca2+ nanodomains are uniquely decoded (e.g. vesicular scaffolding and local activation of calcineurin or Synaptotagmin7); (ii) how TPCs/TRPML1 differentially regulate the life cycle of the phagosome from formation to resolution.

Ca2+ signalling from acidic organelles is ubiquitous across the plant and animal kingdoms and in most cell types so is of universal importance and no scientific backwater. Indeed, the messenger NAADP was first discovered in sea urchins, and TPCs were first defined as acidic store Ca2+ channels in plants. From these humble beginnings, the field is poised to explode across multiple areas of biology. Our multidisciplinary approach from fundamental biology to chemistry and from imaging techniques to physiology, will impact on a large and varied community of scientists. The topic cuts across many disciplines and brings to the field of immune cells a rare insight from cell physiological approaches.

It is essential to understand the new roles of endo-lysosomes as Ca2+ signalling hubs because they impact fundamental cell functions important for health and, conversely, contribute to the pathology and progression of a broad range of diseases that are major global health challenges. The very fact that endo-lysosomes are involved in so many cell processes in varied cell contexts ensures that our findings will have far-reaching consequences beyond the endo-lysosomal Ca2+ signalling field, including neurodegeneration, cardiovascular diseases, inflammation, autoimmunity and infectious diseases.

Researchers including those in the pharmaceutical industry will find relevance and future potential from our findings, with the potential to identify niche therapeutic strategies that selectively target endo-lysosomal Ca2+ without perturbing global Ca2+ signalling. Indeed, endo-lysosomes represent an exquisite example of the local signalling paradigm: a local Ca2+ nanodomain that activates closely apposed targets. Although our focus is Ca2+, this architectural template may be applied to other local signalling modalities at the endo-lysosomal surface.

Our work will:
(a) Bring much-needed new tools that will impact multiple areas of cell biology. These will be available to the wider scientific community and training and advice will be given to those who wish to employ them.
(b) Increase our understanding of the basic cell physiology of endo-lysosomes and their interactions with other systems/organelles.
(c) Illuminate their adaptable Ca2+ signalling roles tailored to each specialized cell type.
(d) Highlight how precision targeting of specific local Ca2+ signalling, especially highlighting endo-lysosomal ion channels, may offer new strategies for therapeutic intervention.

The application is concerned with basic science. However, it is important that the general public is aware that calcium ions play a role in living systems beyond their role in teeth and bones. This may be useful since many may be treated with calcium channel blockers for hypertension or cardiac disorders. Communication of our work outlining our work on the signalling role of calcium will help such people understand how their medicines may work and the central role of this important ion as a universal cellular regulator.
Socio-economically, given that the Pharmaceutical Industry is showing interest in endo-lysosomal mechanisms and TPCs as potential new drug targets, this study will act as a showcase to help steer the industry in this direction.