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In angiosperms, wound-derived signals travel through the vasculature to systemically activate defence responses throughout the plant. In Arabidopsis thaliana, activity of vasculature-specific Clade 3 glutamate receptor-like (GLR) channels is required for the transmission of electrical signals and cytosolic Ca 2+ ([Ca 2+ ] cyt ) waves from wounded leaves to distal tissues, triggering activation of oxylipin-dependent defences. Whether nonvascular plants mount systemic responses upon wounding remains unknown. To explore the evolution of systemic defence responses, we investigated electrical and calcium signalling in the nonvascular plant Marchantia polymorpha. We found that electrical signals and [Ca 2+ ] cyt waves are generated in response to mechanical wounding and propagated to nondamaged distal tissues in M. polymorpha. Functional analysis of MpGLR, the only GLR encoded in the genome of M. polymorpha, indicates that its activity is necessary for the systemic transmission of wound-induced electrical signals and [Ca 2+ ] cyt waves, similar to vascular plants. However, spread of these signals is neither coupled to systemic accumulation of oxylipins nor to a transcriptional defence response in the distal tissues of wounded M. polymorpha plants. Our results suggest that lack of vasculature prevents translocation of additional signalling factors that, together with electrical signals and [Ca 2+ ] cyt waves, contribute to systemic activation of defences in tracheophytes., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Bitter taste receptors (TAS2Rs) expressed in extraoral tissues represent a whole-body sensory system, whose role and mechanisms could be of interest for the identification of new therapeutic targets. It is known that TAS2R46s in pre-contracted airway smooth muscle cells increase mitochondrial calcium uptake, leading to bronchodilation, and that several SNPs have been identified in its gene sequence. There are very few reports on the structure-function analysis of TAS2Rs. Thus, we delved into the subject by using mutagenesis and in silico studies. We generated a cellular model that expresses native TAS2R46 to evaluate the influence of the four most common SNPs on calcium fluxes following the activation of the receptor by its specific ligand absinthin. Then, docking studies were conducted to correlate the calcium flux results to the structural mutation. The analysed SNPs differently modulate the TAS2R46 signal cascade according to the altered protein domain. In particular, the SNP in the sixth transmembrane domain of the receptors did not modulate calcium homeostasis, while the SNPs in the sequence coding for the fourth transmembrane domain completely abolished the mitochondrial calcium uptake. In conclusion, these results indicate the fourth transmembrane domain of TAS2R46 is critical for the intrinsic receptor activity.

Zinc is essential for many physiological functions, with a major role in digestive system, skin health, and learning and memory. On the cellular level, zinc is involved in cell proliferation and cell death. A selective zinc sensing receptor, ZnR/GPR39 is a Gq-coupled receptor that acts via the inositol trisphosphate pathway to release intracellular Ca 2+ . The ZnR/GPR39 serves as a mediator between extracellular changes in Zn 2+ concentration and cellular Ca 2+ signalling. This signalling pathway regulates ion transporters activity and thereby controls the formation of transepithelial gradients or neuronal membrane potential, which play a fundamental role in the physiological function of these tissues. This review focuses on the role of Ca 2+ signalling, and specifically ZnR/GPR39, with respect to the regulation of the Na + /H + exchanger, NHE1, and of the K + /Cl - cotransporters, KCC1-3, and also describes the physiological implications of this regulation., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Store operated Ca 2+ entry (SOCE) is a ubiquitous signalling module with established roles in the immune system, secretion and muscle development. Recent evidence supports a complex role for SOCE in the nervous system. In this review we present an update of the current knowledge on SOCE function in the brain with a focus on its role as a regulator of brain activity and excitability., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Spatio-temporal definition of Ca 2+ signals involves the assembly of signaling complexes within the nano-architecture of contact sites between the sarco/endoplasmic reticulum (SR/ER) and the plasma membrane (PM). While the requirement of precise spatial assembly and positioning of the junctional signaling elements is well documented, the role of the nano-scale membrane architecture itself, as an ion-reflecting confinement of the signalling unit, remains as yet elusive. Utilizing the Na + /Ca 2+ Exchanger-1 / SR/ER Ca 2+ ATPase-2-mediated ER Ca 2+ refilling process as a junctional signalling paradigm, we provide here the first evidence for an indispensable cellular function of the junctional membrane architecture. Our stochastic modeling approach demonstrates that junctional ER Ca 2+ refilling operates exclusively at nano-scale membrane spacing, with a strong inverse relationship between junctional width and signaling efficiency. Our model predicts a breakdown of junctional Ca 2+ signaling with loss of reflecting membrane confinement. In addition we consider interactions between Ca 2+ and the phospholipid membrane surface, which may support interfacial Ca 2+ transport and promote receptor targeting. Alterations in the molecular and nano-scale membrane organization at organelle-PM contacts are suggested as a new concept in pathophysiology., Competing Interests: Declaration of Competing Interest We declare no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

Coordinated events of calcium (Ca 2+ ) released from the endoplasmic reticulum (ER) are key second messengers in excitable cells. In pain-sensing dorsal root ganglion (DRG) neurons, these events can be observed as Ca 2+ sparks, produced by a combination of ryanodine receptors (RyR) and inositol 1,4,5-triphosphate receptors (IP3R1). These microscopic signals offer the neuronal cells with a possible means of modulating the subplasmalemmal Ca 2+ handling, initiating vesicular exocytosis. With super-resolution dSTORM and expansion microscopies, we visualised the nanoscale distributions of both RyR and IP3R1 that featured loosely organised clusters in the subplasmalemmal regions of cultured rat DRG somata. We adapted a novel correlative microscopy protocol to examine the nanoscale patterns of RyR and IP3R1 in the locality of each Ca 2+ spark. We found that most subplasmalemmal sparks correlated with relatively small groups of RyR whilst larger sparks were often associated with larger groups of IP3R1. These data also showed spontaneous Ca 2+ sparks in <30% of the subplasmalemmal cell area but consisted of both these channel species at a 3.8-5 times higher density than in nonactive regions of the cell. Taken together, these observations reveal distinct patterns and length scales of RyR and IP3R1 co-clustering at contact sites between the ER and the surface plasmalemma that encode the positions and the quantity of Ca 2+ released at each Ca 2+ spark.

Egg activation at fertilization in mouse eggs is caused by a series of cytosolic Ca2+ oscillations that are associated with an increase in ATP concentrations driven by increased mitochondrial activity. We have investigated the role of Ca2+ oscillations in these changes in ATP at fertilization by measuring the dynamics of ATP and Ca2+ in mouse eggs. An initial ATP increase started with the first Ca2+ transient at fertilization and then a secondary increase in ATP occurred ∼1 h later and this preceded a small and temporary increase in the frequency of Ca2+ oscillations. Other stimuli that caused Ca2+ oscillations such as PLCz1 or thimerosal, caused smaller or slower changes in ATP that failed to show the distinct secondary rise. Sperm-induced Ca2+ oscillations in the egg also triggered changes in the fluorescence of NADH which followed the pattern of Ca2+ spikes in a similar pattern to oscillations triggered by PLCz1 or thimerosal. When eggs were loaded with low concentrations of the Ca2+ chelator BAPTA, sperm triggered one small Ca2+ increase, but there were still extra phases of ATP increase that were similar to control fertilized eggs. Singular Ca2+ increases caused by thapsigargin were much less effective in elevating ATP levels. Together these data suggest that the secondary ATP increase at fertilization in mouse eggs is not caused by increases in cytosolic Ca2+. The fertilizing sperm may stimulate ATP production in eggs via both Ca2+ and by another mechanism that is independent of PLCz1 or Ca2+ oscillations., (© 2023 The Author(s).)

Our sense of hearing depends on the function of a specialised class of sensory cells, the hair cells, which are found in the organ of Corti of the mammalian cochlea. The unique physiological environment in which these cells operate is maintained by a syncitium of non-sensory supporting cells, which are crucial for regulating cochlear physiology and metabolic homeostasis. Despite their importance for cochlear function, the role of these supporting cells in age-related hearing loss, the most common sensory deficit in the elderly, is poorly understood. Here, we investigated the age-related changes in the expression and function of metabotropic purinergic receptors (P2Y 1 , P2Y 2 and P2Y 4 ) in the supporting cells of the cochlear apical coil. Purinergic signalling in supporting cells is crucial during the development of the organ of Corti and purinergic receptors are known to undergo changes in expression during ageing in several tissues. Immunolabelling and Ca 2+ imaging experiments revealed a downregulation of P2Y receptor expression and a decrease of purinergic-mediated calcium responses after early postnatal stages in the supporting cells. An upregulation of P2Y receptor expression was observed in the aged cochlea when compared to 1 month-old adults. The aged mice also had significantly larger calcium responses and displayed calcium oscillations during prolonged agonist applications. We conclude that supporting cells in the aged cochlea upregulate P2Y 2 and P2Y 4 receptors and display purinergic-induced Ca 2+ responses that mimic those observed during pre-hearing stages of development, possibly aimed at limiting or preventing further damage to the sensory epithelium. KEY POINTS: Age-related hearing loss is associated with lower hearing sensitivity and decreased ability to understand speech. We investigated age-related changes in the expression and function of metabotropic purinergic (P2Y) receptors in cochlear non-sensory supporting cells of mice displaying early-onset (C57BL/6N) and late-onset (C3H/HeJ) hearing loss. The expression of P2Y 1 , P2Y 2 and P2Y 4 receptors in the supporting cells decreased during cochlear maturation, but that of P2Y 2 and P2Y 4 was upregulated in the aged cochlea. P2Y 2 and P2Y 4 receptors were primarily responsible for the ATP-induced Ca 2+ responses in the supporting cells. The degree of purinergic expression upregulation in aged supporting cells mirrored hearing loss progression in the different mouse strains. We propose that the upregulation of purinergic-mediated signalling in the aged cochlea is subsequent to age-related changes in the hair cells and may act as a protective mechanism to limit or to avoid further damage to the sensory epithelium., (© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Plants require the major macronutrients, nitrogen (N), phosphorus (P) and potassium (K) for normal growth and development. Their deficiency in soil directly affects vital cellular processes, particularly root growth and architecture. Their perception, uptake and assimilation are regulated by complex signalling pathways. To overcome nutrient deficiencies, plants have developed certain response mechanisms that determine developmental and physiological adaptations. The signal transduction pathways underlying these responses involve a complex interplay of components such as nutrient transporters, transcription factors and others. In addition to their involvement in cross-talk with intracellular calcium signalling pathways, these components are also engaged in NPK sensing and homeostasis. The NPK sensing and homeostatic mechanisms hold the key to identify and understand the crucial players in nutrient regulatory networks in plants under both abiotic and biotic stresses. In this review, we discuss calcium signalling components/pathways underlying plant responses to NPK sensing, with a focus on the sensors, transporters and transcription factors involved in their respective signalling and homeostasis., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

ATP, as a paracrine signalling molecule, induces intracellular Ca 2+ elevation via the activation of purinergic receptors on the surface of glia-like cochlear supporting cells. These cells, including the Deiters' cells (DCs), are also coupled by gap junctions that allow the propagation of intercellular Ca 2+ waves via diffusion of Ca 2+ mobilising second messenger IP 3 between neighbouring cells. We have compared the ATP-evoked Ca 2+ transients and the effect of two different gap junction (GJ) blockers (octanol and carbenoxolone, CBX) on the Ca 2+ transients in DCs located in the apical and middle turns of the hemicochlea preparation of BALB / c mice (P14-19). Octanol had no effect on Ca 2+ signalling, while CBX inhibited the ATP response, more prominently in the middle turn. Based on astrocyte models and using our experimental results, we successfully simulated the Ca 2+ dynamics in DCs in different cochlear regions. The mathematical model reliably described the Ca 2+ transients in the DCs and suggested that the tonotopical differences could originate from differences in purinoceptor and Ca 2+ pump expressions and in IP 3 -Ca 2+ release mechanisms. The cochlear turn-dependent effect of CBX might be the result of the differing connexin isoform composition of GJs along the tonotopic axis. The contribution of IP 3 -mediated Ca 2+ signalling inhibition by CBX cannot be excluded.

Understanding the molecular mechanism(s) of small compounds in cellular growth control are essential for using those against the disease(s). Oral cancers exhibit a very high mortality rate due to higher metastatic potential. Aberrant EGFR, RAR, HH signalling, enhanced [Ca 2+ ] and oxidative stress are some of the important characteristics of oral cancer. So, we target these for our study. Herein, we tested the effect of fendiline hydrochloride (FH) as an LTCC Ca 2+ -channel inhibitor, erismodegib (a SMO inhibitor of HH-signalling) and all-trans retinoic acid (RA) inducer of RAR signalling that causes cellular differentiation. OCT4 activating compound (OAC1) counters differentiation and induces stemness properties. Cytosine β-D arabinofuranoside (Cyto-BDA), a DNA replication inhibitor was used to reduce high proliferative capacity. Treatment of FaDu cells with OAC1, Cyto-BDA and FH increase G0/G1 population by 3%, 20% and 7% respectively, and lead to reduction of cyclin D1, CDK4/6 levels. Erismodegib arrests the cells in S-phase with reduced cyclin-E1&A1 levels, whereas RA-treatment causes G2/M phase arrest with reduced cyclin-B1. There was a decrease in the expression of EGFR and mesenchymal markers, Snail/Slug/Vim/Zeb/Twist, and increased E-cadherin expression in all the drug treatments, indicating a reduction in proliferative signal and EMT. Enhanced MLL2 (Mll4) and reduced EZH2 expression associated overexpression of p53 and p21 were traced out. We conclude that these drugs impact expression of epigenetic modifiers by modulating signalling pathways and the epigenetic modifiers then controls the expression of cell cycle control genes, including p53 and p21., Competing Interests: Declaration of competing interest None., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Epithelial-mesenchymal transition (EMT) is a form of cellular phenotypic plasticity and is considered a crucial step in the progression of many cancers. The calcium ion (Ca 2+ ) acts as a ubiquitous second messenger and is implicated in many cellular processes, including cell death, migration, invasion and more recently EMT. Throughout this review, the complex interplay between Ca 2+ signalling and EMT will be explored. An overview of the Ca 2+ pathways that are remodelled as a consequence of EMT is provided and the role of Ca 2+ signalling in regulating EMT and its significance is considered. Ca 2+ signalling pathways may represent a therapeutic opportunity to regulate EMT. However, as will be described in this review, the complexity of these signalling pathways represents significant challenges that must be considered if Ca 2+ signalling is to be manipulated with the aim of therapeutic intervention in cancer., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Greg Monteith reports financial support was provided by National Health and Medical Research Council of Australia and the Office of the Assistant Secretary of defence for Health Affairs, through the Breast Cancer Research Program., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, 'calcium sparks', determine the contraction of the myocardium. We adapted a correlative super-resolution microscopy protocol to correlate sub-plasmalemmal spontaneous calcium sparks in rat right ventricular myocytes with the local nanoscale RyR2 positions. This revealed a steep relationship between the integral of a calcium spark and the sum of the local RyR2s. Segmentation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR2 clusters. In myocytes taken from failing right ventricles, RyR2 clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. They also featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyR2s. While fragmented (smaller) RyR2 clusters were rarely observed directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight, non-random clustering of the local RyR2 in both healthy and failing ventricles.

Astrocytes are a heterogenous population of macroglial cells spread throughout the central nervous system with diverse functions, expression signatures, and intricate morphologies. Their subcellular compartments contain a distinct range of mitochondria, with functional microdomains exhibiting widespread activities, such as controlling local metabolism and Ca2+ signaling. Ca2+ is an ion of utmost importance, both physiologically and pathologically, and participates in critical central nervous system processes, including synaptic plasticity, neuron-astrocyte integration, excitotoxicity, and mitochondrial physiology and metabolism. The mitochondrial Ca2+ handling system is formed by the mitochondrial Ca2+ uniporter complex (MCUc), which mediates Ca2+ influx, and the mitochondrial Na+/Ca2+ exchanger (NCLX), responsible for most mitochondrial Ca2+ efflux, as well as additional components, including the mitochondrial permeability transition pore (mtPTP). Over the last decades, mitochondrial Ca2+ handling has been shown to be key for brain homeostasis, acting centrally in physiopathological processes such as astrogliosis, astrocyte-neuron activity integration, energy metabolism control, and neurodegeneration. In this review, we discuss the current state of knowledge regarding the mitochondrial Ca2+ handling system molecular composition, highlighting its impact on astrocytic homeostasis., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Cell membrane repair is a critical process used to maintain cell integrity and survival from potentially lethal chemical, and mechanical membrane injury. Rapid increases in local calcium levels due to a membrane rupture have been widely accepted as a trigger for multiple membrane-resealing models that utilize exocytosis, endocytosis, patching, and shedding mechanisms. Calcium-sensor proteins, such as synaptotagmins (Syt), dysferlin, S100 proteins, and annexins, have all been identified to regulate, or participate in, multiple modes of membrane repair. Dysfunction of membrane repair from inefficiencies or genetic alterations in these proteins contributes to diseases such as muscular dystrophy (MD) and heart disease. The present review covers the role of some of the key calcium-sensor proteins and their involvement in membrane repair., (© 2023 The Author(s).)

Calcium signaling is essential for lymphocyte activation, with genetic disruptions of store-operated calcium (Ca 2+ ) entry resulting in severe immunodeficiency. The inositol 1,4,5-trisphosphate receptor (IP 3 R), a homo- or heterotetramer of the IP 3 R1-3 isoforms, amplifies lymphocyte signaling by releasing Ca 2+ from endoplasmic reticulum stores following antigen stimulation. Although knockout of all IP 3 R isoforms in mice causes immunodeficiency, the seeming redundancy of the isoforms is thought to explain the absence of variants in human immunodeficiency. In this study, we identified compound heterozygous variants of ITPR3 (a gene encoding IP 3 R subtype 3) in two unrelated Caucasian patients presenting with immunodeficiency. To determine whether ITPR3 variants act in a nonredundant manner and disrupt human immune responses, we characterized the Ca 2+ signaling capacity, the lymphocyte response, and the clinical phenotype of these patients. We observed disrupted Ca 2+ signaling in patient-derived fibroblasts and immune cells, with abnormal proliferation and activation responses following T-cell receptor stimulation. Reconstitution of IP 3 R3 in IP 3 R knockout cell lines led to the identification of variants as functional hypomorphs that showed reduced ability to discriminate between homeostatic and induced states, validating a genotype-phenotype link. These results demonstrate a functional link between defective endoplasmic reticulum Ca 2+ channels and immunodeficiency and identify IP 3 Rs as diagnostic targets for patients with specific inborn errors of immunity. These results also extend the known cause of Ca 2+ -associated immunodeficiency from store-operated entry to impaired Ca 2+ mobilization from the endoplasmic reticulum, revealing a broad sensitivity of lymphocytes to genetic defects in Ca 2+ signaling., (© 2022. The Author(s).)

Calcium signalling in platelets through store operated Ca 2+ entry (SOCE) or receptor-operated Ca 2+ entry (ROCE) mechanisms is crucial for platelet activation and function. Orai1 proteins have been implicated in platelet's SOCE. In this study we evaluated the contribution of Orai1 proteins to these processes using washed platelets from adult mice from both genders with platelet-specific deletion of the Orai1 gene (Orai1 flox/flox ; Pf4-Cre termed as Orai1 Plt-KO ) since mice with ubiquitous Orai1 deficiency show early lethality. Platelet aggregation as well as Ca 2+ entry and release were measured in vitro following stimulation with collagen, collagen related peptide (CRP), thromboxane A2 analogue U46619, thrombin, ADP and the sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibitor thapsigargin, respectively. SOCE and aggregation induced by Thapsigargin up to a concentration of 0.3 µM was abrogated in Orai1-deficient platelets. Receptor-operated Ca 2+ -entry and/or platelet aggregation induced by CRP, U46619 or thrombin were partially affected by Orai1 deletion depending on the gender. In contrast, ADP-, collagen- and CRP-induced aggregation was comparable in Orai1 Plt-KO platelets and control cells over the entire concentration range. Our results reinforce the indispensability of Orai1 proteins for SOCE in murine platelets, contribute to understand its role in agonist-dependent signalling and emphasize the importance to analyse platelets from both genders.

cis-[Ru(bpy)2(py)NO2](PF6) (RuBPY) is a ruthenium complex nitric oxide (NO) donor that presents a nitrite in its moiety and has been shown to induce vasodilation in various arteries, as well as arterial pressure reduction with no changes in heart rate. Because vascular tone is highly dependent on the cytosolic calcium concentration ([Ca 2+ ]c), the current study aimed to investigate the effects of RuBPY on the intracellular mobilization of calcium stores of rat aortic vascular smooth muscle cells. Vascular reactivity experiments were performed in isolated aortic rings that were contracted with a high concentration of KCl or phenylephrine (Phe). Moreover, primary cultured vascular smooth muscle cells were used to measure [Ca 2+ ]c by confocal microscopy. The NO donor RuBPY decreased the [Ca 2+ ]c and reduced KCl and Phe-induced contractile responses. The selective inhibitor of sarco-endoplasmic Ca-ATPase (SERCA) with thapsigargin impaired the effect of RuBPY on Phe-induced contractile response. RuBPY also reduced caffeine-induced contraction, and the contraction dependent on the capacitive Ca 2+ influx. Therefore, our results suggest that NO released from RuBPY decreased [Ca 2+ ]c by calcium influx blockade and activation of guanylyl-cyclase-cGMP-GK pathway. These results indicate that RuBPY increases Ca 2+ storage in the sarcoplasmic reticulum by SERCA activation and also by capacitive Ca 2+ influx inhibition, which is dependent on the intracellular release of nitric oxide from this compound., (© 2022 John Wiley & Sons Australia, Ltd.)

Upon depolarization of chromaffin cells (CCs), a prompt release of catecholamines occurs. This event is triggered by a subplasmalemmal high-Ca 2+ microdomain (HCMD) generated by Ca 2+ entry through nearby voltage-activated calcium channels. HCMD is efficiently cleared by local mitochondria that avidly take up Ca 2+ through their uniporter (MICU), then released back to the cytosol through mitochondrial Na + /Ca 2+ exchanger (MNCX). We found that newly synthesized derivative ITH15004 facilitated the release of catecholamines triggered from high K + -depolarized bovine CCs. Such effect seemed to be due to regulation of mitochondrial Ca 2+ circulation because: (i) FCCP-potentiated secretory responses decay was prevented by ITH15004; (ii) combination of FCCP and ITH15004 exerted additive secretion potentiation; (iii) such additive potentiation was dissipated by the MICU blocker ruthenium red (RR) or the MNCX blocker CGP37157 (CGP); (iv) combination of FCCP and ITH15004 produced both additive augmentation of cytosolic Ca 2+ concentrations ([Ca 2+ ] c ) K + -challenged BCCs, and (v) non-inactivated [Ca 2+ ] c transient when exposed to RR or CGP. On pharmacological grounds, data suggest that ITH15004 facilitates exocytosis by acting on mitochondria-controlled Ca 2+ handling during K + depolarization. These observations clearly show that ITH15004 is a novel pharmacological tool to study the role of mitochondria in the regulation of the bioenergetics and exocytosis in excitable cells.

Alighting aphids probe a new host plant by intracellular test punctures for suitability. These induce immediate calcium signals that emanate from the punctured sites and might be the first step in plant recognition of aphid feeding and the subsequent elicitation of plant defence responses. Calcium is also involved in the transmission of non-persistent plant viruses that are acquired by aphids during test punctures. Therefore, we wanted to determine whether viral infection alters calcium signalling. For this, calcium signals triggered by aphids were imaged on transgenic Arabidopsis plants expressing the cytosolic FRET-based calcium reporter YC3.6-NES and infected with the non-persistent viruses cauliflower mosaic (CaMV) and turnip mosaic (TuMV), or the persistent virus, turnip yellows (TuYV). Aphids were placed on infected leaves and calcium elevations were recorded by time-lapse fluorescence microscopy. Calcium signal velocities were significantly slower in plants infected with CaMV or TuMV and signal areas were smaller in CaMV-infected plants. Transmission tests using CaMV-infected Arabidopsis mutants impaired in pathogen perception or in the generation of calcium signals revealed no differences in transmission efficiency. A transcriptomic meta-analysis indicated significant changes in expression of receptor-like kinases in the BAK1 pathway as well as of calcium channels in CaMV- and TuMV-infected plants. Taken together, infection with CaMV and TuMV, but not with TuYV, impacts aphid-induced calcium signalling. This suggests that viruses can modify plant responses to aphids from the very first vector/host contact.

Changes in cellular Ca 2+ levels have major influences on vascular function and blood pressure regulation. Vascular smooth muscle cells (SMCs) and endothelial cells (ECs) orchestrate vascular activity in distinct ways, often involving highly specific fluctuations in Ca 2+ signalling. Ageing is a major risk factor for cardiovascular diseases, but the impact of ageing per se on vascular Ca 2+ signalling has received insufficient attention. We reviewed the literature for age-related changes in Ca 2+ signalling in relation to vascular structure and function. Vascular tone dysregulation in several vascular beds has been linked to abnormal expression or activity of SMC voltage-gated Ca 2+ channels, Ca 2+ -activated K + channels or TRPC6 channels. Some of these effects were linked to altered caveolae density, microRNA expression or 20-HETE abundance. Intracellular store Ca 2+ handling was suppressed in ageing mainly via reduced expression of intracellular Ca 2+ release channels, and Ca 2+ reuptake or efflux pumps. An increase in mitochondrial Ca 2+ uptake, leading to oxidative stress, could also play a role in SMC hypercontractility and structural remodelling in ageing. In ECs, ageing entailed diverse effects on spontaneous and evoked Ca 2+ transients, as well as structural changes at the EC-SMC interface. The concerted effects of altered Ca 2+ signalling on myogenic tone, endothelium-dependent vasodilatation, and vascular structure are likely to contribute to blood pressure dysregulation and blood flow distribution deficits in critical organs. With the increase in the world's ageing population, future studies should be directed at solving specific ageing-induced Ca 2+ signalling deficits to combat the imminent accelerated vascular ageing and increased risk of cardiovascular diseases., (© 2021 The Authors. The Journal of Physiology © 2021 The Physiological Society.)

The Mycobacterium ulcerans exotoxin, mycolactone, is an inhibitor of co-translational translocation via the Sec61 complex. Mycolactone has previously been shown to bind to, and alter the structure of the major translocon subunit Sec61α, and change its interaction with ribosome nascent chain complexes. In addition to its function in protein translocation into the ER, Sec61 also plays a key role in cellular Ca2+ homeostasis, acting as a leak channel between the endoplasmic reticulum (ER) and cytosol. Here, we have analysed the effect of mycolactone on cytosolic and ER Ca2+ levels using compartment-specific sensors. We also used molecular docking analysis to explore potential interaction sites for mycolactone on translocons in various states. These results show that mycolactone enhances the leak of Ca2+ ions via the Sec61 translocon, resulting in a slow but substantial depletion of ER Ca2+. This leak was dependent on mycolactone binding to Sec61α because resistance mutations in this protein completely ablated the increase. Molecular docking supports the existence of a mycolactone-binding transient inhibited state preceding translocation and suggests mycolactone may also bind Sec61α in its idle state. We propose that delayed ribosomal release after translation termination and/or translocon 'breathing' during rapid transitions between the idle and intermediate-inhibited states allow for transient Ca2+ leak, and mycolactone's stabilisation of the latter underpins the phenotype observed., (© 2021 The Author(s).)

Duchenne muscular dystrophy (DMD) leads to disability and death in young men. This disease is caused by mutations in the DMD gene encoding diverse isoforms of dystrophin. Loss of full-length dystrophins is both necessary and sufficient for causing degeneration and wasting of striated muscles, neuropsychological impairment, and bone deformities. Among this spectrum of defects, abnormalities of calcium homeostasis are the common dystrophic feature. Given the fundamental role of Ca 2+ in all cells, this biochemical alteration might be underlying all the DMD abnormalities. However, its mechanism is not completely understood. While abnormally elevated resting cytosolic Ca 2+ concentration is found in all dystrophic cells, the aberrant mechanisms leading to that outcome have cell-specific components. We probe the diverse aspects of calcium response in various affected tissues. In skeletal muscles, cardiomyocytes, and neurons, dystrophin appears to serve as a scaffold for proteins engaged in calcium homeostasis, while its interactions with actin cytoskeleton influence endoplasmic reticulum organisation and motility. However, in myoblasts, lymphocytes, endotheliocytes, and mesenchymal and myogenic cells, calcium abnormalities cannot be clearly attributed to the loss of interaction between dystrophin and the calcium toolbox proteins. Nevertheless, DMD gene mutations in these cells lead to significant defects and the calcium anomalies are a symptom of the early developmental phase of this pathology. As the impaired calcium homeostasis appears to underpin multiple DMD abnormalities, understanding this alteration may lead to the development of new therapies. In fact, it appears possible to mitigate the impact of the abnormal calcium homeostasis and the dystrophic phenotype in the total absence of dystrophin. This opens new treatment avenues for this incurable disease., Competing Interests: The authors declare no conflicts of interest.

TMEM16A Ca 2+ -activated chloride channels are involved in multiple cellular functions and are proposed targets for diseases such as hypertension, stroke, and cystic fibrosis. This therapeutic endeavor, however, suffers from paucity of selective and potent modulators. Here, exploiting a synthetic small molecule with a biphasic effect on the TMEM16A channel, anthracene-9-carboxylic acid (A9C), we shed light on sites of the channel amenable for pharmacological intervention. Mutant channels with the intracellular gate constitutively open were generated. These channels were entirely insensitive to extracellular A9C when intracellular Ca 2+ was omitted. However, when physiological Ca 2+ levels were reestablished, the mutants regained sensitivity to A9C. Thus, intracellular Ca 2+ is mandatory for the channel response to an extracellular modulator. The underlying mechanism is a conformational change in the outer pore that enables A9C to enter the pore to reach its binding site. The explanation of this structural rearrangement highlights a critical site for pharmacological intervention and reveals an aspect of Ca 2+ gating in the TMEM16A channel., Competing Interests: The authors declare no competing interest.

Novel molecular pinball machines of the plasma membrane control cytosolic Ca 2+ levels that regulate plant metabolism. The essential components involve: 1. an auxin-activated proton pump; 2. arabinogalactan glycoproteins (AGPs); 3. Ca 2+ channels; 4. auxin-efflux "PIN" proteins. Typical pinball machines release pinballs that trigger various sound and visual effects. However, in plants, "proton pinballs" eject Ca 2+ bound by paired glucuronic acid residues of numerous glycomodules in periplasmic AGP-Ca 2+ . Freed Ca 2+ ions flow down the electrostatic gradient through open Ca 2+ channels into the cytosol, thus activating numerous Ca 2+ -dependent activities. Clearly, cytosolic Ca 2+ levels depend on the activity of the proton pump, the state of Ca 2+ channels and the size of the periplasmic AGP-Ca 2+ capacitor; proton pump activation is a major regulatory focal point tightly controlled by the supply of auxin. Auxin efflux carriers conveniently known as "PIN" proteins (null mutants are pin-shaped) pump auxin from cell to cell. Mechanosensitive Ca 2+ channels and their activation by reactive oxygen species (ROS) are yet another factor regulating cytosolic Ca 2+ . Cell expansion also triggers proton pump/pinball activity by the mechanotransduction of wall stress via Hechtian adhesion, thus forming a Hechtian oscillator that underlies cycles of wall plasticity and oscillatory growth. Finally, the Ca 2+ homeostasis of plants depends on cell surface external storage as a source of dynamic Ca 2+ , unlike the internal ER storage source of animals, where the added regulatory complexities ranging from vitamin D to parathormone contrast with the elegant simplicity of plant life. This paper summarizes a sixty-year Odyssey.

We analysed spatiotemporal properties of Ca 2+ signals in protoplasmic astrocytes in the CA1 stratum radiatum of hippocampal slices from young (2-3 months old) mice housed in control conditions or exposed to a caloric restriction (CR) diet for one month. The astrocytic Ca 2+ events became shorter in duration and smaller in size; they also demonstrated reduced velocity of expansion and shrinkage following CR. At the same time, Ca 2+ signals in the astrocytes from the CR animals demonstrated higher amplitude and the faster rise and decay rates. These changes can be attributed to CR-induced morphological remodelling and uncoupling of astrocytes described in our previous study. CR-induced changes in the parameters of Ca 2+ activity were partially reversed by inhibition of gap junctions/hemichannels with carbenoxolone (CBX). The effect of CBX on Ca 2+ activity in CR-animals was unexpected because the diet already decreases gap junctional coupling in astrocytic syncytia. It may reflect the blockade of hemichannels also sensitive to this drug. Thus, CR-induced morphological remodelling of astrocytes is at least partly responsible for changes in the pattern of Ca 2+ activity in the astrocytic network. How such changes in spatiotemporal Ca 2+ landscape can translate into astrocytic physiology and neuron-glia interactions remains a matter for future studies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aims: The cardiac ryanodine receptor (RyR2), which mediates intracellular Ca2+ release to trigger cardiomyocyte contraction, participates in development of acquired and inherited arrhythmogenic cardiac disease. This study was undertaken to characterize the network of inter- and intra-subunit interactions regulating the activity of the RyR2 homotetramer., Methods and Results: We use mutational investigations combined with biochemical assays to identify the peptide sequence bridging the β8 with β9 strand as the primary determinant mediating RyR2 N-terminus self-association. The negatively charged side chains of two aspartate residues (D179 and D180) within the β8-β9 loop are crucial for the N-terminal inter-subunit interaction. We also show that the RyR2 N-terminus domain interacts with the C-terminal channel pore region in a Ca2+-independent manner. The β8-β9 loop is required for efficient RyR2 subunit oligomerization but it is dispensable for N-terminus interaction with C-terminus. Deletion of the β8-β9 sequence produces unstable tetrameric channels with subdued intracellular Ca2+ mobilization implicating a role for this domain in channel opening. The arrhythmia-linked R176Q mutation within the β8-β9 loop decreases N-terminus tetramerization but does not affect RyR2 subunit tetramerization or the N-terminus interaction with C-terminus. RyR2R176Q is a characteristic hypersensitive channel displaying enhanced intracellular Ca2+ mobilization suggesting an additional role for the β8-β9 domain in channel closing., Conclusion: These results suggest that efficient N-terminus inter-subunit communication mediated by the β8-β9 loop may constitute a primary regulatory mechanism for both RyR2 channel activation and suppression., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Fungal diseases are responsible for the deaths of over 1.5 million people worldwide annually. Antifungal peptides represent a useful source of antifungals with novel mechanisms-of-action, and potentially provide new methods of overcoming resistance. Here we investigate the mode-of-action of the small, rationally designed synthetic antifungal peptide PAF26 using the model fungus Neurospora crassa. Here we show that the cell killing activity of PAF26 is dependent on extracellular Ca 2+ and the presence of fully functioning fungal Ca 2+ homeostatic/signaling machinery. In a screen of mutants with deletions in Ca 2+ -signaling machinery, we identified three mutants more tolerant to PAF26. The Ca 2+ ATPase NCA-2 was found to be involved in the initial interaction of PAF26 with the cell envelope. The vacuolar Ca 2+ channel YVC-1 was shown to be essential for its accumulation and concentration within the vacuolar system. The Ca 2+ channel CCH-1 was found to be required to prevent the translocation of PAF26 across the plasma membrane. In the wild type, Ca 2+ removal from the medium resulted in the peptide remaining trapped in small vesicles as in the Δyvc-1 mutant. It is, therefore, apparent that cell killing by PAF26 is complex and unusually dependent on extracellular Ca 2+ and components of the Ca 2+ -regulatory machinery., (© 2020 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Understanding the relationship between shape and function of dendritic spines is an elusive topic. Several modelling approaches have been used to investigate the interplay between spine geometry, calcium diffusion and electric signalling. We here use a second order finite element method to solve the Poisson-Nernst-Planck equations and describe electrodiffusion in dendritic spines. With this, we obtain relationships between dendritic geometry and calcic as well as electric responses to synaptic events. Our findings support the hypothesis that spine geometry plays a role shaping the electrical responses to synaptic events. Our method was also able to reveal the fine scale distribution of calcium in spines with irregular shapes.

Nitrate, the major source of inorganic nitrogen for plants, is a critical signal controlling nutrient transport and assimilation and adaptive growth responses throughout the plant. Understanding how plants perceive nitrate and how this perception is transduced into responses that optimize growth are important for the rational improvement of crop productivity and for mitigating pollution from the use of fertilizers. This review highlights recent findings that reveal key roles of cytosolic-nuclear calcium signalling and dynamic protein phosphorylation via diverse mechanisms in the primary nitrate response (PNR). Nitrate-triggered calcium signatures as well as the critical functions of subgroup III calcium-sensor protein kinases, a specific protein phosphatase 2C, and RNA polymerase II C-terminal domain phosphatase-like 3 are discussed. Moreover, genome-wide meta-analysis of nitrate-regulated genes encoding candidate protein kinases and phosphatases for modulating critical phosphorylation events in the PNR are elaborated. We also consider how phosphoproteomics approaches can contribute to the identification of putative regulatory protein kinases in the PNR. Exploring and integrating experimental strategies, new methodologies, and comprehensive datasets will further advance our understanding of the molecular and cellular mechanisms underlying the complex regulatory processes in the PNR., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Calcium (Ca 2+ ) signals initiate egg activation across the animal kingdom and in at least some plants. These signals are crucial for the success of development and, in the case of mammals, health of the offspring. The mechanisms associated with fertilization that trigger these signals and the molecules that regulate their characteristic patterns vary widely. With few exceptions, a major contributor to fertilization-induced elevation in cytoplasmic Ca 2+ is release from endoplasmic reticulum stores through the IP3 receptor. In some cases, Ca 2+ influx from the extracellular space and/or release from alternative intracellular stores contribute to the rise in cytoplasmic Ca 2+ . Following the Ca 2+ rise, the reuptake of Ca 2+ into intracellular stores or efflux of Ca 2+ out of the egg drive the return of cytoplasmic Ca 2+ back to baseline levels. The molecular mediators of these Ca 2+ fluxes in different organisms include Ca 2+ release channels, uptake channels, exchangers and pumps. The functions of these mediators are regulated by their particular activating mechanisms but also by alterations in their expression and spatial organization. We discuss here the molecular basis for modulation of Ca 2+ signalling at fertilization, highlighting differences across several animal phyla, and we mention key areas where questions remain.

Many reptiles and some fish determine offspring sex by environmental cues such as incubation temperature. The mechanism by which environmental signals are captured and transduced into specific sexual phenotypes has remained unexplained for over 50 years. Indeed, environmental sex determination (ESD) has been viewed as an intractable problem because sex determination is influenced by a myriad of genes that may be subject to environmental influence. Recent demonstrations of ancient, conserved epigenetic processes in the regulatory response to environmental cues suggest that the mechanisms of ESD have a previously unsuspected level of commonality, but the proximal sensor of temperature that ultimately gives rise to one sexual phenotype or the other remains unidentified. Here, we propose that in ESD species, environmental cues are sensed by the cell through highly conserved ancestral elements of calcium and redox (CaRe) status, then transduced to activate ubiquitous signal transduction pathways, or influence epigenetic processes, ultimately to drive the differential expression of sex genes. The early evolutionary origins of CaRe regulation, and its essential role in eukaryotic cell function, gives CaRe a propensity to be independently recruited for diverse roles as a 'cellular sensor' of environmental conditions. Our synthesis provides the first cohesive mechanistic model connecting environmental signals and sex determination pathways in vertebrates, providing direction and a framework for developing targeted experimentation., (© 2020 Cambridge Philosophical Society.)

Programmed cell death (PCD) is the destruction of unwanted cells through an intracellularly mediated process. Perforation formation in the lace plant (Aponogeton madagascariensis) provides an excellent model for studying developmentally regulated PCD. Ca 2+ fluxes have previously been identified as important signals for PCD in plants and mammals. The fundamental goal of this project was to determine the influence of Ca 2+ on the rate of cell death and perforation formation during leaf development in the lace plant. This was investigated using the application of various known calcium modulators including lanthanum III chloride (LaCl 3 ), ruthenium red and calcium ionophore A23187. Detached lace plant leaves at an early stage of development were treated with these modulators in both short- and long-term exposure assays and analysed using live cell imaging. Results from this study indicate that calcium plays a vital role in developmentally regulated PCD in the lace plant as application of the modulators significantly altered the rate of cell death and perforation formation during leaf development. In conclusion, this study exemplifies the suitability of the lace plant for live cell imaging and detached leaf experiments to study cell death and provides insight into the importance of Ca 2+ in developmentally regulated PCD in planta., (© 2019 Royal Microscopical Society.)

Astroglia regulate neurovascular coupling while engaging in signal exchange with neurons. The underlying cellular machinery is thought to rely on astrocytic Ca 2+ signals, but what controls their amplitude and waveform is poorly understood. Here, we employ time-resolved two-photon excitation fluorescence imaging in acute hippocampal slices and in cortex in vivo to find that resting [Ca 2+ ] predicts the scale (amplitude) and the maximum (peak) of astroglial Ca 2+ elevations. We bidirectionally manipulate resting [Ca 2+ ] by uncaging intracellular Ca 2+ or Ca 2+ buffers and use ratiometric imaging of a genetically encoded Ca 2+ indicator to establish that alterations in resting [Ca 2+ ] change co-directionally the peak level and anti-directionally the amplitude of local Ca 2+ transients. This relationship holds for spontaneous and for induced (for instance by locomotion) Ca 2+ signals. Our findings uncover a basic generic rule of Ca 2+ signal formation in astrocytes, thus also associating the resting Ca 2+ level with the physiological "excitability" state of astroglia., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

During development, disease or in response to changes in local environmental and/or nutrient supply, cellular metabolism is substantially remodeled. Reduced mitochondrial Ca 2+ uptake was recently reported to induce metabolic remodeling, which through stimulating alterations in the epigenome causes changes in gene expression associated with fibroblast to myofibroblast differentiation., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Axon degeneration and axonal loss is a feature of neurodegenerative disease and injury and occurs via programmed pathways that are distinct from cell death pathways. While the pathways of axonal loss following axon severing are well described, less is known about axonal loss following other neurodegenerative insults. Here we use primary mouse cortical neuron cultures grown in compartmentalized chambers to investigate the role of calcium in the degeneration of axons that occurs following a somal insult by the excitotoxin kainic acid. Calcium influx has been implicated in both excitotoxicity and axon degeneration mechanisms, however the link between a somal insult and axonal calcium increase is unclear. Live imaging of axons demonstrated that pharmacologically preventing intracellular calcium increases through the endoplasmic reticulum or mitochondria significantly (p < 0.05) reduced axon degeneration. Live calcium-imaging with the Ca 2+ indicator Fluo-4 demonstrated that kainic acid exposure to the soma resulted in a rapid, and transient, increase in calcium in the axon, which occured even at low kainic acid concentrations that do not cause axon degeneration within 24 h. However, this calcium transient was followed by a gradual increase in axonal calcium, which was associated with axonal loss. Furthermore, treatment with a range of doses of the microtubule stabilizing drug taxol, which protects against axon fragmentation in this model, prevented this gradual calcium increase, suggesting that the intra-axonal calcium changes are downstream of microtubule associated events. Biochemical analysis of taxol treated neurons demonstrated a shift in microtubule post-translational modifications, with a significant (p < 0.05) increase in acetylated tubulin and a significant (p < 0.05) decrease in tyrosinated tubulin, suggestive of a more stable microtubule pool. Together our results suggest that axonal degeneration following excitotoxicity is dependent on an increase in axonal calcium, which is downstream of a microtubule-dependent event., (© 2019 International Society for Neurochemistry.)

Oxaliplatin is a widely used chemotherapeutic drug and represents the cornerstone of colorectal cancer therapy, in combination with 5-fluorouracil and folinic acid. As with many chemotherapeutic agents, its use is associated with a number of side effects, ranging from hypersensitivity reactions to haematological dyscrasias. Oxaliplatin also induces acute and chronic peripheral neuropathy. While it is likely that the haematological side effects are associated with its anti-proliferative effects and with the ability to form DNA adducts, the molecular mechanisms underlying peripheral neuropathy and hypersensitivity reactions are poorly understood, and therefore the choice of adequate supportive therapies is largely empirical. Here we show that an acute low dose oxaliplatin application on DRG neurons is able to induce an increase in intracellular calcium that is dependent on the Histamine 1 receptor (H1). Oxaliplatin-induced intracellular calcium rises are blocked by two selective H1 antagonist, as well as by U73122, a PLC inhibitor, and by 2-APB, a non-specific IP 3 receptor blocker. Moreover, expression of the H1 receptor on HEK293 t cells unmasks an oxaliplatin-induced Ca 2+ -rise. Last, activation of H1 via either histamine or oxaliplatin activates TRPV1 receptors, a mechanism that has been associated with itch. These data, together with literature data that has shown that anti-histamine agents reduce the incidence of oxaliplatin-induced hypersensitivity, may provide a molecular mechanism of this side effect in oncological patients., Competing Interests: Declaration of Competing Interest The authors declares no conflict of interest., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Type I interferons are effector cytokines essential for the regulation of the innate immunity. A key effector of the type I interferon response that is dysregulated in autoimmunity and cancer is the cGAS-STING signalling axis. Recent work suggests that calcium and associated signalling proteins can regulate both cGAS-STING and autoimmunity. How calcium regulates STING activation is complex and involves both stimulatory and inhibitory mechanisms. One of these is calmodulin-mediated signalling that is necessary for STING activation. The alterations in calcium flux that occur during STING activation can also regulate autophagy, which in turn plays a role in innate immunity through the clearance of intracellular pathogens. Also connected to calcium signalling pathways is the cGAS inhibitor TREX1, a cytoplasmic exonuclease linked to several autoimmune diseases including systemic lupus erythematosus (SLE). In this review, we summarize these and other findings that indicate a regulatory role for calcium signalling in innate and autoimmunity through the cGAS-STING pathway., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The past two decades have seen the identification of important roles for calcium signalling in many of the hallmarks of cancer. One of the cancer types that has been a particular focus of such studies is breast cancer. The breast is intrinsically linked to the calcium ion due to the importance of milk calcium in neonatal growth and development. Indeed, some of the calcium channels and pumps involved in transporting calcium ions into milk also have altered expression in some breast cancers. However, altered expression is not confined to channels and pumps important in lactation, other calcium channels and pumps may also be modulated and may even be specific to breast cancer molecular subtypes. This review considers calcium signalling in the context of breast cancer and provides an overview of the roles that have been attributed to specific regulators of cellular calcium levels in processes relevant to breast cancer progression. Emerging areas in the study of calcium signalling in breast cancer are considered, such as the intersection between calcium signalling, the tumour microenvironment and breast cancer., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Increasing crop yields by using ecofriendly practices is of high priority to tackle problems regarding food security and malnutrition worldwide. A sustainable crop production requires a limited use of fertilizer and the employment of plant varieties with improved ability to acquire nutrients from soil. To reach these goals, the scientific community aims to understand plant nutrients homeostasis by deciphering the nutrient sensing and signalling mechanisms of plants. Several lines of evidence about the involvement of Ca 2+ as the signal of an impaired nutrient availability have been reported. Ca 2+ signalling is a tightly regulated process that requires specific protein toolkits to perceive external stimuli and to induce the specific responses in the plant needed to survive. Here, we summarize both older and recent findings concerning the involvement of Ca 2+ signalling in the homeostasis of nutrients. In this review, we present new emerging technologies, based on the use of genetically encoded Ca 2+ sensors and advanced microscopy, which offer the chance to perform in planta analyses of Ca 2+ dynamics at cellular resolution. The harnessing of these technologies with different genetic backgrounds and subjected to different nutritional stresses will provide important insights to the still little-known mechanisms of nutrient sensing in plants., (© 2019 John Wiley & Sons Ltd.)

Twenty-five years have elapsed since the calcium-sensing receptor (CaSR) was first identified in bovine parathyroid and the receptor is now recognized as a fundamental contributor to extracellular Ca2+ (Ca2+ e) homeostasis, regulating parathyroid hormone release and urinary calcium excretion. The CaSR is a class C G-protein-coupled receptor (GPCR) that is functionally active as a homodimer and couples to multiple G-protein subtypes to activate intracellular signalling pathways. The importance of the CaSR in the regulation of Ca2+ e has been highlighted by the identification of >400 different germline loss- and gain-of-function CaSR mutations that give rise to disorders of Ca2+ e homeostasis. CaSR-inactivating mutations cause neonatal severe hyperparathyroidism, characterised by marked hypercalcaemia, skeletal demineralisation and failure to thrive in early infancy; and familial hypocalciuric hypercalcaemia, an often asymptomatic disorder associated with mild-moderately elevated serum calcium concentrations. Activating mutations are associated with autosomal dominant hypocalcaemia, which is occasionally associated with a Bartter's-like phenotype. Recent elucidation of the CaSR extracellular domain structure enabled the locations of CaSR mutations to be mapped and has revealed clustering in locations important for structural integrity, receptor dimerisation and ligand binding. Moreover, the study of disease-causing mutations has demonstrated that CaSR signals in a biased manner and have revealed specific residues important for receptor activation. This review presents the current understanding of the genetic landscape of CaSR mutations by summarising findings from clinical and functional studies of disease-associated mutations. It concludes with reflections on how recently uncovered signalling pathways may expand the understanding of calcium homeostasis disorders.

Calcium signalling is one of the most important mechanisms of information propagation in the body. In embryogenesis the interplay between calcium signalling and mechanical forces is critical to the healthy development of an embryo but poorly understood. Several types of embryonic cells exhibit calcium-induced contractions and many experiments indicate that calcium signals and contractions are coupled via a two-way mechanochemical feedback mechanism. We present a new analysis of experimental data that supports the existence of this coupling during apical constriction. We then propose a simple mechanochemical model, building on early models that couple calcium dynamics to the cell mechanics and we replace the hypothetical bistable calcium release with modern, experimentally validated calcium dynamics. We assume that the cell is a linear, viscoelastic material and we model the calcium-induced contraction stress with a Hill function, i.e. saturating at high calcium levels. We also express, for the first time, the "stretch-activation" calcium flux in the early mechanochemical models as a bottom-up contribution from stretch-sensitive calcium channels on the cell membrane. We reduce the model to three ordinary differential equations and analyse its bifurcation structure semi-analytically as two bifurcation parameters vary-the [Formula: see text] concentration, and the "strength" of stretch activation, [Formula: see text]. The calcium system ([Formula: see text], no mechanics) exhibits relaxation oscillations for a certain range of [Formula: see text] values. As [Formula: see text] is increased the range of [Formula: see text] values decreases and oscillations eventually vanish at a sufficiently high value of [Formula: see text]. This result agrees with experimental evidence in embryonic cells which also links the loss of calcium oscillations to embryo abnormalities. Furthermore, as [Formula: see text] is increased the oscillation amplitude decreases but the frequency increases. Finally, we also identify the parameter range for oscillations as the mechanical responsiveness factor of the cytosol increases. This work addresses a very important and not well studied question regarding the coupling between chemical and mechanical signalling in embryogenesis.

Key Points: Sympathetic outflow and circulating glucogenic hormones both regulate liver function by increasing cytosolic calcium, although how these calcium signals are integrated at the tissue level is currently unknown. We show that stimulation of hepatic nerve fibres or perfusing the liver with physiological concentrations of vasopressin only will evoke localized cytosolic calcium oscillations and modest increases in hepatic glucose production. The combination of these stimuli acted synergistically to convert localized and asynchronous calcium responses into co-ordinated intercellular calcium waves that spread throughout the liver lobule and elicited a synergistic increase in hepatic glucose production. The results obtained in the present study demonstrate that subthreshold levels of one hormone can create an excitable medium across the liver lobule, which allows global propagation of calcium signals in response to local sympathetic innervation and integration of metabolic regulation by multiple hormones. This enables the liver lobules to respond as functional units to produce full-strength metabolic output at physiological levels of hormone., Abstract: Glucogenic hormones, including catecholamines and vasopressin, induce frequency-modulated cytosolic Ca 2+ oscillations in hepatocytes, and these propagate as intercellular Ca 2+ waves via gap junctions in the intact liver. We investigated the role of co-ordinated Ca 2+ waves as a mechanism for integrating multiple endocrine and neuroendocrine inputs to control hepatic glucose production in perfused rat liver. Sympathetic nerve stimulation elicited localized Ca 2+ increases that were restricted to hepatocytes in the periportal zone. During perfusion with subthreshold vasopressin, sympathetic stimulation converted asynchronous Ca 2+ signals in a limited number of hepatocytes into co-ordinated intercellular Ca 2+ waves that propagated across entire lobules. A similar synergism was observed between physiological concentrations of glucagon and vasopressin, where glucagon also facilitated the recruitment of hepatocytes into a Ca 2+ wave. Hepatic glucose production was significantly higher with intralobular Ca 2+ waves. We propose that inositol 1,4,5-trisphosphate (IP 3 )-dependent Ca 2+ signalling gives rise to an excitable medium across the functional syncytium of the hepatic lobule, co-ordinating and amplifying the metabolic responses to multiple hormonal inputs., (© 2019 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Key Points: A computational model of P2X channel activation in microglia was developed that includes downfield Ca 2+ -dependent signalling pathways. This model provides quantitative insights into how diverse signalling pathways in microglia converge to control microglial function., Abstract: Microglia function is orchestrated through highly coupled signalling pathways that depend on calcium (Ca 2+ ). In response to extracellular ATP, transient increases in intracellular Ca 2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis. Although the steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic purinergic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumour necrosis factor α (TNFα) cytokine. In addition to Ca 2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P2X4 and P2X7 activation, activation of nuclear factor of activated T-cells (NFAT) transcription factors, and TNFα production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNFα production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNFα production. Our results suggest that pulsatile stimulation of P2X4 via micromolar ATP may be sufficient to promote TNFα production, whereas high-amplitude ATP exposure is necessary for production via P2X7. Furthermore, under conditions that increase P2X4 expression, for instance, following activation by pathogen-associated molecular factors, P2X4-associated TNFα production is greatly enhanced. Given that Ca 2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)