Polycystin-2 Mediated Calcium Signalling in the Dictyostelium Model for Autosomal Dominant Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) occurs when the proteins Polycystin-1 (PC1, PKD1) and Polycystin-2 (PC2, PKD2) contain mutations. PC1 is a large membrane receptor that can interact and form a complex with the calcium-permeable cation channel PC2. This complex localizes to the plasma membrane, primary cilia and ER. Dysregulated calcium signalling and consequential alterations in downstream signalling pathways in ADPKD are linked to cyst formation and expansion; however, it is not completely understood how PC1 and PC2 regulate calcium signalling. We have studied Polycystin-2 mediated calcium signalling in the model organism Dictyostelium discoideum by overexpressing and knocking down the expression of the endogenous Polycystin-2 homologue, Polycystin-2. Chemoattractant-stimulated cytosolic calcium response magnitudes increased and decreased in overexpression and knockdown strains, respectively, and analysis of the response kinetics indicates that Polycystin-2 is a significant contributor to the control of Ca2+ responses. Furthermore, basal cytosolic calcium levels were reduced in Polycystin-2 knockdown transformants. These alterations in Ca2+ signalling also impacted other downstream Ca2+-sensitive processes including growth rates, endocytosis, stalk cell differentiation and spore viability, indicating that Dictyostelium is a useful model to study Polycystin-2 mediated calcium signalling.

Exploring the intricacies of calcium dysregulation in ischemic stroke: Insights into neuronal cell death and therapeutic strategies.

Calcium ion (Ca2+) dysregulation is one of the main causes of neuronal cell death and brain damage after cerebral ischemia. During ischemic stroke, the ability of neurons to maintain Ca2+ homeostasis is compromised. Ca2+ regulates various functions of the nervous system, including neuronal activity and adenosine triphosphate (ATP) production. Disruptions in Ca2+ homeostasis can trigger a cascade of events, including activation of the unfolded protein response (UPR) pathway, which is associated with endoplasmic reticulum (ER) stress and mitochondrial dysfunction. This response occurs when the cell is unable to manage protein folding within the ER due to various stressors, such as a high influx of Ca2+. Consequently, the UPR is initiated to restore ER function and alleviate stress, but prolonged activation can lead to mitochondrial dysfunction and, ultimately, cell death. Hence, precise regulation of Ca2+ within the cell is mandatory. The ER and mitochondria are two such organelles that maintain intracellular Ca2+ homeostasis through various calcium-operating channels, including ryanodine receptors (RyRs), inositol trisphosphate receptors (IP3Rs), sarco/endoplasmic reticulum calcium ATPases (SERCAs), the mitochondrial Na+/Ca2+ exchanger (NCLX), the mitochondrial calcium uniporter (MCU) and voltage-dependent anion channels (VDACs). These channels utilize Ca2+ sequestering and release mechanisms to maintain intracellular Ca2+ homeostasis and ensure proper cellular function and survival. The present review critically evaluates the significance of Ca2+ and its physiological role in cerebral ischemia. We have compiled recent findings on calcium's role and emerging treatment strategies, particularly targeting mitochondria and the endoplasmic reticulum, to address Ca2+ overload in cerebral ischemia.

Calcium signaling in endothelial and vascular smooth muscle cells: sex differences and the influence of estrogens and androgens.

Calcium signaling in vascular endothelial cells (ECs) and smooth muscle cells (VSMCs) is essential for the regulation of vascular tone. However, the changes to intracellular Ca2+ concentrations are often influenced by sex differences. Furthermore, a large body of evidence shows that sex hormone imbalance leads to dysregulation of Ca2+ signaling and this is a key factor in the pathogenesis of cardiovascular diseases. In this review, the effects of estrogens and androgens on vascular calcium-handling proteins are discussed, with emphasis on the associated genomic or nongenomic molecular mechanisms. The experimental models from which data were collected were also considered. The review highlights 1) in female ECs, transient receptor potential vanilloid 4 (TRPV4) and mitochondrial Ca2+ uniporter (MCU) enhance Ca2+-dependent nitric oxide (NO) generation. In males, only transient receptor potential canonical 3 (TRPC3) plays a fundamental role in this effect. 2) Female VSMCs have lower cytosolic Ca2+ levels than males due to differences in the activity and expression of stromal interaction molecule 1 (STIM1), calcium release-activated calcium modulator 1 (Orai1), calcium voltage-gated channel subunit-α1C (CaV1.2), Na+-K+-2Cl- symporter (NKCC1), and the Na+/K+-ATPase. 3) When compared with androgens, the influence of estrogens on Ca2+ homeostasis, vascular tone, and incidence of vascular disease is better documented. 4) Many studies use supraphysiological concentrations of sex hormones, which may limit the physiological relevance of outcomes. 5) Sex-dependent differences in Ca2+ signaling mean both sexes ought to be included in experimental design.

Model of Calcium Dynamics Regulating [Formula: see text], ATP and Insulin Production in a Pancreatic [Formula: see text]-Cell.

The calcium signals regulate the production and secretion of many signaling molecules like inositol trisphosphate ([Formula: see text]) and adenosine triphosphate (ATP) in various cells including pancreatic [Formula: see text]-cells. The calcium signaling mechanisms regulating [Formula: see text], ATP and insulin responsible for various functions of [Formula: see text]-cells are still not well understood. Any disturbance in these mechanisms can alter the functions of [Formula: see text]-cells leading to diabetes and metabolic disorders. Therefore, a mathematical model is proposed by incorporating the reaction-diffusion equation for calcium dynamics and a system of first-order differential equations for [Formula: see text], ATP-production and insulin secretion with initial and boundary conditions. The model incorporates the temporal dependence of [Formula: see text]-production and degradation, ATP production and insulin secretion on calcium dynamics in a [Formula: see text]-cell. The piecewise linear finite element method has been used for the spatial dimension and the Crank-Nicolson scheme for the temporal dimension to obtain numerical results. The effect of changes in source influxes and buffers on calcium dynamics and production of [Formula: see text], ATP and insulin levels in a [Formula: see text]-cell has been analyzed. It is concluded that the dysfunction of source influx and buffers can cause significant variations in calcium levels and dysregulation of [Formula: see text], ATP and insulin production, which can lead to various metabolic disorders, diabetes, obesity, etc. The proposed model provides crucial information about the changes in mechanisms of calcium dynamics causing proportionate disturbances in [Formula: see text], ATP and insulin levels in pancreatic cells, which can be helpful for devising protocols for diagnosis and treatment of various metabolic diseases.

HERPUD1 governs tumor cell mitochondrial function via inositol 1,4,5-trisphosphate receptor-mediated calcium signaling.

The intricate relationship between calcium (Ca2+) homeostasis and mitochondrial function is crucial for cellular metabolic adaptation in tumor cells. Ca2+-initiated signaling maintains mitochondrial respiratory capacity and ATP synthesis, influencing critical cellular processes in cancer development. Previous studies by our group have shown that the homocysteine-inducible ER Protein with Ubiquitin-Like Domain 1 (HERPUD1) regulates inositol 1,4,5-trisphosphate receptor (ITPR3) levels and intracellular Ca2+ signals in tumor cells. This study explores the role of HERPUD1 in regulating mitochondrial function and tumor cell migration by controlling ITPR3-dependent Ca2+ signals. We found HERPUD1 levels correlated with mitochondrial function in tumor cells, with HERPUD1 deficiency leading to enhanced mitochondrial activity. HERPUD1 knockdown increased intracellular Ca2+ release and mitochondrial Ca2+ influx, which was prevented using the ITPR3 antagonist xestospongin C or the Ca2+ chelator BAPTA-AM. Furthermore, HERPUD1 expression reduced tumor cell migration by controlling ITPR3-mediated Ca2+ signals. HERPUD1-deficient cells exhibited increased migratory capacity, which was attenuated by treatment with xestospongin C or BAPTA-AM. Additionally, HERPUD1 deficiency led to reactive oxygen species-dependent activation of paxillin and FAK proteins, which are associated with enhanced cell migration. Our findings highlight the pivotal role of HERPUD1 in regulating mitochondrial function and cell migration by controlling intracellular Ca2+ signals mediated by ITPR3. Understanding the interplay between HERPUD1 and mitochondrial Ca2+ regulation provides insights into potential therapeutic targets for cancer treatment and other pathologies involving altered energy metabolism.

Orai1 Ca2+ channel modulators as therapeutic tools for treating cancer: Emerging evidence!

In non-excitable cells, Orai proteins represent the main channel for Store-Operated Calcium Entry (SOCE), and also mediate various store-independent Calcium Entry (SICE) pathways. Deregulation of these pathways contribute to increased tumor cell proliferation, migration, metastasis, and angiogenesis. Among Orais, Orai1 is an attractive therapeutic target explaining the development of specific modulators. Therapeutic trials using Orai1 channel inhibitors have been evaluated for treating diverse diseases such as psoriasis and acute pancreatitis, and emerging data suggest that Orai1 channel modulators may be beneficial for cancer treatment. This review discusses herein the importance of Orai1 channel modulators as potential therapeutic tools and the added value of these modulators for treating cancer.

Store-operated calcium entry inhibits primary ciliogenesis via the activation of Aurora A.

The primary cilium is an antenna-like organelle protruding from the cell surface that can detect physical and chemical stimuli in the extracellular space to activate specific signaling pathways and downstream gene expressions. Calcium ion (Ca2+ ) signaling regulates a wide spectrum of cellular processes, including fertilization, proliferation, differentiation, muscle contraction, migration, and death. This study investigated the effects of the regulation of cytosolic Ca2+ levels on ciliogenesis using chemical, genetic, and optogenetic approaches. We found that ionomycin-induced Ca2+ influx inhibited ciliogenesis and Ca2+ chelator BATPA-AM-induced Ca2+ depletion promoted ciliogenesis. In addition, store-operated Ca2+ entry and the endoplasmic reticulum Ca2+ sensor stromal interaction molecule 1 (STIM1) negatively regulated ciliogenesis. Moreover, an optogenetic platform was used to create different Ca2+ oscillation patterns by manipulating lighting parameters, including density, frequency, exposure time, and duration. Light-activated Ca2+ -translocating channelrhodopsin (CatCh) is activated by 470-nm blue light to induce Ca2+ influx. Our results show that high-frequency Ca2+ oscillations decrease ciliogenesis. Furthermore, the inhibition of cilia formation induced by Ca2+ may occur via the activation of Aurora kinase A. Cilia not only induce Ca2+ signaling but also regulate cilia formation by Ca2+ signaling.

Development of chemical tools based on GSK-7975A to study store-operated calcium entry in cells.

Many physiological functions, such as cell differentiation, proliferation, muscle contraction, neurotransmission and fertilisation, are regulated by changes of Ca2+ levels. The major Ca2+ store in cells is the endoplasmic reticulum (ER). Certain cellular processes induce ER store depletion, e.g. by activating IP3 receptors, that in turn induces a store refilling process known as store-operated calcium entry (SOCE). This refilling process entails protein-protein interactions between Ca2+ sensing stromal interaction molecules (STIM) in the ER membrane and Orai proteins in the plasma membrane. Fully assembled STIM/Orai complexes then form highly selective Ca2+ channels called Ca2+ release-activated Ca2+ Channels (CRAC) through which Ca2+ ions flow into the cytosol and subsequently are pumped into the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Abnormal SOCE has been associated with numerous human diseases and cancers, and therefore key players STIM and Orai have attracted significant therapeutic interest. Several potent experimental and clinical candidate compounds have been developed and have helped to study SOCE in various cell types. We have synthesized multiple novel small-molecule probes based on the known SOCE inhibitor GSK-7975A. Here we present GSK-7975A derivatives, which feature photo-caging, photo-crosslinking, biotin and clickable moieties, and also contain deuterium labels. Evaluation of these GSK-7975A probes using a fluorometric imaging plate reader (FLIPR)-Tetra-based Ca2+ imaging assay showed that most synthetic modifications did not have a detrimental impact on the SOCE inhibitory activity. The photo-caged GSK-7975A was also used in patch-clamp electrophysiology experiments. In summary, we have developed a number of active, GSK-7975A-based molecular probes that have interesting properties and therefore are useful experimental tools to study SOCE in various cells and settings.

Sending positive signals and good (calcium) vibes.

In this issue of JEM, Yada et al. (https://doi.org/10.1084/jem.20222178) demonstrate that effective antibody affinity selection in germinal centers relies on the store-operated calcium entry (SOCE) component of the B cell receptor (BCR) signaling network. Therefore, active BCR signaling is as relevant to positive selection as the function of BCRs as endocytic receptors, answering a question that had puzzled experts for a while. These findings transform our understanding of the mechanisms supporting adaptive immune responses (to vaccines, for example) and have important implications for interpreting the genomics and pathogenesis of germinal center-derived B cell lymphomas.

Cracking the Endothelial Calcium (Ca2+) Code: A Matter of Timing and Spacing.

A monolayer of endothelial cells lines the innermost surface of all blood vessels, thereby coming into close contact with every region of the body and perceiving signals deriving from both the bloodstream and parenchymal tissues. An increase in intracellular Ca2+ concentration ([Ca2+]i) is the main mechanism whereby vascular endothelial cells integrate the information conveyed by local and circulating cues. Herein, we describe the dynamics and spatial distribution of endothelial Ca2+ signals to understand how an array of spatially restricted (at both the subcellular and cellular levels) Ca2+ signals is exploited by the vascular intima to fulfill this complex task. We then illustrate how local endothelial Ca2+ signals affect the most appropriate vascular function and are integrated to transmit this information to more distant sites to maintain cardiovascular homeostasis. Vasorelaxation and sprouting angiogenesis were selected as an example of functions that are finely tuned by the variable spatio-temporal profile endothelial Ca2+ signals. We further highlighted how distinct Ca2+ signatures regulate the different phases of vasculogenesis, i.e., proliferation and migration, in circulating endothelial precursors.

The ER stress sensor IRE1 interacts with STIM1 to promote store-operated calcium entry, T cell activation, and muscular differentiation.

Store-operated Ca2+ entry (SOCE) mediated by stromal interacting molecule (STIM)-gated ORAI channels at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites maintains adequate levels of Ca2+ within the ER lumen during Ca2+ signaling. Disruption of ER Ca2+ homeostasis activates the unfolded protein response (UPR) to restore proteostasis. Here, we report that the UPR transducer inositol-requiring enzyme 1 (IRE1) interacts with STIM1, promotes ER-PM contact sites, and enhances SOCE. IRE1 deficiency reduces T cell activation and human myoblast differentiation. In turn, STIM1 deficiency reduces IRE1 signaling after store depletion. Using a CaMPARI2-based Ca2+ genome-wide screen, we identify CAMKG2 and slc105a as SOCE enhancers during ER stress. Our findings unveil a direct crosstalk between SOCE and UPR via IRE1, acting as key regulator of ER Ca2+ and proteostasis in T cells and muscles. Under ER stress, this IRE1-STIM1 axis boosts SOCE to preserve immune cell functions, a pathway that could be targeted for cancer immunotherapy.

Orai1α and Orai1ß support calcium entry and mammosphere formation in breast cancer stem cells.

Orai1 is the pore-forming subunit of the Ca2+-release activated Ca2+ channels that mediate store-operated Ca2+ entry (SOCE) in excitable and non-excitable cells. Two Orai1 forms have been identified in mammalian cells, the full-length variant Orai1α, and the short form Orai1ß, lacking the N-terminal 63 amino acids. Stem cells were isolated from non-tumoral breast epithelial cells of the MCF10A cell line, and the most representative ER+ , HER2 or triple negative breast cancer cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Orai and TRPC family members expression was detected by RT-PCR and Western blotting. Changes in cytosolic Ca2+ concentration were analyzed by confocal microscopy using Fluo 4 and the spheroid-forming ability and self-renewal was estimated in culture plates coated with pHEMA using a cell imaging system. Here, we have characterized the expression of Orai family members and several TRPC channels at the transcript level in breast stem cells (BSC) derived from the non-tumoral breast epithelial cell line MCF10A and breast cancer stem cells (BCSC) derived from the well-known estrogen receptor positive (ER+), HER2 and triple negative cell lines MCF7, SKBR3 and MDA-MB-231, respectively. Furthermore, we have evaluated the mammosphere formation efficiency and self-renewal of the BSC and BCSC. Next, through a combination of Orai1 knockdown by iRNA and the use of MDA-MB-231 KO cells, missing the native Orai1, transfected with plasmids encoding for either Orai1α or Orai1ß, we show that Orai1 is essential for mammosphere formation and self-renewal efficiency in BCSC derived from triple negative and HER2 subtypes cell cultures, while this channel has a negligible effect in BCSC derived from ER+ cells as well as in non-tumoral BSC. Both, Orai1α, and Orai1ß support SOCE in MDA-MB-231-derived BCSC with similar efficiency, as well as COX activation and mammosphere formation. These findings provide evidence of the functional role of Orai1α and Orai1ß in spheroid forming efficiency and self-renewal in breast cancer stem cells.

AAV-compatible optogenetic tools for activating endogenous calcium channels in vivo.

Calcium ions (Ca2+) play pivotal roles in regulating diverse brain functions, including cognition, emotion, locomotion, and learning and memory. These functions are intricately regulated by a variety of Ca2+-dependent cellular processes, encompassing synaptic plasticity, neuro/gliotransmitter release, and gene expression. In our previous work, we developed 'monster OptoSTIM1' (monSTIM1), an improved OptoSTIM1 that selectively activates Ca2+-release-activated Ca2+ (CRAC) channels in the plasma membrane through blue light, allowing precise control over intracellular Ca2+ signaling and specific brain functions. However, the large size of the coding sequence of monSTIM1 poses a limitation for its widespread use, as it exceeds the packaging capacity of adeno-associated virus (AAV). To address this constraint, we have introduced monSTIM1 variants with reduced coding sequence sizes and established AAV-based systems for expressing them in neurons and glial cells in the mouse brain. Upon expression by AAVs, these monSTIM1 variants significantly increased the expression levels of cFos in neurons and astrocytes in the hippocampal CA1 region following non-invasive light illumination. The use of monSTIM1 variants offers a promising avenue for investigating the spatiotemporal roles of Ca2+-mediated cellular activities in various brain functions. Furthermore, this toolkit holds potential as a therapeutic strategy for addressing brain disorders associated with aberrant Ca2+ signaling.

Computational insight into energy control balance by Ca2+ and cAMP-PKA signaling in pacemaker cells.

Cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling controls sinoatrial node cell (SANC) function by affecting the degree of coupling between Ca2+ and membrane clocks. PKA is known to phosphorylate ionic channels, Ca2+ pump and release from the sarcoplasmic reticulum, and enzymes controlling ATP production in the mitochondria. While the PKA cytosolic targets in SANC have been extensively explored, its mitochondrial targets and its ability to maintain SANC energetic balance remain to be elucidated. To investigate the role of PKA in SANC energetics, we tested three hypotheses: (i) PKA is an important regulator of the ATP supply-to-demand balance, (ii) Ca2+ regulation of energetics is important for maintenance of NADH level and (iii) abrupt reduction in ATP demand first reduces the AP firing rate and, after dropping below a certain threshold, leads to a reduction in ATP. To gain mechanistic insights into the ATP supply-to-demand matching regulators, a modified model of mitochondrial energy metabolism was integrated into our coupled-clock model that describes ATP demand. Experimentally, increased ATP demand was accompanied by maintained ATP and NADH levels. Ca2+ regulation of energetics was found by the model to be important in the maintenance of NADH and PKA regulation was found to be important in the maintenance of intracellular ATP and the increase in oxygen consumption. PKA inhibition led to a biphasic reduction in AP firing rate, with the first phase being rapid and ATP-independent, while the second phase was slow and ATP-dependent. Thus, SANC energy balance is maintained by both Ca2+ and PKA signaling.

Physiological functions of calcium signaling via Orai1 in cancer.

Intracellular calcium (Ca2+) signaling regulates many cellular functions, including cell proliferation and migration, in both normal cells and cancer cells. Store-operated Ca2+ entry (SOCE) is a major mechanism by which Ca2+ is imported from the extracellular space to the intracellular space, especially in nonexcitable cells. Store-operated Ca2+ entry (SOCE) is also a receptor-regulated Ca2+ entry pathway that maintains Ca2+ homeostasis by sensing reduced Ca2+ levels in the endoplasmic reticulum (ER). In general, the activation of G protein-coupled receptors (GPCRs) or immunoreceptors, such as T-cell, B-cell and Fc receptors, results in the production of inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors located in the ER membrane. The, IP3 receptors in the ER membrane trigger a rapid and transient release of Ca2+ from the ER store. The resulting depletion of ER Ca2+ concentrations is sensed by the EF-hand motif of stromal interaction molecule (STIM), i.e., calcium sensor, which then translocates to the plasma membrane (PM). STIM interacts with Orai Ca2+ channel subunits (also known as CRACM1) on the PM, leading to Ca2+ influx from the extracellular space to increase intracellular Ca2+ concentrations. The physiological functions of Orai and STIM have been studied mainly with respect to their roles in the immune system. Based on numerous previous studies, Orai channels (Orai1, Orai2 and Orai3 channels) control Ca2+ release-activated Ca2+ (CRAC) currents and contribute to SOCE currents in other types of cells, including various cancer cells. There are many reports that Orai1 is involved in cell proliferation, migration, metastasis, apoptosis and epithelial-mesenchymal transition (EMT) in various cancers. We previously found that Orai1 plays important roles in cell apoptosis and migration in melanoma. Recently, we reported novel evidence of Orai1 in human oral squamous cell carcinoma (OSCC) cells and human cardiac fibroblasts (HCFs). In this review, we present multiple physiological functions of Orai1 in various cancer cells and cardiac fibroblasts, including our findings.

Fine tuning calcium dynamics by inhibition of Store-operated Calcium Entry as a novel therapeutic approach for the treatment of chronic pancreatitis.

Chronic pancreatitis (CP) is a complex inflammatory disorder characterized by progressive fibrosis, leading to pancreatic dysfunction, reduced quality of life and an elevated pancreatic cancer risk. Current therapeutic options for CP are restricted to symptomatic treatment. Using ex vivo and in vivo preclinical disease models, Szabó et al. now explored for the first time the involvement of Store-operated Ca2+ entry (SOCE) in the progression of CP and propose that a selective pharmacological inhibition of the SOCE signaling component Orai1 might serve as specific treatment option for CP[1,2].

Numerical simulation of calcium dynamics dependent ATP degradation, IP3 and NADH production due to obesity in a hepatocyte cell.

Calcium (Ca[Formula: see text]) signals have a crucial role in regulating various processes of almost every cell to maintain its structure and function. Calcium dynamics has been studied in various cells including hepatocytes by many researchers, but the mechanisms of calcium signals involved in regulation and dysregulation of various processes like ATP degradation rate, IP[Formula: see text] and NADH production rate respectively in normal and obese cells are still poorly understood. In this paper, a reaction diffusion equation of calcium is employed to propose a model of calcium dynamics by coupling ATP degradation rate, IP[Formula: see text] and NADH production rate in hepatocyte cells under normal and obese conditions. The processes like source influx, buffer, endoplasmic reticulum (ER), mitochondrial calcium uniporters (MCU) and Na[Formula: see text]/Ca[Formula: see text] exchanger (NCX) have been incorporated in the model. Linear finite element method is used along spatial dimension, and Crank-Nicolson method is used along temporal dimension for numerical simulation. The results have been obtained for the normal hepatocyte cells and for cells due to obesity. The comparative study of these results reveal significant difference caused due to obesity in Ca[Formula: see text] dynamics as well as in ATP degradation rate, IP[Formula: see text] and NADH production rate.

Simulation of biochemical dynamics of [Formula: see text] and [Formula: see text] in fibroblast cell.

Calcium dynamics is not only responsible for maintaining the framework and functions of the cell but also plays a role in the dynamics of other biochemical systems in the cell. Phospholipase C-[Formula: see text] l ([Formula: see text]) has a crucial role in the function of fibroblast cells. Experiments have shown that [Formula: see text] and [Formula: see text] have interdependent dynamics in fibroblast cells. However, no reaction-diffusion model exists for the two-way feedback system dynamics of [Formula: see text] and [Formula: see text] in fibroblasts till date. The computational model is designed to investigate the impact of variations in several processes, such as the [Formula: see text] pump, buffer process, source inflow, etc., on the system dynamics of [Formula: see text] and [Formula: see text] in fibroblast cells. The computational findings are obtained using finite element techniques, and the consequences of dysregulation in various processes on the spatiotemporal calcium and [Formula: see text] dynamics in fibroblasts are investigated. The results lead to the conclusion that the effects of buffer, source influx, diffusion, and [Formula: see text] pump can cause fluctuations in the dynamics of [Formula: see text] and [Formula: see text] in fibroblasts. Disruptions in these constitutive processes can result in changes in the dynamics of calcium and [Formula: see text]. Thus, the current model provides new/novel information regarding the precise dysregulatory constitutive systems that regulate calcium and [Formula: see text] kinetics, such as source inflow, diffusion, [Formula: see text], and buffer, can be responsible for excessive calcium and [Formula: see text] concentrations leading to fibrotic illnesses such as cancer and fibrosis.

Functional Interrogation of Ca2+ Signals in Human Cancer Cells In Vitro and Ex Vivo by Fluorescent Microscopy and Molecular Tools.

Genetically encoded calcium indicators (GECIs) and high-resolution confocal microscopy enable dynamic visualization of calcium signals in cells and tissues. Two-dimensional and 3D biocompatible materials mimic the mechanical microenvironments of tumor and healthy tissues in a programmable manner. Cancer xenograft models and ex vivo functional imaging of tumor slices reveal physiologically relevant functions of calcium dynamics in tumors at different progression stages. Integration of these powerful techniques allows us to quantify, diagnose, model, and understand cancer pathobiology. Here, we describe detailed materials and methods used to establish this integrated interrogation platform, from generating transduced cancer cell lines that stably express CaViar (GCaMP5G + QuasAr2) to in vitro and ex vivo calcium imaging of the cells in 2D/3D hydrogels and tumor tissues. These tools open the possibility for detailed explorations of mechano-electro-chemical network dynamics in living systems.

Astrocyte Calcium Signaling Shifts the Polarity of Presynaptic Plasticity.

Astrocytes have been increasingly acknowledged to play active roles in regulating synaptic transmission and plasticity. Through a variety of metabotropic and ionotropic receptors expressed on their surface, astrocytes detect extracellular neurotransmitters, and in turn, release gliotransmitters to modify synaptic strength, while they can also alter neuronal membrane excitability by modulating extracellular ionic milieu. Given the seemingly large repertoire of synaptic modulation, when, where and how astrocytes interact with synapses remain to be fully understood. Previously, we have identified a role for astrocyte NMDA receptor and L-VGCCs signaling in heterosynaptic presynaptic plasticity and promoting the heterogeneity of presynaptic strengths at hippocampal synapses. Here, we have sought to further clarify the mode by which astrocytes regulate presynaptic plasticity by exploiting a reduced culture system to globally evoke NMDA receptor-dependent presynaptic plasticity. Recording from a postsynaptic neuron intracellularly loaded with BAPTA, briefly bath applying NMDA and glycine induces a stable decrease in the rate of spontaneous glutamate release, which requires the presence of astrocytes and the activation of A1 adenosine receptors. Upon preventing astrocyte calcium signaling or blocking L-VGCCs, NMDA + glycine application triggers an increase, rather than a decrease, in the rate of spontaneous glutamate release, thereby shifting the presynaptic plasticity to promote an increase in strength. Our findings point to a crucial and surprising role of astrocytes in controlling the polarity of NMDA receptor and adenosine-dependent presynaptic plasticity. Such a pivotal mechanism unveils the power of astrocytes in regulating computations performed by neural circuits and is expected to profoundly impact cognitive processes.