Intercellular Calcium Signaling Research Articles

Induced Pluripotent Stem Cell-Derived Parathyroid Organoids Resemble Parathyroid Morphology and Function.

The primary role of the parathyroid glands is to maintain calcium homeostasis through the secretion of parathyroid hormone (PTH). The limited proliferative capacity and differentiation of parathyroid cells hinder the generation of cell therapy options. In this study, parathyroid organoids are successfully generated from human-induced pluripotent stem cells (hiPSCs). At the end of the 20 days of differentiation, the parathyroid organoids exhibited distinct parathyroid morphology. Stereomicroscope, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) analysis demonstrated the 3D arrangement of the cell layers in which intracellular structures of parathyroid cells resemble human parathyroid cellular morphology. Comprehensive molecular analyses, including RNA sequencing (RNA-Seq) and liquid chromatography/mass spectrometry (LC-MS/MS), confirmed the expression of key parathyroid-related markers. Protein expression of CasR, CxCr4, Gcm2, and PTH are observed in parathyroid organoids. Parathyroid organoids secrete PTH, demonstrate active intercellular calcium signaling, and induce osteogenic differentiation via their secretome. The tissue integration potential of parathyroid organoids is determined by transplantation into parathyroidectomized rats. The organoid transplanted animals showed significant elevations in PTH-related markers (CasR, CxCr4, Foxn1, Gcm2, and PTH). PTH secretion is detected in organoid-transplanted animals. The findings represent a significant advancement in parathyroid organoid culture and may offer a cellular therapy for treating PTH-related diseases, including hypoparathyroidism.

Post-gastrulation transition from whole-body to tissue-specific intercellular calcium signaling in the appendicularian tunicate Oikopleuradioica

We recently described calcium signaling in the appendicularian tunicate Oikopleura dioica during pre-gastrulation stages, and showed that regularly occurring calcium waves progress throughout the embryo in a characteristic spatiotemporal pattern from an initiation site in muscle lineage blastomeres. Here, we have extended our observations to the period spanning from gastrulation to post-hatching stages. We find that repetitive Ca2+ waves persist throughout this developmental window, albeit with a gradual increase in frequency. The initiation site of the waves shifts from muscle cells at gastrulation and early tailbud stages, to the central nervous system at late tailbud and post-hatching stages, indicating a transition from muscle-driven to neurally driven events as tail movements emerge. At these later stages, both the voltage gated Na+ ​channel blocker tetrodotoxin (TTX) and the T-type Ca2+ channel blocker and nAChR antagonist mecamylamine eliminate tail movements. At late post-hatching stages, mecamylamine blocks Ca2+ signals in the muscles but not the central nervous system. Post-gastrulation Ca2+ signals also arise in epithelial cells, first in a haphazard pattern in scattered cells during tailbud stages, evolving after hatching into repetitive rostrocaudal waves with a different frequency than the nervous system-to-muscle waves, and insensitive to mecamylamine. The desynchronization of Ca2+ waves arising in different parts of the body indicates a shift from whole-body to tissue/organ-specific Ca2+ signaling dynamics as organogenesis occurs, with neurally driven Ca2+ signaling dominating at the later stages when behavior emerges.

Encoding, transmission, decoding, and specificity of calcium signals in plants.

Calcium acts as a signal and transmits information in all eukaryotes. Encoding machinery consisting of calcium channels, stores, buffers, and pumps can generate a variety of calcium transients in response to external stimuli, thus shaping the calcium signature. Mechanisms for the transmission of calcium signals have been described, and a large repertoire of calcium binding proteins exist that can decode calcium signatures into specific responses. Whilst straightforward as a concept, mysteries remain as to exactly how such information processing is biochemically implemented. Novel developments in imaging technology and genetically encoded sensors (such as calcium indicators), in particular for multi-signal detection, are delivering exciting new insights into intra- and intercellular calcium signaling. Here, we review recent advances in characterizing the encoding, transmission, and decoding mechanisms, with a focus on long-distance calcium signaling. We present technological advances and computational frameworks for studying the specificity of calcium signaling, highlight current gaps in our understanding and propose techniques and approaches for unravelling the underlying mechanisms.

From spikes to intercellular waves: Tuning intercellular calcium signaling dynamics modulates organ size control.

Information flow within and between cells depends significantly on calcium (Ca2+) signaling dynamics. However, the biophysical mechanisms that govern emergent patterns of Ca2+ signaling dynamics at the organ level remain elusive. Recent experimental studies in developing Drosophila wing imaginal discs demonstrate the emergence of four distinct patterns of Ca2+ activity: Ca2+ spikes, intercellular Ca2+ transients, tissue-level Ca2+ waves, and a global "fluttering" state. Here, we used a combination of computational modeling and experimental approaches to identify two different populations of cells within tissues that are connected by gap junction proteins. We term these two subpopulations "initiator cells," defined by elevated levels of Phospholipase C (PLC) activity, and "standby cells," which exhibit baseline activity. We found that the type and strength of hormonal stimulation and extent of gap junctional communication jointly determine the predominate class of Ca2+ signaling activity. Further, single-cell Ca2+ spikes are stimulated by insulin, while intercellular Ca2+ waves depend on Gαq activity. Our computational model successfully reproduces how the dynamics of Ca2+ transients varies during organ growth. Phenotypic analysis of perturbations to Gαq and insulin signaling support an integrated model of cytoplasmic Ca2+ as a dynamic reporter of overall tissue growth. Further, we show that perturbations to Ca2+ signaling tune the final size of organs. This work provides a platform to further study how organ size regulation emerges from the crosstalk between biochemical growth signals and heterogeneous cell signaling states.

Effects of store-operated and receptor-operated calcium channels on synchronization of calcium oscillations in astrocytes

Intercellular calcium signaling allows cells to communicate with each other and to interact with adjacent cells. Gap junction is the most common and important way for cellular communication. Recently, mathematical models have been widely used to gain a precise and quantitative understanding of the dynamics of intracellular calcium ions (Ca2＋). In this paper, we establish a mathematical model considering the gap junction permeable to Ca2＋ and to IP3 for describing the calcium oscillations in coupled astrocytes. Store-operated calcium entry (SOCE) is viewed as the main process which controls the non-excitable cells, hence, we focus on the effect of store-operated calcium channel (SOCC) and receptor-operated calcium channel (ROCC) on the intercellular synchronization, respectively. By employing bifurcation analysis on this model, the dynamic behaviors of the coupled system with different physiological state cells is obtained with changes in the maximum capacity of the SOCC and the ROCC. The synchronization boundaries for different conditions are gained in the two parameters space of the channel parameters and the coupling strength. The results suggest that the variation of the maximum flow for different calcium channels determines the stable oscillations of the coupled system, as well as for the frequency and amplitude of oscillations. The SOCC has an expected effect on the change of the oscillatory interval while the ROCC demonstrated the influence on the amplitude modulation. Furthermore, the coupling strength and channel parameters could induce 1:1 locking of intercellular Ca2＋ oscillations and the synchronization region like Arnol’d tongue is found.

The Synergistic Effect of Anabolic Steroid and Loading on Intercellular Calcium Signaling Via Gap Junctions in Human Supraspinatus Tendon Cells

Objective: We hypothesized that anabolic steroid administration would act synergistically with substrate strain in two-dimensional cultures of human supraspinatus tendon cells, to upregulate the expression of connexin-43 and to increase the Ca2+ wave propagation through gap junctions. Methods: Supraspinatus tendon cells were isolated intra-operatively from human specimens during shoulder arthroscopy. Cells were plated in two-dimensional spot cultures and arranged into four experimental groups: 1) non-load, non-steroid (NLNS, n=12 wells); 2) non-load, steroid (NLS, n=12 wells); 3) load, non-steroid (LNS, n=12 wells); and 4) load, steroid (LS, n=12 wells) in order to produce bioartificial tendons (BATs). The load groups were stretched in culture plates and the steroid groups were given nandrolone decanoate. When BATs were macro- and microscopically mature, at five days, they were evaluated with immunocytochemistry for connexin-43 staining, fluorescence microscopy for calcium imaging and mechanical stimulation with a micropipette tip manipulation for calcium propagation. Dose response test was performed in order to establish any relation between nandrolone decanoate dose and calcium signaling response. ATP was applied to the spot culture cells from all groups and all patients to determine if the cells were sensitive to extracellular ATP. Results: Load-steroid group demonstrated the greatest density of cnx43 in comparison to all other groups. There were no significant differences between the groups considering the percentage of cells responding after mechanical stimulation (cell recruitment). The cells of load-steroid group showed a significantly greater mean peak [Ca2+]ic compared to the values of the other groups (p<0.05). The propagation time was significantly decreased in the LS group compared with the other groups (p<0.05). There were no significant differences between the groups considering the number of cells that were responding spontaneously prior to stimulation or the number of responding cells that were oscillating after the stimulation. Conclusion: Nandrolone decanoate and loading seem to have a synergistic effect on the upregulation of the gap junction protein cxn43 enhancing calcium signaling via gap junctions. Consecutively, anabolic steroid administration and load may enhance the formation of a better-organized cytoskeleton and particularly the actin stress monofilaments.

Bottom-Up Assembly of Functional Intracellular Synthetic Organelles by Droplet-Based Microfluidics.

Bottom-up synthetic biology has directed most efforts toward the construction of artificial compartmentalized systems that recreate living cell functions in their mechanical, morphological, or metabolic characteristics. However, bottom-up synthetic biology also offers great potential to study subcellular structures like organelles. Because of their intricate and complex structure, these key elements of eukaryotic life forms remain poorly understood. Here, the controlled assembly of lipid enclosed, organelle-like architectures is explored by droplet-based microfluidics. Three types of giant unilamellar vesicles (GUVs)-based synthetic organelles (SOs) functioning within natural living cells are procedured: (A) synthetic peroxisomes supporting cellular stress-management, mimicking an organelle innate to the host cell by using analogous enzymatic modules; (B) synthetic endoplasmic reticulum (ER) as intracellular light-responsive calcium stores involved in intercellular calcium signalling, mimicking an organelle innate to the host cell but utilizing a fundamentally different mechanism; and (C) synthetic magnetosomes providing eukaryotic cells with a magnetotactic sense, mimicking an organelle that is not natural to the host cell but transplanting its functionality from other branches of the phylogenetic tree. Microfluidic assembly of functional SOs paves the way for high-throughput generation of versatile intracellular structures implantable into living cells. This in-droplet SO design may support or expand cellular functionalities in translational nanomedicine.

Does Neuronal Activity Promote Glioma Progression?

Excessive glutamate release by glioma cells induces pharmacologically accessible neuronal hyperexcitation, including epilepsy. Two recent reports by Venkataramani etal. and Venkatesh etal. suggest that neuronal hyperexcitation stimulates bona fide glutamatergic synapses on glioma cells. Ionotropic glutamate receptors activate intercellular calcium signaling networks to orchestrate glioma cell growth and invasion, presumably by facilitating oncogenic signaling cascades and cytoskeletal remodeling.

Neurogenesis-on-Chip: Electric field modulated transdifferentiation of human mesenchymal stem cell and mouse muscle precursor cell coculture

A number of bioengineering strategies, using biophysical stimulation, are being explored to guide the human mesenchymal stem cells (hMScs) into different lineages. In this context, we have limited understanding on the transdifferentiation of matured cells to another functional-cell type, when grown with stem cells, in a constrained cellular microenvironment under biophysical stimulation. While addressing such aspects, the present work reports the influence of the electric field (EF) stimulation on the phenotypic and functionality modulation of the coculture of murine myoblasts (C2C12) with hMScs [hMSc:C2C12=1:10] in a custom designed polymethylmethacrylate (PMMA) based microfluidic device with in-built metal electrodes. The quantitative and qualitative analysis of the immunofluorescence study confirms that the cocultured cells in the conditioned medium with astrocytic feed, exhibit differentiation towards neural-committed cells under biophysical stimulation in the range of the endogenous physiological electric field strength (8 ± 0.06 mV/mm). The control experiments using similar culture protocols revealed that while C2C12 monoculture exhibited myotube-like fused structures, the hMScs exhibited the neurosphere-like clusters with SOX2, nestin, βIII-tubulin expression. The electrophysiological study indicates the significant role of intercellular calcium signalling among the differentiated cells towards transdifferentiation. Furthermore, the depolarization induced calcium influx strongly supports neural-like behaviour for the electric field stimulated cells in coculture. The intriguing results are explained in terms of the paracrine signalling among the transdifferentiated cells in the electric field stimulated cellular microenvironment. In summary, the present study establishes the potential for neurogenesis on-chip for the coculture of hMSc and C2C12 cells under tailored electric field stimulation, in vitro.

Intercellular Calcium Signaling Induced by ATP Potentiates Macrophage Phagocytosis

SummaryExtracellular ATP is a signaling molecule exploited by the immune cells for both autocrine regulation and paracrine communication. By performing live calcium imaging experiments, we show that triggered mouse macrophages are able to propagate calcium signals to resting bystander cells by releasing ATP. ATP-based intercellular communication is mediated by P2X4 and P2X7 receptors and is a feature of pro-inflammatory macrophages. In terms of functional significance, ATP signaling is required for efficient phagocytosis of pathogen-derived molecules and apoptotic cells and may represent a target for macrophage regulation by CD39-expressing cells. These results highlight a cell-to-cell communication mechanism tuning innate immunity.

Vitamin K2-Dependent GGCX and MGP Are Required for Homeostatic Calcium Regulation of Sperm Maturation

SummaryA low-calcium microenvironment is essential for spermatozoa to mature in the epididymis; however, it remains unclear how dysregulation of epididymal luminal calcium is associated with male infertility. Using a warfarin-induced vitamin K2 deficiency rat model, we found that vitamin-K-dependent γ-glutamyl carboxylase (GGCX) and matrix Gla protein (MGP) were essential in extracellular calcium signaling of the intercellular communication required for epididymal sperm maturation. We found that GGCX and MGP co-localized in the vesicular structures of epididymal cells and spermatozoa. Calcium-regulated MGP binds to proteins in a biphasic manner; sub-millimolar calcium enhances, whereas excessive calcium inhibits, the binding. Bioinformatic analysis of the calcium-dependent MGP-bound proteome revealed that vesicle-mediated transport and membrane trafficking underlie the intercellular communication networks. We also identified an SNP mutation, rs699664, in the GGCX gene of infertile men with asthenozoospermia. Overall, we revealed that the GGCX-MGP system is integrated with the intercellular calcium signaling to promote sperm maturation.

Inner Ear Connexin Channels: Roles in Development and Maintenance of Cochlear Function.

Connexin 26 and connexin 30 are the prevailing isoforms in the epithelial and connective tissue gap junction systems of the developing and mature cochlea. The most frequently encountered variants of the genes that encode these connexins, which are transcriptionally coregulated, determine complete loss of protein function and are the predominant cause of prelingual hereditary deafness. Reducing connexin 26 expression by Cre/loxP recombination in the inner ear of adult mice results in a decreased endocochlear potential, increased hearing thresholds, and loss of >90% of outer hair cells, indicating that this connexin is essential for maintenance of cochlear function. In the developing cochlea, connexins are necessary for intercellular calcium signaling activity. Ribbon synapses and basolateral membrane currents fail to mature in inner hair cells of mice that are born with reduced connexin expression, even though hair cells do not express any connexin. In contrast, pannexin 1, an alternative mediator of intercellular signaling, is dispensable for hearing acquisition and auditory function.

A NEW REGISTRATION APPROACH FOR DYNAMIC ANALYSIS OF CALCIUM SIGNALS IN ORGANS.

Wing disc pouches of fruit flies are a powerful genetic model for studying physiological intercellular calcium (Ca 2+) signals for dynamic analysis of cell signaling in organ development and disease studies. A key to analyzing spatial-temporal patterns of Ca 2+ signal waves is to accurately align the pouches across image sequences. However, pouches in different image frames may exhibit extensive intensity oscillations due to Ca 2+ signaling dynamics, and commonly used multimodal non-rigid registration methods may fail to achieve satisfactory results. In this paper, we develop a new two-phase non-rigid registration approach to register pouches in image sequences. First, we conduct segmentation of the region of interest. (i.e., pouches) using a deep neural network model. Second, we use a B-spline based registration to obtain an optimal transformation and align pouches across the image sequences. Evaluated using both synthetic data and real pouch data, our method considerably outperforms the state-of-the-art non-rigid registration methods.

Precision guided microfluidic chemical perfusion strategy for cell dynamic imaging

Cell signaling has been recognized as a highly dynamic process. How to capture such dynamics with changes of chemical microenvironment at single cell resolution among cells population without interfering with adjacent cells has been still an immense challenge. Here, we proposed a precision guided microfluidic chemical perfusion strategy for investigating chemical-activated cell signaling via dynamic imaging with high temporal (∼12 ms), spatial resolutions and precision. Theory for the microfluidic operation was first established and validated by both numerical simulations and experiments. A computer-aided multi-channel pressure control system was constructed to automatically implement chemical perturbations with various microperfusion times, relaxation times and number of cycles. The width and the position of the chemical microflow could also be precisely controlled with high reliability and accuracy. Investigations of intracellular and intercellular calcium signaling were successfully demonstrated using the proposed method. These results revealed that the precision guided microfluidic chemical perfusion strategy provided a versatile means for cell signaling analysis, which is potentially meaningful in chemical biology, cell biology and pharmacology.

Modulation of multiple differentiation potential in mesenchymal stem cells via an intercellular calcium signaling pathway

Modulation of multiple differentiation potential in mesenchymal stem cells via an intercellular calcium signaling pathway

Patterns of calcium signaling: A link between chronic emotions and cancer.

Intra and inter-cellular calcium signaling is present in all types of cells and body tissues. In the human brain, calcium currents and waves are related to mental activities, including emotions. We present a theoretical interpretation of these phenomena suggesting their involvement in chronic emotional patterns and in the pathology of cancer. Recent developments on biophysics, translational biology and psychoneuroendocrinoimmunology (PNEI) can support explanatory hypotheses about the link between emotional stresses and the origin and development of different types of tumor cells. Chronic stresses may cause perturbations of rhythms of the PNEI system, excessive activation of HPA axis and abnormal activation of calcium signals in somatic tissues, with deleterious effects on different parts of the body. The increasing of calcium signaling inside cells may lead to a deregulation of different pathways and epigenetic systems that promote the production of genomic mutations in a second phase. In particular, the hyperactivation of the transcription nuclear factor kappaB (NF-κB), if is not counterbalanced by the following activation of the nuclear factor (erythroid-derived2)-like 2 (NFE2L2 or Nrf2), increases the production of oxidative catabolites, as the advanced glycation end products (AGE), which play a key role in the progression of different types of cancer and other degenerative diseases. Cortisol binding to glucocorticoid receptor (GR) reduces the activity of both NF-κB and Nrf2 inside the cells but inhibits the cellular immunity and the anabolic processes of tissue regeneration. The tissue atrophy and the defective anti-ageing mechanisms promotes the tumoral cells growth and their escape from the immune-surveillance.

Calcium spikes, waves and oscillations in a large, patterned epithelial tissue

While calcium signaling in excitable cells, such as muscle or neurons, is extensively characterized, calcium signaling in epithelial tissues is little understood. Specifically, the range of intercellular calcium signaling patterns elicited by tightly coupled epithelial cells and their function in the regulation of epithelial characteristics are little explored. We found that in Drosophila imaginal discs, a widely studied epithelial model organ, complex spatiotemporal calcium dynamics occur. We describe patterns that include intercellular waves traversing large tissue domains in striking oscillatory patterns as well as spikes confined to local domains of neighboring cells. The spatiotemporal characteristics of intercellular waves and oscillations arise as emergent properties of calcium mobilization within a sheet of gap-junction coupled cells and are influenced by cell size and environmental history. While the in vivo function of spikes, waves and oscillations requires further characterization, our genetic experiments suggest that core calcium signaling components guide actomyosin organization. Our study thus suggests a possible role for calcium signaling in epithelia but importantly, introduces a model epithelium enabling the dissection of cellular mechanisms supporting the initiation, transmission and regeneration of long-range intercellular calcium waves and the emergence of oscillations in a highly coupled multicellular sheet.

Stochastic left-right neuronal asymmetry in Caenorhabditis elegans.

Left-right asymmetry in the nervous system is observed across species. Defects in left-right cerebral asymmetry are linked to several neurological diseases, but the molecular mechanisms underlying brain asymmetry in vertebrates are still not very well understood. The Caenorhabditis elegans left and right amphid wing 'C' (AWC) olfactory neurons communicate through intercellular calcium signalling in a transient embryonic gap junction neural network to specify two asymmetric subtypes, AWCOFF (default) and AWCON (induced), in a stochastic manner. Here, we highlight the molecular mechanisms that establish and maintain stochastic AWC asymmetry. As the components of the AWC asymmetry pathway are highly conserved, insights from the model organism C. elegans may provide a window onto how brain asymmetry develops in humans.This article is part of the themed issue 'Provocative questions in left-right asymmetry'.

A New System for Profiling Drug-Induced Calcium Signal Perturbation in Human Embryonic Stem Cell–Derived Cardiomyocytes

The emergence of human stem cell–derived cardiomyocyte (hSCCM)–based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling “fingerprint” in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca2+ signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.

Cooperative effects of inherent stochasticity and random long-range connections on synchronization and coherence resonance in diffusively coupled calcium oscillators

The cooperative effects of inherent stochasticity and random long-range connections (RLRCs) on synchronization and coherence resonance in networks of calcium oscillators have been investigated. Two different types of collective behaviors, coherence resonance (CR) and synchronization, have been studied numerically in the context of chemical Langevin equations (CLEs). In the CLEs, the reaction steps are all stochastic, including the exchange of calcium ions between adjacent and non-adjacent cells through the gap junctions. The calcium oscillators’ synchronization was characterized by the standard deviation of the cytosolic calcium concentrations. Meanwhile, the temporal coherence of the calcium spike train was characterized by the reciprocal coefficient of variance (RCV). Synchronization induced by RLRCs was observed, namely, the exchange of calcium ions between non-adjacent cells can promote the synchronization of the cells. Moreover, it was found that the RCV shows a clear peak when both inherent stochasticity and RLRCs are optimal, indicating the existence of CR. Since inherent stochasticity and RLRCs are two essential ingredients of cellular processes, synchronization and CR are also important for cells’ functions. The results reported in this paper are expected to be useful for understanding the dynamics of intercellular calcium signaling processes in vivo.