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Supplemental Movie 1: Stochastic Ca2+ transients in ICC-DMP.

Supplemental Movie 1: Stochastic Ca2+ transients in ICC-DMP.  Movie of intracellular Ca2+ transients in ICC-DMP labelled with the genetically encoded Ca2+ indicator GCaMP3.  The top left FOV shows elongated ICC-DMP at 60x and the top right FOV shows ICC-DMP at 100x magnification.  Note the lack of coincidence of Ca2+ transients between the blue bit-masked cell and the non-bit-masked cell in the 100x FOV.  The blue bit-masked ICC-DMP in the 100x FOV was used to construct a spatio-temporal map of Ca2+-induced fluorescence intensity along the length of the cell (lower panel).  Note the stochastic firing of spontaneous Ca2+ transients in ICC-DMP.  Copied with permission from reference (127).    <a href="https://doi.org/10.1152/physrev.00036.2022">https://doi.org/10.1152/physrev.00036.2022</a>

Supplemental Movie 2: Clustered Ca2+ transients (CTCs) in gastric ICC-MY occur from multiple firing sites.

Supplemental Movie 2: Clustered Ca2+ transients (CTCs) in gastric ICC-MY occur from multiple firing sites.  ICC-MY in the gastric antrum firing of CTCs and imaged at high resolution with a spinning disk confocal microscope using a 60x objective. Ca2+ signals were monitored in a gastric muscle from a mouse with the genetically encoded Ca2+ indicator, GCaMP6f, expressed exclusively in ICC. The left panel shows typical stellate-shaped ICC-MY with multiple interconnecting processes. The middle panel shows the Ca2+ particle (PTCL) activity, color coded in blue for raw PTCLs, and the centroids of particles are indicated in purple and green indicates Ca2+ firing sites. There are multiple sites firing Ca2+ transients during the CTCs.  The right panel shows an occurrence map of color-coded initiation/firing sites. The pattern of firing sites Ca2+ activity was temporally clustered as activation of Ca2+ transients swept through the network of ICC-MY.  The onset of the CTCs was explosive, and then asynchronous firing of occurred at multiple sites and was sustained for more than 2 sec.  Note also the complete quiescence of firing immediately upon conclusion of a CTC (absolute refractory period) and then sporadic initiation of firing with time.  It is the re-initiation of firing that sets off the next CTC by activating ANO1 channels, depolarization and activation of voltage-dependent Ca2+ current (see text for details).  Reformatted with permission from reference (106).

Supplemental Movie 4: Subtypes of ICC-IM with different Ca2+ firing patterns in the IAS.

Supplemental Movie 4:  Subtypes of ICC-IM with different Ca2+ firing patterns in the IAS Video from the distal edge of the internal anal sphincter (IAS) from a mouse expressing GCaMP6f exclusively in ICC using a 20x objective. Active ICC-IM show 2 patterns of Ca2+ transients.  Type I cells (* and green text) displayed stochastic Ca2+ transients with short distances of spatial spread.  Type II cells (* and yellow text) showed whole-cell flashes of activity. The still image and spatio-temporal (ST) maps (derived from the highlighted cells) and Ca2+ traces shown in Fig. 20A-E were generated from this recording.  Data correspond to figure in reference (136).

Supplemental Movie 5: Pattern of slow wave activation in intact stomach as viewed by imaging of Ca2+ transients in ICC-MY.

Supplemental Movie 5:  Pattern of slow wave activation in intact stomach as viewed by imaging of Ca2+ transients in ICC-MY  Ca2+ transients were monitored in canulated whole stomachs taken from mice expressing GCaMP3 exclusively in ICC (upper left panel). The whole stomach from fundus through the pylorus is visualized in the FOV.  Right panel shows differentiated image in which background (unchanged pixels) remains black and active pixels (ICC) are intensity-coded to white.  Activation of Ca2+ waves (which activate currents in ICC-MY and generate slow waves in gastric muscles) develop into a spiral pattern of activation from a dominant pacemaker region near the greater curvature of the corpus.  Ca2+ transients in the whole stomach are plotted as a spatio-temporal map, displaying proximal to distal propagation of Ca2+ waves along the length of the stomach (lower panel). (Provided by Dr. Grant Henning)

opencc-by-4.0Jan 2022View details →

Terminal density reversal and the role of Ca2+ in red blood cells clearance of healthy individuals

Density reversal of senescent red blood cells (RBCs) has been known for more than ten years, yet the identity of the candidate protein(s) is still elusive. While performing Percoll density gradient separation of RBCs from healthy individuals and their subsequent characterization, we identified a fraction of cells in the low-density fraction (~0.025% compared to total RBCs population) which shows reversal in their densities along with the characteristics of cellular senescence such as loss of membrane Band 3 protein and the phosphatidylserine exposure to the outer membrane leaflet. Our subsequent analysis showed that these cells are overloaded with Ca2+. We further measured intracellular [Na+] in individual RBCs by flow cytometry utilizing the dye CoroNa Green-AM.  Our findings showed that the cells with senescent characteristics lost their transmembrane Na+ gradient despite maintaining the membrane integrity. Consequently, these findings lead us to designate these cells as “senescent-like” cells. Our data further demonstrated altered activities of nonselective cation channels and pumps in these cells. In addition to a facilitated Na+ extrusion by Na+, K+-ATPase, our findings indicated altered ion transport via Piezo1 in these cells. Pharmacological modulation of Piezo1 with Yoda1/GsMTx4 showed that Piezo1 and, possibly, other nonselective cation channels by promiscuously transporting Na+ and Ca2+ play an important role in producing these low density “senescent like” cells.

opencc-zeroJan 2022View details →

Ca2+ activity maps of astrocytes tagged by axo-astrocytic AAV transfer

Astrocytes exhibit localized Ca2+ microdomain (MD) activity thought to be actively involved in information processing in the brain. However, functional organization of Ca2+ MDs in space and time in relationship to behavior and neuronal activity is poorly understood. Here, we first show that Adeno-Associated Virus (AAV) particles transfer anterogradely from axons to astrocytes. Then we use this axo-astrocytic AAV transfer to express genetically encoded Ca2+ indicators at high contrast circuit-specifically. In combination with two-photon microscopy and unbiased, event-based analysis we investigated cortical astrocytes embedded in the vibrissal thalamocortical circuit. We found a wide range of Ca2+ MD signals, some of which were ultrafast (≤300 ms). Frequency and size of signals were extensively increased by locomotion but only subtly with sensory stimulation. The overlay of these signals resulted in behavior dependent maps with characteristic Ca2+ activity hotspots, maybe representing memory engrams. These functional subdomains are stable over days, suggesting subcellular specialization.

opencc-by-4.0Apr 2022View details →

p53 alters intracellular Ca2+ signaling through regulation of TRPM4

Altered expression of transient receptor potential channel melastatin 4 (TRPM4) contributes to several malignancies, including cardiac conduction diseases, immune diseases, and cancer. Yet the underlying mechanisms of TRPM4 expression changes remain elusive.In this study, we report that loss of tumor suppressor protein p53 or p63γ function or mutation of a putative p53 response element in the TRPM4 promoter region increase TRPM4 promoter activity in the colorectal cancer cell line HCT 116. In cells that lack p53 expression, we observed increased TRPM4 mRNA and protein levels and TRPM4-mediated Na+ currents. This phenotype can be reversed by transient overexpression of p53. In the prostate cancer cell line LNCaP, which expresses p53 endogenously, p53 overexpression decreases TRPM4-mediated currents. As in other cancer cells, CRISPRcas9 mediated knockout of TRPM4 in p53 deficient HCT 116 cells results in increased store-operated Ca2+entry. The effect of the TRPM4 knockout is mimicked by p53 mediated suppression of TRPM4 in the parental cell line expressing TRPM4. In addition, a TRPM4 knockout-mediated shift in cell cycle is abolished upon loss of p53.Taken together, these findings indicate that p53 represses TRPM4 expression, thereby altering cellular Ca2+ signaling and that TRPM4 adds to cell cycle shift dependent on p53 signaling.

opencc-zeroMay 2022View details →

β-cell-specific deletion of Zfp148 improves nutrient-stimulated β-cell Ca2+ responses

Insulin secretion from pancreatic β-cells is essential for glucose homeostasis. An insufficient response to the demand for insulin results in diabetes. We previously showed that β-cell-specific deletion of Zfp148 (β-Zfp148KO) improves glucose tolerance and insulin secretion in mice. Here, we performed Ca2+ imaging of islets from β‑Zfp148KO and control mice on both a chow and a Western-style diet. β-Zfp148KO islets demonstrate improved sensitivity and sustained Ca2+ oscillations in response to elevated glucose. β-Zfp148KO islets also exhibit elevated sensitivity to amino acid-induced Ca2+ influx under low glucose conditions, suggesting enhanced mitochondrial phosphoenolpyruvate (PEP)-dependent KATP channel closure, independent of glycolysis. RNA sequencing and proteomics of β-Zfp148KO islets revealed altered levels of enzymes involved in amino acid metabolism (SLC3A2, SLC7A8, GLS, GLS2, PSPH, PHGDH, PSAT1) and intermediary metabolism (GOT1, PCK2), consistent with altered PEP cycling. In agreement with this, β-Zfp148KO islets displayed enhanced insulin secretion in response to L-glutamine and activation of glutamate dehydrogenase. Understanding pathways controlled by ZFP148 may provide promising strategies for improving β-cell function that are robust to the metabolic challenge imposed by a Western diet.

opencc-by-4.0May 2024View details →

Dataset of molecular structures of PNAS article " Ca2+ permeation through C-terminal cleaved, but not full-length human Pannexin1 hemichannels, mediates cell death"

The PMFWT_91_80.tar.gz file contains the WT molecular system described in the cited article. Briefly, the package contains the structure and topology files for AMBER software, along with configuration files to run Umbrella Sampling method and calculation of PMF of a Ca+2 ion traslocating the human pannexin channel (WT).  The TRUCWT_91_80.tar.gz file contains the truncated molecular system described in the cited article. Briefly, the package contains the structure and topology files for AMBER software, along with configuration files to run Umbrella Sampling method and calculation of PMF of a Ca+2 ion traslocating the truncated human pannexin channel as described in the article. Two NetCDF trajectories (*.nc) of a single PMF window are provided for each system.

opencc-by-4.0Jun 2024View details →

Association between Dysregulated Expression of Ca2+ and ROS-Related Gene Pairs and Breast Cancer Patient Survival

This file is composed of two documents: <ul> <li>Supplementary File 1 containing three Excel files with cumulative proportion survival from redox-related genes, calcium-related genes, and redox and calcium-related genes.</li> <li>Supplementary Table 3 including an Excel file with a functional enrichment analysis using redox and calcium correlated genes. Cell cycle regulation (sheet 1) and Cell adhesion and projection (sheet 2) were the biological processes more enriched.</li> </ul> Both supplementary tables belongs to the study Association between Dysregulated Expression of Ca2+ and ROS-Related Gene Pairs and Breast Cancer Patient Survival, published in Molecular Diagnosis and therapy

opencc-by-4.0Jul 2018View details →

Axial Tubule Junctions Activate Atrial Ca2+ Release across Species

Repository of "Axial Tubule Junctions Activate Atrial Ca2+ Release across Species" in Front Physiol: Regions of interest (ROI) from intact isolated mouse atrial myocytes stained with the membrane dye Chol-PEG-KK114 (5 µM), which were used for TAT network analysis in Figure 3. Please see methods section for detailed information.

opencc-zeroFeb 2023View details →

Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants

We illustrate the use of induced pluripotent stem cells (iPSCs) as platforms for investigating cardiomyocyte phenotypes in a human family pedigree exemplified by novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants occurring alone and in combination. The proband, a four-month-old boy, presented with polymorphic ventricular tachycardia (PVT). Genetic tests revealed double novel heterozygous RYR2-A1855D and SCN10A-Q1362H variants inherited from his father (F) and mother (M) respectively. His father showed ventricular premature beats (VPB); his mother was asymptomatic. Molecular biological characterisations demonstrated greater TNNT2 mRNA expression in the iPSCs-induced cardiomyocytes (iPS-CMs) than in the iPSCs. cTNTs became progressively organised, but cytoplasmic RYR2 and SCN10A aggregations occurred in the iPS-CMs. Proband-specific iPS-CMs showed decreased RYR2 and SCN10A mRNA expression. The RYR2-A1855D variant resulted in premature spontaneous sarcoplasmic reticular (SR) Ca2+ transients (PCTs), Ca2+ oscillations (COs), and increased action potential durations (APDs). SCN10A-Q1362H did not confer any specific phenotype. However, the combined heterozygous RYR2-A1855D and SCN10A-Q1362H variants in the proband iPS-CMs resulted in accentuated Ca2+ homeostasis disorders, AP prolongation and susceptibility to early afterdepolarisations (EADs) at high stimulus frequencies. These findings attribute the clinical phenotype in the proband to effects of the heterozygous RYR2 variant exacerbated by heterozygous SCN10A modification.

opencc-zeroMar 2023View details →

Genetic variation in mouse islet Ca2+ oscillations reveals novel regulators of islet function

Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca2+ into β-cells triggers insulin release. Since genetics strongly influences variation in islet secretory responses, we surveyed islet Ca2+ dynamics in eight genetically diverse mouse strains. We found high strain variation in response to four conditions: 1) 8 mM glucose; 2) 8 mM glucose plus amino acids; 3) 8 mM glucose, amino acids, plus 10nM GIP; and 4) 2 mM glucose. These stimuli interrogate β-cell function, α-cell to β-cell signaling, and incretin responses. We then correlated components of the Ca2+ waveforms to islet protein abundances in the same strains used for the Ca2+ measurements. To focus on proteins relevant to human islet function, we identified human orthologues of correlated mouse proteins that are proximal to glycemic-associated SNPs in human GWAS. Several orthologues have previously been shown to regulate insulin secretion (e.g. ABCC8, PCSK1, and GCK), supporting our mouse-to-human integration as a discovery platform. By integrating these data, we nominated novel regulators of islet Ca2+ oscillations and insulin secretion with potential relevance for human islet function. We also provide a resource for identifying appropriate mouse strains in which to study these regulators.

opencc-by-4.0May 2023View details →

Database of Ca2+ transients from single cell E. coli measurements

Database configuration To efficiently compare conditions from over 7 Tb of image data, we constructed an HDF5 database with the extracted fluorescent traces for each experiment.  The database was configured such that the fluorophore specifies the first group (e.g., '/gcamp'). The second group is the unique experimental identifier generated at the time of the experiment (e.g., '/20210322_5 Hz_gcamp_90pts'). The third group specifies the variable name (See Table below for description).  The database is also available from https://bitbucket.org/meyerct6/soc_bacteria_repo/src/master/ in (Database/soc_bacteria_database.h5) and can be used to recreate most figures in the manuscript. <table> <caption>Description of HDF5 database variables for each experiment</caption> <tbody> <tr> <td>/meanintMatG</td> <td>Matrix of mean intensity within each segmented cell (rows) at all time points (columns).</td> </tr> <tr> <td>/corrIntMatG</td> <td>Matrix of mean intensity corrected for photobleaching.</td> </tr> <tr> <td>/fitRsqr</td> <td>Vector of R-squared fit values for photobleach equation.</td> </tr> <tr> <td> /timeMat</td> <td>Matrix of acquisition times for each cell (rows) at every timepoint (columns).</td> </tr> <tr> <td>/heat_kill</td> <td>Boolean vector denoting whether the cell was heat killed</td> </tr> </tbody> </table>

opencc-zeroOct 2023View details →

Data from: Scn2a insufficiency alters spontaneous neuronal Ca2+ activity in somatosensory cortex during wakefulness

SCN2A protein-truncating variants (PTV) can result in neurological disorders such as autism spectrum disorder and intellectual disability, but they are less likely to cause epilepsy in comparison to missense variants. While in vitro studies showed PTV reduce action potential firing, consequences at in vivo network level remain elusive. Here, we generated a mouse model of Scn2a insufficiency using antisense oligonucleotides (Scn2a ASO mice), which recapitulated key clinical feature of SCN2A PTV disorders. Simultaneous two-photon Ca2+ imaging and electrocorticography (ECoG) in awake mice showed that spontaneous Ca2+ transients in somatosensory cortical neurons, as well as their pairwise co-activities were generally decreased in Scn2a ASO mice during spontaneous awake state and induced seizure state. The reduction of neuronal activities and paired co-activity are mechanisms associated with motor, social and cognitive deficits observed in our mouse model of severe Scn2a insufficiency, indicating these are likely mechanisms driving SCN2A PTV pathology.

publicFeb 2023View details →

Patient-specific induced pluripotent stem cell properties implicate Ca2+-homeostasis in clinical arrhythmia associated with combined heterozygous RYR2 and SCN10A variants

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publicJan 2022View details →

Ca2+ activity maps of astrocytes tagged by axo-astrocytic AAV transfer

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