Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells
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ABSTRACT:
SUBMITTER: Chen Z
PROVIDER: S-EPMC11023258 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
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Project description:The purpose of this study was to determine if transient cell membrane disruptions (TPMDs) in single keratocytes can trigger signaling events in neighboring keratocytes. Stromal cells were cultured from human corneas (HCSC) and mouse corneas (MCSC). TPMDs were produced using a multiphoton microscope in Cal-520-AM loaded cells. TPMD-induced calcium increases (Ca++i) were measured in Ca++-containing and Ca++-free solutions containing thapsigargin, ryanodine, BAPTA-AM, 18-α-glycyrrhetinic acid (18α-GA), apyrase, BCTC, AMG 9810, or AMTB. Fluorescence intensity was recorded as the number of cells responding and the area under the fluorescence versus time curve. The maximum distance of responding neighboring cells in ex vivo human corneas was measured. Connexin 43 protein in HCSC and MCSC was examined using immunofluorescence staining, and corneal rubbing was applied to confirm whether TPMDs occur following mechanical manipulation. Our results demonstrate that single cell TPMDs result in Ca++ waves in neighboring keratocytes both in culture and within ex vivo corneas. The source of Ca++ is both intra-and extra-cellular, and the signal can be mediated by ATP and/or gap junctions, and is species dependent. Stromal rubbing confirmed that TPMDs do occur following mechanical manipulation. Keratocyte TPMDs and their associated signaling events are likely common occurrences following minor or major corneal trauma.
| S-EPMC7029045 | biostudies-literature
Project description:Plasma membrane calcium-ATPases (PMCAs) play a critical role in regulating intracellular calcium concentration. Four genes encode PMCA proteins with alternative splicing of transcripts at three sites (A, B and C) serving to increase isoform diversity. Our previous work shows that all four PMCAs are expressed and have specific locations in human corneal epithelium (hCE). The present work examined which splice variants of PMCAs are expressed in hCE. Total RNA was extracted from hCE scraped from cadaver corneas of five different donors (two females and three males, age range 55-76 years). RT-PCR was performed using PMCA isoform-specific primers designed to amplify transcripts that included either splice site A or splice sites B and C. PMCA cDNAs were sequenced or cloned, and then sequenced. There was uniformity in the PMCA1 and PMCA4 expression profile among the five donors. Specifically, every donor expressed PMCA4 transcripts (4x at site A and 4b at site B/C). Every donor also expressed PMCA1 transcripts at sites B/C, specifically PMCA1b and PMCA1kb. In contrast, PMCA2 and PMCA3 expression varied; PCR DNAs were detected in two of five donors. One donor expressed PMCA2a and a novel PMCA2 variant we termed PMCA2((i)). PMCA3a transcript was demonstrated in a different donor. Finally, for all the donors, bands encoding site A transcripts for PMCA4 were obtained but no PCR transcripts were detected at site A for PMCA1, PMCA2 and PMCA3. This investigation showed that hCE expressed multiple splice variants of PMCA isoforms. Furthermore, this study documented the expression of the PMCA1k variant (PMCA1kb) previously only described in intestine and pancreatic beta cells and describes a novel PMCA2((i)) variant. Finally, this study suggests that the molecular configuration of PMCA1, PMCA2 and PMCA3 in the region of splice site A in hCE must be different than in other tissues since the same primers that produced site A transcripts in several other tissues were ineffective in priming PCR in hCE.
| S-EPMC2174367 | biostudies-literature
Project description:The plasma membrane of the cell is a complex, tightly regulated, heterogeneous environment shaped by proteins, lipids, and small molecules. Ca2+ ions are important cellular messengers, spatially separated from anionic lipids. After cell injury, disease, or apoptotic events, anionic lipids are externalized to the outer leaflet of the plasma membrane and encounter Ca2+, resulting in dramatic changes in the plasma membrane structure and initiation of signaling cascades. Despite the high chemical and biological significance, the structures of lipid-Ca2+ nanoclusters are still not known. Previously, we demonstrated by solid-state nuclear magnetic resonance (NMR) spectroscopy that upon binding to Ca2+, individual phosphatidylserine lipids populate two distinct yet-to-be-characterized structural environments. Here, we concurrently employ extensive all-atom molecular dynamics (MD) simulations with our accelerated membrane mimetic and detailed NMR measurements to identify lipid-Ca2+ nanocluster conformations. We find that major structural characteristics of these nanoclusters, including interlipid pair distances and chemical shifts, agree with observable NMR parameters. Simulations reveal that lipid-ion nanoclusters are shaped by two characteristic, long-lived lipid structures induced by divalent Ca2+. Using ab initio quantum mechanical calculations of chemical shifts on MD-captured lipid-ion complexes, we show that computationally observed conformations are validated by experimental NMR data. Both NMR measurements of diluted specifically labeled lipids and MD simulations reveal that the basic structural unit that reshapes the membrane is a Ca2+-coordinated phosphatidylserine tetramer. Our combined computational and experimental approach presented here can be applied to other complex systems in which charged membrane-active molecular agents leave structural signatures on lipids.
| S-EPMC6475452 | biostudies-literature
Project description:Thermosensitive transient receptor potential (TRP) proteins such as TRPV1-TRPV4 are all heat-activated non-selective cation channels that are modestly permeable to Ca(2+). TRPV1, TRPV3, and TRPV4 functional expression were previously identified in human corneal epithelial cells (HCEC). However, the membrane currents were not described underlying their activation by either selective agonists or thermal variation. This study characterized the membrane currents and [Ca(2+)](i) transients induced by thermal and agonist TRPV1 and 4 stimulation. TRPV1 and 4 expressions were confirmed by RT-PCR and TRPV2 transcripts were also detected. In fura2-loaded HCEC, a TRPV1-3 selective agonist, 100 µM 2-aminoethoxydiphenyl borate (2-APB), induced intracellular Ca(2+) transients and an increase in non-selective cation outward currents that were suppressed by ruthenium-red (RuR) (10-20 µM), a non-selective TRPV channel blocker. These changes were also elicited by rises in ambient temperature from 25 to over 40 °C. RuR (5 µM) and a selective TRPV1 channel blocker capsazepine CPZ (10 µM) or another related blocker, lanthanum chloride (La(3+)) (100 µM) suppressed these temperature-induced Ca(2+) increases. Planar patch-clamp technique was used to characterize the currents underlying Ca(2+) transients. Increasing the temperature to over 40 °C induced reversible rises in non-selective cation currents. Moreover, a hypotonic challenge (25%) increased non-selective cation currents confirming TRPV4 activity. We conclude that HCEC possess in addition to thermosensitive TRPV3 activity TRPV1, TRPV2, and TRPV4 activity. Their activation confers temperature sensitivity at the ocular surface, which may protect the cornea against such stress.
| S-EPMC3072442 | biostudies-literature
Project description:Pattern recognition receptors (PRRs) at the plasma membrane promote plant immunity through the detection of conserved microbe-associated molecular patterns (MAMPs). In plants, the PRR for bacterial flagellin (flg22) is encoded by the receptor kinase FLS2. One of the earliest MAMP responses is the rapid and transient increase of cytosolic calcium (Ca2+) ions, which is required for many of the well-described downstream responses, e.g. generation of reactive oxygen species (ROS) and the transcriptional activation of defence-associated genes. Despite its relevance, the molecular components regulating the Ca2+ burst remain largely unknown. Here, we show that the plasma membrane P2B-type Ca2+ ATPase ACA8 forms a dynamic complex with the PRR FLS2. ACA8 and its closest homologue ACA10 are required for immunity against virulent bacteria. Mutant aca8 aca10 plants are reduced in the flg22-induced Ca2+ burst, show reduced ROS production and exhibit altered transcriptional reprogramming. In particular, flg22-induced gene expression is elevated downstream of signalling mitogen-activated protein (MAP) kinases, but reduced downstream of the calcium-dependent protein (CDP) kinase cascade. These results demonstrate that the fine regulation of Ca2+ fluxes in the cytosol is critical for the coordination of the downstream MAMP responses and provide for the first time a link between the FLS2 receptor complex and signalling kinases via the secondary messenger Ca2+. ACA8 also interacted with the BRI1 and CLV1 receptor kinases, which correlated with the developmental phenotypes of aca8 aca10 mutants suggesting a broader role for Ca2+ ATPases in receptor-mediated signalling. We used Affymetrix Arabidopsis Tiling 1.0R Array to compare global transcript levels in 7 days-old sterile grown seedlings. Steady-state mRNA levels in total RNA samples of 7 days old sterile seedlings
2012-04-24 | E-GEOD-31517 | biostudies-arrayexpress
Project description:PurposeTo investigate the corneal epithelial thickness topography with optical coherence tomography (OCT) and its relationship with vision quality in epithelial basement membrane dystrophy (EBMD).Methods45 eyes of EBMD patients, 26 eyes of dry eye (DED) patients and 22 eyes of normal subjects were enrolled. All participants were subjected to 9-mm corneal epithelial mapping with OCT and vision quality was assessed with the optical quality analysis system using the objective scatter index (OSI). Central, superior, inferior, minimum, maximum, and standard deviation of epithelium thickness (Irregularity), were analysed and correlations with the OSI were calculated.ResultsThe mean (±SD) central, inferior and maximum epithelial thicknesses of the EBMD patients (respectively, 56.4 (±8.1) μm, 58.9 (±6.4) μm, and 67.1 (±8.3) μm) were thicker compared to DED patients (P<0.05) and normal subjects (P<0.05). We found greater irregularity of epithelial thickness in EBMD (5.1±2.5 μm) compared to DED patients (2.6±1.0 μm) (P = 4.4.10-6) and normal subjects (2.1±0.7 μm) (P = 7.6.10-7). The mean OSI was worse in EBMD patients than in DED patients (P = 0.01) and compared to normal subjects (P = 0.02). The OSI correlated with the epithelial thickness irregularity (Spearman coefficient = 0.54; P = 2.65.10-5).ConclusionsThe OCT pachymetry map demonstrated that EBMD patients had thicker corneal epithelium in the central and inferior region. These changes were correlated with objective measurements of vision quality. This OCT characterisation of the EMBD provides a better understanding of the epithelial behaviour in this dystrophy and its role in vision quality.
| S-EPMC7688121 | biostudies-literature
Project description:Plasma membrane and underlying actin network are connected to a functional unit that by non-linear interactions is capable of forming patterns. For instance, in cell motility and chemotaxis, cells polarize to form a protruding front and a retracting tail. Here we address dynamic patterns that are formed on a planar substrate surface and are therefore easily accessible to optical recording. In these patterns two distinct areas of the membrane and actin cortex are interconverted at the site of circular actin waves. The inner territory circumscribed by a wave is distinguished from the external area by a high PIP3 content and high Ras activity. In contrast, the external area is occupied with the PIP3-degrading phosphatase PTEN. In the underlying cortex, these areas differ in the proteins associated with the actin network. Actin waves can be formed at zones of increasing as well as decreasing Ras activity. Both types of waves are headed by myosin IB. When waves collide, they usually extinguish each other, and their decay is accompanied by the accumulation of coronin. No membrane patterns have been observed after efficient depolymerization of actin, suggesting that residual actin filaments are necessary for the pattern generating system to work. Where appropriate, we relate the experimental data obtained with Dictyostelium to human normal and malignant cell behavior, in particular to the role of Ras-GAP as an enhancer of macropinocytosis, to mutations in the tumor suppressor PTEN, to frustrated phagocytosis, and to the role of coronin in immune cells and neurons.
| S-EPMC6747932 | biostudies-literature
Project description:Pattern recognition receptors (PRRs) at the plasma membrane promote plant immunity through the detection of conserved microbe-associated molecular patterns (MAMPs). In plants, the PRR for bacterial flagellin (flg22) is encoded by the receptor kinase FLS2. One of the earliest MAMP responses is the rapid and transient increase of cytosolic calcium (Ca2+) ions, which is required for many of the well-described downstream responses, e.g. generation of reactive oxygen species (ROS) and the transcriptional activation of defence-associated genes. Despite its relevance, the molecular components regulating the Ca2+ burst remain largely unknown. Here, we show that the plasma membrane P2B-type Ca2+ ATPase ACA8 forms a dynamic complex with the PRR FLS2. ACA8 and its closest homologue ACA10 are required for immunity against virulent bacteria. Mutant aca8 aca10 plants are reduced in the flg22-induced Ca2+ burst, show reduced ROS production and exhibit altered transcriptional reprogramming. In particular, flg22-induced gene expression is elevated downstream of signalling mitogen-activated protein (MAP) kinases, but reduced downstream of the calcium-dependent protein (CDP) kinase cascade. These results demonstrate that the fine regulation of Ca2+ fluxes in the cytosol is critical for the coordination of the downstream MAMP responses and provide for the first time a link between the FLS2 receptor complex and signalling kinases via the secondary messenger Ca2+. ACA8 also interacted with the BRI1 and CLV1 receptor kinases, which correlated with the developmental phenotypes of aca8 aca10 mutants suggesting a broader role for Ca2+ ATPases in receptor-mediated signalling. We used Affymetrix Arabidopsis Tiling 1.0R Array to compare global transcript levels in 7 days-old sterile grown seedlings.
2012-04-25 | GSE31517 | GEO
Project description:Purpose:Contact lenses, osmotic stressors, and chemical burns may trigger severe discomfort and vision loss by damaging the cornea, but the signaling mechanisms used by corneal epithelial cells (CECs) to sense extrinsic stressors are not well understood. We therefore investigated the mechanisms of swelling, temperature, strain, and chemical transduction in mouse CECs. Methods:Intracellular calcium imaging in conjunction with electrophysiology, pharmacology, transcript analysis, immunohistochemistry, and bioluminescence assays of adenosine triphosphate (ATP) release were used to track mechanotransduction in dissociated CECs and epithelial sheets isolated from the mouse cornea. Results:The transient receptor potential vanilloid (TRPV) transcriptome in the mouse corneal epithelium is dominated by Trpv4, followed by Trpv2, Trpv3, and low levels of Trpv1 mRNAs. TRPV4 protein was localized to basal and intermediate epithelial strata, keratocytes, and the endothelium in contrast to the cognate TRPV1, which was confined to intraepithelial afferents and a sparse subset of CECs. The TRPV4 agonist GSK1016790A induced cation influx and calcium elevations, which were abolished by the selective blocker HC067047. Hypotonic solutions, membrane strain, and moderate heat elevated [Ca2+]CEC with swelling- and temperature-, but not strain-evoked signals, sensitive to HC067047. GSK1016790A and swelling evoked calcium-dependent ATP release, which was suppressed by HC067027 and the hemichannel blocker probenecid. Conclusions:These results demonstrate that cation influx via TRPV4 transduces osmotic and thermal but not strain inputs to CECs and promotes hemichannel-dependent ATP release. The TRPV4-hemichannel-ATP signaling axis might modulate corneal pain induced by excessive mechanical, osmotic, and chemical stimulation.
| S-EPMC7401707 | biostudies-literature
Project description:We examined whether p38 MAPK plays role in calcium oxalate monohydrate (COM) crystal-induced tight junction disruption. Polarized MDCK cells were pretreated with or without 20 ?M SB239063 (p38 MAPK inhibitor) for 2-h, and then incubated with 100 ?g/ml COM crystals for up to 48-h. Western blotting showed increased level of phospho-p38, not total p38, in COM-treated cells, whereas SB239063 pretreatment successfully maintained phospho-p38 at its basal level. COM crystals also caused decreased levels of two tight junction proteins, zonula occludens-1 (ZO-1) and occludin. Immunofluorescence study revealed disruption of tight junction, redistribution, and dissociation of ZO-1 and occludin. Moreover, transepithelial resistance (TER) showed defective barrier function, whereas Western blotting for Na(+)/K(+)-ATPase-?1 revealed defective fence function of tight junction in COM-treated cells. All these expression and functional defects were successfully prevented by SB239063 pretreatment. These findings indicate that COM crystals cause tight junction disruption in distal renal tubular epithelial cells through p38 MAPK activation.
| S-EPMC3540397 | biostudies-literature