Magnetic fields applied to collagen-coated ferric oxide beads induce stretch-activated Ca2+ flux in fibroblasts

1995 ◽  
Vol 269 (5) ◽  
pp. C1093-C1104 ◽  
Author(s):  
M. Glogauer ◽  
J. Ferrier ◽  
C. A. McCulloch
Keyword(s):  
Cell Membrane ◽  
Magnetic Force ◽  
Calcium Influx ◽  
Single Cells ◽  
Cell Spreading ◽  
Calcium Flux ◽  
Filamentous Actin ◽  
Physical Force ◽  
Physical Stimuli ◽  
Applied Forces

The ability to apply controlled forces to the cell membrane may enable elucidation of the mechanisms and pathways involved in signal transduction in response to applied physical stimuli. We have developed a magnetic particle-electromagnet model that allows the application of controlled forces to the plasma membrane of substrate-attached fibroblasts. The system allows applied forces to be controlled by the magnitude of the magnetic field and by the surface area of cell membrane covered with collagen-coated ferric beads. Analysis by single-cell ratio fluorimetry of fura 2-loaded cells demonstrated large calcium transients (50-300 nM) in response to the magnetic force applications. Experiments using either the stretch-activated channel blocker gadolinium chloride or ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to eliminate external calcium ions, or addition of extracellular manganese ions, indicated that there was a calcium influx through putative stretch-activated channels. The probability of a calcium influx in single cells was increased by higher surface bead loading and the degree of cell spreading. Depolymerization of actin filaments by cytochalasin D increased the amplitude of calcium response twofold. The regulation of calcium flux by filamentous actin content and by cell spreading indicates a possible modulatory role for the cytoskeleton in channel sensitivity. Magnetic force application to beads on single cells provides a controlled model to study mechanisms and heterogeneity in physical force stimulation of cation-permeable channels.

Null Mutation of Pleckstrin2, a PI3K Effector, Impairs Platelet Secretion.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3522-3522
Author(s):  
Yanfeng Wang ◽  
Lurong Lian ◽  
Charles S. Abrams
Keyword(s):  
Cell Membrane ◽  
Inositol Phosphates ◽  
Cell Spreading ◽  
Lipid Binding ◽  
Jurkat Cells ◽  
Null Mutation ◽  
Filamentous Actin ◽  
Dense Granules ◽  
Ph Domains ◽  
Platelet Secretion

Abstract Pleckstrin-2, a widely expressed paralog of pleckstrin, is composed of two Pleckstrin Homology (PH) domains and Disheveled-Egl 10-Pleckstrin (DEP) domain. Although the activity of pleckstrin is regulated by its’ phosphorylation state, pleckstrin-2 is not a phospho-protein suggesting that it possesses a different mechanism of regulation. Previous reports have shown that many PH domains mediate protein binding to inositol phosphates and phospholipids, thus regulating protein function. Therefore, we speculated that localized production of specific polyphosphatidylinositols might bind, and activate, pleckstrin-2. Using a lipid-binding assay, we found that pleckstrin-2 bound with greatest affinity to the products of phosphatidylinositol 3-kinase and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bound the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. GFP-tagged pleckstrin-2 had a cytoplasmic distribution in non-adherent Jurkat cells, but through a pathway dependent on the phospholipid-binding pocket of its PH domains, rapidly moved to the cell membrane following adhesion to immobilized fibronectin. Once bound to the cell membrane, pleckstrin-2 enhanced Jurkat cell spreading 2-fold and increased membrane ruffling. The membrane association of pleckstrin-2, and its resultant cell spreading, were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. To investigate the role of this protein within platelets, we generated mice containing a null mutation within the pleckstrin-2 gene. Pleckstrin-2 null mice were born at the expected frequency, and had no overt spontaneous hemorrhagic events. Mice lacking pleckstrin-2 had normal platelet counts and morphologic appearance of their megakaryocytes. Following stimulation of the PAR-4 (thrombin) receptor, pleckstrin-2 knockout platelets displayed normal assembly of filamentous actin. However, pleckstrin-2 null platelets had impaired aggregation following stimulation by collagen or submaximal doses of the PAR-4 activating peptide. Since pleckstrin-2 deficient platelets aggregated normally in response to ADP, these results suggested that these platelets might have an impaired ability to secrete dense granules. Accordingly, we found that pleckstrin-2 null platelets had a defect in their ability to secrete ATP in response to stimulation by 5μM collagen or 200μM of the PAR-4 activating peptide. However, pleckstrin-2 knockout platelets did incorporate 14C-serotonin as efficiently as wild type platelets. This latter observation suggested that the secretion defect in pleckstrin-2 null platelets was not attributable to a deficiency of dense granules, but instead is due to a defect in exocytosis of granules. Together, these data suggest that the PH domains of pleckstrin-2 cooperatively bind PI3K generated phospholipids on the cell membrane, and help mediate platelet secretion.

scholarly journals Voltage-gated calcium flux mediates Escherichia coli mechanosensation

2017 ◽  
Vol 114 (35) ◽  
pp. 9445-9450 ◽  
Author(s):  
Giancarlo N. Bruni ◽  
R. Andrew Weekley ◽  
Benjamin J. T. Dodd ◽  
Joel M. Kralj
Keyword(s):  
Escherichia Coli ◽  
Calcium Influx ◽  
Single Cells ◽  
Calcium Flux ◽  
Excitable Cells ◽  
Intracellular Signals ◽  
Downstream Effects ◽  
New Findings ◽  
Dynamic Voltage ◽  
Induced Changes

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli, including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.

scholarly journals Investigation of an Optical Imaging Platform Integrated with an Ultrasound Application System for In Vitro Verification of Ultrasound-Mediated Drug Delivery

2021 ◽  
Vol 11 (6) ◽  
pp. 2846
Author(s):  
Jong-ryul Choi ◽  
Juyoung Park
Keyword(s):  
Drug Delivery ◽  
Cervical Cancer ◽  
Cell Membrane ◽  
Optical Imaging ◽  
Cancer Cell ◽  
Fluorescent Imaging ◽  
Calcium Flux ◽  
Cervical Cancer Cell ◽  
Application System ◽  
Ultrasound Application

Techniques that increase the permeability of the cell membrane and transfer drugs or genes to cells have been actively developed as effective therapeutic modalities. Also, in line with the development of these drug delivery techniques, the establishment of tools to verify the techniques at the cellular level is strongly required. In this study, we demonstrated an optical imaging platform integrated with an ultrasound application system to verify the feasibility of safe and efficient drug delivery through the cell membrane using ultrasound-microbubble cavitation. To examine the potential of the platform, fluorescence images of both Fura-2 AM and propidium iodide (PI) to measure calcium flux changes and intracellular PI delivery, respectively, during and after the ultrasound-microbubble cavitation in the cervical cancer cell were acquired. Using the optical imaging platform, we determined that calcium flux increased immediately after the ultrasound-microbubble cavitation and were restored to normal levels, and fluorescence signals from intracellular PI increased gradually after the cavitation. The results acquired by the platform indicated that ultrasound-microbubble cavitation can deliver PI into the cervical cancer cell without irreversible damage of the cell membrane. The application of an additional fluorescent imaging module and high-speed imaging modalities can provide further improvement of the performance of this platform. Also, as additional studies in ultrasound instrumentations to measure real-time cavitation signals progress, we believe that the ultrasound-microbubble cavitation-based sonoporation can be employed for safe and efficient drug and gene delivery to various cancer cells.

scholarly journals TRPC6 channel as an emerging determinant of the podocyte injury susceptibility in kidney diseases

2015 ◽  
Vol 309 (5) ◽  
pp. F393-F397 ◽  
Author(s):  
Daria V. Ilatovskaya ◽  
Alexander Staruschenko
Keyword(s):  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Kidney Diseases ◽  
Critical Role ◽  
Receptor Potential ◽  
Calcium Flux ◽  
Calcium Handling ◽  
Podocyte Injury ◽  
Trpc6 Channel ◽  
Transient Receptor

Podocytes (terminally differentiated epithelial cells of the glomeruli) play a key role in the maintenance of glomerular structure and permeability and in the incipiency of various renal abnormalities. Injury to podocytes is considered a major contributor to the development of kidney disease as their loss causes proteinuria and progressive glomerulosclerosis. The physiological function of podocytes is critically dependent on proper intracellular calcium handling; excessive calcium influx in these cells may result in the effacement of foot processes, apoptosis, and subsequent glomeruli damage. One of the key proteins responsible for calcium flux in the podocytes is transient receptor potential cation channel, subfamily C, member 6 (TRPC6); a gain-of-function mutation in TRPC6 has been associated with the onset of the familial forms of focal segmental glomerulosclerosis (FSGS). Recent data also revealed a critical role of this channel in the onset of diabetic nephropathy. Therefore, major efforts of the research community have been recently dedicated to unraveling the TRPC6-dependent effects in the initiation of podocyte injury. This mini-review focuses on the TRPC6 channel in podocytes and colligates recent data in an attempt to shed some light on the mechanisms underlying the pathogenesis of TRPC6-mediated glomeruli damage and its potential role as a therapeutic target for the treatment of chronic kidney diseases.

Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching

1997 ◽  
Vol 110 (1) ◽  
pp. 11-21 ◽  
Author(s):  
M. Glogauer ◽  
P. Arora ◽  
G. Yao ◽  
I. Sokholov ◽  
J. Ferrier ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Ions ◽  
Calcium Influx ◽  
Magnetic Bead ◽  
Mechanical Stimuli ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Force Application ◽  
Membrane Rigidity ◽  
In Cells

The actin-dependent sensory and response elements of stromal cells that are involved in mechanical signal transduction are poorly understood. To study mechanotransduction we have described previously a collagen-magnetic bead model in which application of well-defined forces to integrins induces an immediate (< 1 second) calcium influx. In this report we used the model to determine the role of calcium ions and tyrosine-phosphorylation in the regulation of force-mediated actin assembly and the resulting change in membrane rigidity. Collagen-beads were bound to cells through the focal adhesion-associated proteins talin, vinculin, alpha 2-integrin and beta-actin, indicating that force application was mediated through cytoskeletal elements. When force (2 N/m2) was applied to collagen beads, confocal microscopy showed a marked vertical extension of the cell which was counteracted by an actin-mediated retraction. Immunoblotting showed that force application induced F-actin accumulation at the bead-membrane complex but vinculin, talin and alpha 2-integrin remained unchanged. Atomic force microscopy showed that membrane rigidity increased 6-fold in the vicinity of beads which had been exposed to force. Force also induced tyrosine phosphorylation of several cytoplasmic proteins including paxillin. The force-induced actin accumulation was blocked in cells loaded with BAPTA/AM or in cells preincubated with genistein, an inhibitor of tyrosine phosphorylation. Repeated force application progressively inhibited the amplitude of force-induced calcium ion flux. As force-induced actin reorganization was dependent on calcium and tyrosine phosphorylation, and as progressive increases of filamentous actin in the submembrane cortex were correlated with increased membrane rigidity and dampened calcium influx, we suggest that cortical actin regulates stretch-activated cation permeable channel activity and provides a desensitization mechanism for cells exposed to repeated long-term mechanical stimuli. The actin response may be cytoprotective since it counteracts the initial force-mediated membrane extension and potentially strengthens cytoskeletal integrity at force-transfer points.

Detection of Similar Elastic Properties Using a Magnetic Force Controlled Afm

1996 ◽  
Vol 436 ◽  
Author(s):  
Shin-Ichi Yamamoto ◽  
Hirofumi Yamada ◽  
Suzanne P. Jarvis ◽  
Makoto Motomatsu ◽  
Hiroshi Tokumoto
Keyword(s):  
Magnetic Field ◽  
External Magnetic Field ◽  
Elastic Properties ◽  
Magnetic Force ◽  
Contact Stiffness ◽  
Regional Variations ◽  
Young’S Moduli ◽  
Local Contact ◽  
Applied Forces ◽  
Afm Cantilever

AbstractWe have investigated regional variations of elastic properties using a magnetic force controlled AFM. A piece of small magnet was fixed at the end of the backside of the AFM cantilever so as to apply forces directly to the tip through the external magnetic field of an electromagnet. By modulating the applied forces to the tip and measuring the resulting amplitude of oscillation, a sensitive measurement of the local contact stiffness can be made. We have applied this technique to phase-separated films of polystyrene/polymethylmethacrylate (PS-PMMA) which have almost identical Young's moduli.

Human placental lactogen release in vitro: paradoxical effects of calcium

1981 ◽  
Vol 240 (5) ◽  
pp. E550-E555
Author(s):  
S. Handwerger ◽  
P. M. Conn ◽  
J. Barrett ◽  
S. Barry ◽  
A. Golander
Keyword(s):  
Calcium Influx ◽  
Extracellular Calcium ◽  
Calcium Flux ◽  
Placental Lactogen ◽  
Human Placental Lactogen ◽  
Paradoxical Effects ◽  
Subsequent Exposure ◽  
Normal Calcium

To study the effects of calcium on the release of human placental lactogen (hPL), placental explants were exposed to media containing lower or higher concentrations of calcium than normally available to the placenta. Explants exposed for 2 h to calcium-poor medium or medium containing either 2 mM EDTA or 2 mM EGTA released 160, 248, and 253% more hPL, respectively, than control explants. In contrast, explants exposed to medium containing higher than normal calcium concentrations released the same amounts of hPL as the control explants. At lower than normal extracellular calcium concentrations, the increased hPL release was inversely proportional to the calcium concentration. The increased release in calcium-poor medium was inhibited by subsequent exposure of the explants to medium containing calcium and was prevented by either barium or magnesium. Changes in barium or magnesium concentrations, however, had no effects on hPL release in the presence of normal extracellular calcium concentrations. Methoxyverapamil (D 600), an inhibitor of calcium flux, stimulated hPL release. Because low extracellular calcium and methoxyverapamil both inhibit calcium influx, these experiments suggest that calcium influx inhibits hPL release. The role of calcium in the regulation of hPL release therefore appears to be different from that reported in other release systems.

Calcium influx determines the muscular response to electrotransfer

2012 ◽  
Vol 302 (4) ◽  
pp. R446-R453 ◽  
Author(s):  
Pernille Hojman ◽  
Camilla Brolin ◽  
Hanne Gissel
Keyword(s):  
Cell Membrane ◽  
High Voltage ◽  
Voltage Pulse ◽  
Low Voltage ◽  
Calcium Influx ◽  
High Voltage Pulse ◽  
Electric Pulses ◽  
Tnf Α ◽  
Cell Membrane Permeabilization ◽  
The Times

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca2+-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca2+ was assessed using 45Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca2+ influx and muscular response. Moderate Ca2+ influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca2+ influx. Larger Ca2+ influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca2+ influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca2+ may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.

Influence of Mir-155 On B-Cell Receptor Signaling in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2343-2343
Author(s):  
Liguang Chen ◽  
Bing Cui ◽  
George Chen ◽  
Michelle Salcedo ◽  
Carlo M. Croce ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Calcium Influx ◽  
Critical Role ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Lymphocytic Leukemia ◽  
Calcium Flux ◽  
Expression Levels ◽  
Bcr Signaling

Abstract Abstract 2343 Poster Board II-320 B-cell receptor (BCR) signaling arguably plays an important role in the pathogenesis and/or progression of chronic lymphocytic leukemia. Ligation of the BCR by F(ab)2 anti-μ can induce phosphorylation of p72Syk, BLNK, phospholipase C-gamma (PLCγ) and other downstream adapter/signaling molecules, inducing intracellular calcium flux and cellular activation. Prior studies found that CLL cells that expressed unmutated Ig heavy-chain variable region genes (IGHV) and the zeta-associated protein of 70 kD (ZAP-70) generally experienced greater levels of activation following treatment with anti-μ than did CLL cells that lacked expression of ZAP-70. However, we found unusual cases that lacked expression of ZAP-70 that also responded vigorously to treatment with anti-μ, suggesting that other factors contribute to the noted differences in BCR-signaling. Analyses for expression of microRNAs by microarray revealed that CLL cells that used unmutated IGHV and that expressed ZAP-70 expressed higher levels of certain microRNAs than did cases that used mutated IGHV and that lacked expression of ZAP-70. One of such microRNA, miR-155, was found to target mRNA encoding SHIP-1, a phosphatase that plays a critical role in modulating the level of BCR signaling in normal B cells. Using quantitative assays for miR-155 we found high-level expression of this microRNA was associated with proficient BCR signaling in CLL. To examine whether miR-155 could modulate the levels of SHIP-1 and/or BCR signaling in CLL cells we transfected primary leukemia cells from each of multiple patients with control oligo-RNAs, miR-155, or a specific inhibitor of miR-155 (miR-155 inhibitor). Twenty-four hours later the cells were stimulated with anti-μ or control antibody and then examined 10 minutes later for expression of SHIP-1, induced calcium influx, or phosphorylation of kinases and adapter proteins that are involved in BCR signaling. CLL cells that had low expression levels of miR-155 and that were poorly responsive BCR had significantly higher levels of calcium influx and phosphorylated p72Syk, BLNK, and PLCγ in response to anti-μ following transfection with miR-155 than following mock transfection or transfection with control oligo-RNA. Conversely, CLL cells that had high expression levels of miR-155 and highly responsive BCR were made to have significantly higher amounts of SHIP-1 protein and to have significantly lower relative levels of phosphorylated protein and calcium influx in response to anti-μ following transfection with the miR-155 inhibitor than did mock transfected CLL cells. These results identify miR-155 as a factor that can modulate BCR signaling in CLL in part by regulating the relative expression level of SHIP-1. These results demonstrate that differential expression of microRNAs in CLL can influence physiologic features that potentially contribute to disease progression. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium.

1993 ◽  
Vol 120 (4) ◽  
pp. 1003-1010 ◽  
Author(s):  
M A Schwartz
Keyword(s):  
Endothelial Cells ◽  
Calcium Channels ◽  
Intracellular Ph ◽  
Time Course ◽  
Single Cells ◽  
State Level ◽  
Cell Spreading ◽  
Free Calcium ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels

Intracellular calcium ([Ca2+]i) was measured in FURA 2-loaded endothelial cells plated on fibronectin or vitronectin. Average values for [Ca2+]i increased to approximately twofold above basal levels by approximately 1 h after plating, and then declined. The increase in [Ca2+]i required extracellular calcium. Substituting potassium for sodium in the medium reduced the elevation of [Ca2+]i, a result that rules out the involvement of Na-Ca exchangers or voltage-dependent calcium channels, but that is consistent with the involvement of voltage-independent calcium channels. Plating cells on an anti-integrin beta 1 subunit antibody gave a similar [Ca2+]i response, but clustering beta 1 integrins with the same antibody, or occupying integrins with RGD (arg-gly-asp) peptides had no effect. Time course measurements on single cells revealed that in each cell [Ca2+]i rose abruptly at some point during spreading, from the basal level to a higher steady-state level that was maintained for some time. The elevated [Ca2+]i was unrelated to previously observed changes in intracellular pH, because chelating the Ca2+ in the medium failed to inhibit the elevation of pHi that occurred during cell spreading. In conclusion, these results show that integrin-mediated cell spreading can regulate [Ca2+]i, and the pathways involved are distinct from those that regulate intracellular pH.

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