Cytosolic Free Calcium Research Articles

Store-operated calcium entry compensates fast ER calcium loss in resting hippocampal neurons

The endoplasmic reticulum (ER) acts as a dynamic calcium store and is involved in the generation of specific patterns of calcium signals in neurons. Calcium is mobilized from the ER store by multiple signaling cascades, and neuronal activity is known to regulate ER calcium levels. We asked how neurons regulate ER calcium levels in the resting state. Direct ER calcium imaging showed that ER calcium was lost quite rapidly from the somatic and dendritic ER when resting neurons were transiently kept under calcium-free conditions. Interestingly, free ER and free cytosolic calcium was lost continuously across the plasma membrane and was not held back in the cytosol, implying the presence of a prominent calcium influx mechanism to maintain ER calcium levels at rest. When neurons were treated acutely with inhibitors of store-operated calcium entry (SOCE), an immediate decline in ER calcium levels was observed. This continuous SOCE-like calcium entry did not require the activation of a signaling cascade, but was rather a steady-state phenomenon. The SOCE-like mechanism maintains medium–high ER calcium levels at rest and is essential for balanced resting calcium levels in the ER and cytosol.

The expression of the truncated isoform of somatostatin receptor subtype 5 associates with aggressiveness in medullary thyroid carcinoma cells

The truncated somatostatin receptor variant sst5TMD4 associates with increased invasiveness and aggressiveness in breast cancer. We previously found that sst5 activation may counteract sst2 selective agonist effects in a medullary thyroid carcinoma (MTC) cell line, the TT cells, and that sst5TMD4 is overexpressed in poorly differentiated thyroid cancers. The purpose of this study is to evaluate sst5TMD4 expression in a series of human MTC and to explore the functional role of sst5TMD4 in TT cells. We evaluated sst5TMD4 and sst5 expression in 36 MTC samples. Moreover, we investigated the role of sst5TMD4 in TT cells evaluating cell number, DNA synthesis, free cytosolic calcium concentration ([Ca(2+)]i), calcitonin and vascular endothelial growth factor levels, cell morphology, protein expression, and invasion. We found that in MTC the balance between sst5TMD4 and sst5 expression influences disease stage. sst5TMD4 overexpression in TT cells confers a greater growth capacity, blocks sst2 agonist-induced antiproliferative effects, modifies the cell phenotype, decreases E-cadherin and phosphorylated β-catenin levels, increases vimentin, total β-catenin and phosphorylated GSK3B levels (in keeping with the development of epithelial to mesenchymal transition), and confers a greater invasion capacity. This is the first evidence indicating that sst5TMD4 is expressed in human MTC cells, where it associates with more aggressive behavior, suggesting that sst5TMD4 might play a functionally relevant role.

A novel, rapid method to quantify intraplatelet calcium dynamics by ratiometric flow cytometry.

Cytosolic free calcium ions represent important second-messengers in platelets. Therefore, quantitative measurement of intraplatelet calcium provides a popular and very sensitive tool to evaluate platelet activation and reactivity. Current protocols for determination of intracellular calcium concentrations in platelets have a number of limitations. Cuvette-based methods do not allow measurement of calcium flux in complex systems, such as whole blood, and therefore require isolation steps that potentially interfere with platelet activation. Flow cytometry has the potential to overcome this limitation, but to date the application of calibrated, quantitative readout of calcium kinetics has only been described for Indo-1. As excitation of Indo-1 requires a laser in the ultraviolet range, such measurements cannot be performed with a standard flow cytometer. Here, we describe a novel, rapid calibration method for ratiometric calcium measurement in platelets using both Ar+-laser excited fluorescence dyes Fluo-4 and Fura Red. We provide appropriate equations that allow rapid quantification of intraplatelet calcium fluxes by measurement of only two standardisation buffers. We demonstrate that this method allows quantitative calcium measurement in platelet rich plasma as well as in whole blood. Further, we show that this method prevents artefacts due to platelet aggregate formation and is therefore an ideal tool to determine basal and agonist induced calcium kinetics.

Renal autoregulation in health and disease.

Intrarenal autoregulatory mechanisms maintain renal blood flow (RBF) and glomerular filtration rate (GFR) independent of renal perfusion pressure (RPP) over a defined range (80-180 mmHg). Such autoregulation is mediated largely by the myogenic and the macula densa-tubuloglomerular feedback (MD-TGF) responses that regulate preglomerular vasomotor tone primarily of the afferent arteriole. Differences in response times allow separation of these mechanisms in the time and frequency domains. Mechanotransduction initiating the myogenic response requires a sensing mechanism activated by stretch of vascular smooth muscle cells (VSMCs) and coupled to intracellular signaling pathways eliciting plasma membrane depolarization and a rise in cytosolic free calcium concentration ([Ca(2+)]i). Proposed mechanosensors include epithelial sodium channels (ENaC), integrins, and/or transient receptor potential (TRP) channels. Increased [Ca(2+)]i occurs predominantly by Ca(2+) influx through L-type voltage-operated Ca(2+) channels (VOCC). Increased [Ca(2+)]i activates inositol trisphosphate receptors (IP3R) and ryanodine receptors (RyR) to mobilize Ca(2+) from sarcoplasmic reticular stores. Myogenic vasoconstriction is sustained by increased Ca(2+) sensitivity, mediated by protein kinase C and Rho/Rho-kinase that favors a positive balance between myosin light-chain kinase and phosphatase. Increased RPP activates MD-TGF by transducing a signal of epithelial MD salt reabsorption to adjust afferent arteriolar vasoconstriction. A combination of vascular and tubular mechanisms, novel to the kidney, provides for high autoregulatory efficiency that maintains RBF and GFR, stabilizes sodium excretion, and buffers transmission of RPP to sensitive glomerular capillaries, thereby protecting against hypertensive barotrauma. A unique aspect of the myogenic response in the renal vasculature is modulation of its strength and speed by the MD-TGF and by a connecting tubule glomerular feedback (CT-GF) mechanism. Reactive oxygen species and nitric oxide are modulators of myogenic and MD-TGF mechanisms. Attenuated renal autoregulation contributes to renal damage in many, but not all, models of renal, diabetic, and hypertensive diseases. This review provides a summary of our current knowledge regarding underlying mechanisms enabling renal autoregulation in health and disease and methods used for its study.

Two Dimensional Finite Element Model to Study Calcium Distribution in Oocytes

Cytosolic free calcium concentration is a key regulatory factor and perhaps the most widely used means of controlling cellular function. Calcium can enter cells through different pathways which are activated by specific stimuli including membrane depolarization, chemical signals and calcium depletion of intracellular stores. One of the important components of oocyte maturation is differentiation of the Ca 2+ signaling machinery which is essential for egg activation after fertilization. Eggs acquire the ability to produce the fertilization-specific calcium signal during oocyte maturation. The calcium concentration patterns required during different stages of oocyte maturation are still not completely known. Also the mechanisms involved in calcium dynamics in oocyte cell are still not well understood. In view of above a two dimensional FEM model has been proposed to study calcium distribution in an oocyte cell. The parameters such as buffers, ryanodine receptor, SERCA pump and voltage gated calcium channel are incorporated in the model. Based on the biophysical conditions the initial and boundary conditions have been framed. The model is transformed into variational form and Ritz finite element method has been employed to obtain the solution. A program has been developed in MATLAB 7.10 for the entire problem and executed to obtain numerical results. The numerical results have been used to study the effect of buffers, RyR, SERCA pump and VGCC on calcium distribution in an oocyte cell.

Different NaCl-induced calcium signatures in the Arabidopsis thaliana ecotypes Col-0 and C24.

A common feature of stress signalling pathways are alterations in the concentration of cytosolic free calcium ([Ca2+]cyt), which allow the specific and rapid transmission of stress signals through a plant after exposure to a stress, such as salinity. Here, we used an aequorin based bioluminescence assay to compare the NaCl-induced changes in [Ca2+]cyt of the Arabidopsis ecotypes Col-0 and C24. We show that C24 lacks the NaCl specific component of the [Ca2+]cyt signature compared to Col-0. This phenotypic variation could be exploited as a screening methodology for the identification of yet unknown components in the early stages of the salt signalling pathway.

A role for calcium in the regulation of ATP-binding cassette, sub-family C, member 3 (ABCC3) gene expression in a model of epidermal growth factor-mediated breast cancer epithelial–mesenchymal transition

Epithelial–mesenchymal transition (EMT), a process implicated in cancer metastasis, is associated with the transcriptional regulation of members of the ATP-binding cassette superfamily of efflux pumps, and drug resistance in breast cancer cells. Epidermal growth factor (EGF)-induced EMT in MDA-MB-468 breast cancer cells is calcium signal dependent. In this study induction of EMT was shown to result in the transcriptional up-regulation of ATP-binding cassette, subfamily C, member 3 (ABCC3), a member of the ABC transporter superfamily, which has a recognized role in multidrug resistance. Buffering of cytosolic free calcium inhibited EGF-mediated ABCC3 increases, indicating a calcium-dependent mode of regulation. Silencing of TRPM7 (an ion channel involved in EMT associated vimentin induction) did not inhibit ABCC3 up-regulation. Silencing of the store operated calcium entry (SOCE) pathway components ORAI1 and STIM1 also did not alter ABCC3 induction by EGF. However, the calcium permeable ion channel transient receptor potential cation channel, subfamily C, member 1 (TRPC1) appears to contribute to the regulation of both basal and EGF-induced ABCC3 mRNA. Improved understanding of the relationship between calcium signaling, EMT and the regulation of genes important in therapeutic resistance may help identify novel therapeutic targets for breast cancer.

Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance.

Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca(2+)/H(+) exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca(2+)/H(+) exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca(2+) concentration ([Ca(2+)](c)) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)-survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca(2+)](c), carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

Calcium signals inhibition sensitizes ovarian carcinoma cells to anti-Bcl-xL strategies through Mcl-1 down-regulation.

Ovarian carcinoma is the leading cause of death from gynecologic cancer in the developed world and is characterized by acquired chemoresistance leading to an overall 5-year survival rate of about 30 %. We previously showed that Bcl-xL and Mcl-1 cooperatively protect platinum-resistant ovarian cancer cells from apoptosis. Despite BH3-mimetics represent promising drugs to target Bcl-xL, anti-Mcl-1 strategies are still in pre-clinical studies and required new investigations. Calcium is a universal second messenger and dysregulation of calcium signal is often observed during carcinogenesis. As change in cytosolic free calcium concentration [Ca2+]i is known to control the fate of the cell by regulating Bcl-2 family members, we wonder if calcium signal could impact on Mcl-1 expression and if its pharmacological inhibition could be useful to sensitize ovarian carcinoma cells to anti-Bcl-xL strategies. We therefore studied the effect of different calcium signals inhibitors in ovarian carcinoma cell lines SKOV3 and IGROV1-R10 and analysed their effects on proliferation and Mcl-1 expression. We also exposed these cells to these inhibitors in combination with anti-Bcl-xL strategies (siRNA or BH3-mimetic: ABT-737). We found that calcium signaling regulates Mcl-1 through translational events and a calmodulin-mediated pathway. BAPTA-AM and calmodulin inhibitor combination with ABT-737 leads to apoptosis, a process that is reversed by Mcl-1 enforced expression. As Mcl-1 represents a crucial hurdle to the success of chemotherapy, these results could open to new area of investigation using calcium modulators to directly or indirectly target Mcl-1 and thus efficiently sensitize ovarian carcinoma cells to anti-Bcl-xL strategies.Electronic supplementary materialThe online version of this article (doi:10.1007/s10495-015-1095-3) contains supplementary material, which is available to authorized users.

Calcium Transport across Plasma Membrane in Early Stages of Chronic Kidney Disease – Impact of Vitamin D3 Supplementation

Mini review summarizes the results of studies focused on elucidating pathophysiological mechanisms of altered intracellular calcium homeostasis in the peripheral blood mononuclear cells of patients with early stages of chronic kidney disease. The basic mechanisms of calcium entry as well as those of its removal are impaired by the disease. These disturbances cause an increase concentration of free cytosolic calcium which can result in a change of broad range of cellular processes and expression patterns of various signaling molecules. Vitamin D3 supplementation is the standard treatment of frequent vitamin D3 insufficiency or deficiency in these patients. It can be assumed that vitamin D3 through the pleiotropic effects may participate in modulation of intracellular calcium homeostasis. Vitamin D3 supplementation resulted in a reduction of cytosolic free calcium affecting some of the transport systems involved in cell calcium entry as well as calcium exit. Normalization of the cytosolic free calcium concentration can have a beneficial effect on intracellular signaling. The mechanisms for regulating and controlling intracellular calcium homeostasis in chronic kidney disease patients are currently still under investigation.

Soybean and casein hydrolysates induce grapevine immune responses and resistance against Plasmopara viticola.

Plasmopara viticola, the causal agent of grapevine downy mildew, is one of the most devastating grape pathogen in Europe and North America. Although phytochemicals are used to control pathogen infections, the appearance of resistant strains and the concern for possible adverse effects on environment and human health are increasing the search for alternative strategies. In the present investigation, we successfully tested two protein hydrolysates from soybean (soy) and casein (cas) to trigger grapevine resistance against P. viticola. On Vitis vinifera cv. Marselan plants, the application of soy and cas reduced the infected leaf surface by 76 and 63%, as compared to the control, respectively. Since both hydrolysates might trigger the plant immunity, we investigated their ability to elicit grapevine defense responses. On grapevine cell suspensions, a different free cytosolic calcium signature was recorded for each hydrolysate, whereas a similar transient phosphorylation of two MAP kinases of 45 and 49 kDa was observed. These signaling events were followed by transcriptome reprogramming, including the up-regulation of defense genes encoding pathogenesis-related (PR) proteins and the stilbene synthase enzyme responsible for the biosynthesis of resveratrol, the main grapevine phytoalexin. Liquid chromatography analyses confirmed the production of resveratrol and its dimer metabolites, δ- and ε-viniferins. Overall, soy effects were more pronounced as compared to the cas ones. Both hydrolysates proved to act as elicitors to enhance grapevine immunity against pathogen attack.

When isolated at full receptivity, in vitro fertilized wheat (Triticum aestivum, L.) egg cells reveal [Ca2+]cyt oscillation of intracellular origin.

During in vitro fertilization of wheat (Triticum aestivum, L.) in egg cells isolated at various developmental stages, changes in cytosolic free calcium ([Ca2+]cyt) were observed. The dynamics of [Ca2+]cyt elevation varied, reflecting the difference in the developmental stage of the eggs used. [Ca2+]cyt oscillation was exclusively observed in fertile, mature egg cells fused with the sperm cell. To determine how [Ca2+]cyt oscillation in mature egg cells is generated, egg cells were incubated in thapsigargin, which proved to be a specific inhibitor of the endoplasmic reticulum (ER) Ca2+-ATPase in wheat egg cells. In unfertilized egg cells, the addition of thapsigargin caused an abrupt transient increase in [Ca2+]cyt in the absence of extracellular Ca2+, suggesting that an influx pathway for Ca2+ is activated by thapsigargin. The [Ca2+]cyt oscillation seemed to require the filling of an intracellular calcium store for the onset of which, calcium influx through the plasma membrane appeared essential. This was demonstrated by omitting extracellular calcium from (or adding GdCl3 to) the fusion medium, which prevented [Ca2+]cyt oscillation in mature egg cells fused with the sperm. Combined, these data permit the hypothesis that the first sperm-induced transient increase in [Ca2+]cyt depletes an intracellular Ca2+ store, triggering an increase in plasma membrane Ca2+ permeability, and this enhanced Ca2+ influx results in [Ca2+]cyt oscillation.

Silencing Mutations in NOTCH1 Activate Calcium Signaling in B Cells

The NOTCH1 pathway is known to be up-regulated in many cancer types including non Hodgkin lymphomas (NHLs), colorectal, breast and pancreatic cancers. The precise downstream effects of NOTCH1 mutations in lymphoma are not known.We applied high throughput sequencing in three different non Hodgkin lymphomas including diffuse large B cell lymphoma (DLBCL, N=94), mantle cell lymphoma (MCL, N=58) and Burkitt lymphoma (BL, N=59). We also sequenced mutations from 208 patients with immune deficiency. In all, we identified 31 mutations in NOTCH1. Five of these mutations were either nonsense or frameshift mutations. Silencing mutations in the PEST domain have been reported in MCLs, and many other silencing mutations were observed in the other lymphoma cohorts as well as immune deficiency cases. The finding of silencing mutations was unexpected, given that in most cancers, NOTCH1 and its pathway are often constitutively active.Calcium signaling is an important mechanism for activating B cells. Given the shared mutations between NHLs and immune deficiencies, we investigated the role of silencing NOTCH1 mutations in altering calcium signaling by engineering lymphoma cells with a deletion of the transmembrane region resulting in silencing of NOTCH1 using the CRISPR system.We designed guide RNAs that spanned a 57 base pair region from exon 28 that encodes the transmembrane domain of the NOTCH1 gene. Each guide RNA was ligated into a pX458 Cas9 GFP expressing plasmid. Namalwa NHL cells were transfected, FACS-sorted for GFP positivity and subcloned. Successful deletion of the target region in Cas9 NOTCH1 cells was confirmed by Sanger sequencing and Western blotting.Cells that were wild type, Cas9 control, or Cas9 NOTCH1 were incubated in a calcium free buffer containing Fluo-4 for detection of intracellular free calcium. Cells were washed and subjected to flow sorting where baseline calcium was recorded. The cells were stimulated with anti-IgM (Fab’)2 and recorded again for response. To test extracellular calcium influx, 4mM CaCl2 was added to the calcium free buffer containing cells and recorded again for response.Upon stimulation with anti-IgM (Fab’)2, we observed a significant increase (p<10-6) of free cytosolic Calcium in the Cas9 NOTCH1 cells compared to both wild type and Cas9 control. Similar results were obtained when measuring extracellular Calcium influx upon addition of CaCl2to the buffered solution of cells.These findings indicate that silencing of NOTCH1 has an activating role in calcium signaling that could contribute to B cell survival and function in NHLs and immune deficiencies. Similar roles have been reported for activating mutations in PIK3CD and STAT3 that contribute to both immune deficiency and cancer. Our study is the first to identify the role of silencing mutations of NOTCH1 in NHLs and immune deficiencies. DisclosuresNo relevant conflicts of interest to declare.

Nicotinic α7 receptor activation selectively potentiates the function of NMDA receptors in glutamatergic terminals of the nucleus accumbens.

We here provide functional and immunocytochemical evidence supporting the co-localization and functional interaction between nicotinic acetylcholine receptors (nAChRs) and N-methyl-D-aspartic acid receptors (NMDARs) in glutamatergic terminals of the nucleus accumbens (NAc). Immunocytochemical studies showed that a significant percentage of NAc terminals were glutamatergic and possessed GluN1 and α7-containing nAChR. A short-term pre-exposure of synaptosomes to nicotine (30 µM) or choline (1 mM) caused a significant potentiation of the 100 µM NMDA-evoked [3H]D-aspartate ([3H]D-Asp) outflow, which was prevented by α-bungarotoxin (100 nM). The pre-exposure to nicotine (100 µM) or choline (1 mM) also enhanced the NMDA-induced cytosolic free calcium levels, as measured by FURA-2 fluorescence imaging in individual NAc terminals, an effect also prevented by α-bungarotoxin. Pre-exposure to the α4-nAChR agonists 5IA85380 (10 nM) or RJR2429 (1 µM) did not modify NMDA-evoked ([3H]D-Asp) outflow and calcium transients. The NMDA-evoked ([3H]D-Asp) overflow was partially antagonized by the NMDAR antagonists MK801, D-AP5, 5,7-DCKA and R(-)CPP and unaffected by the GluN2B-NMDAR antagonists Ro256981 and ifenprodil. Notably, pre-treatment with choline increased GluN2A biotin-tagged proteins. In conclusion, our results show that the GluN2A-NMDA receptor function can be positively regulated in NAc terminals in response to a brief incubation with α7 but not α4 nAChRs agonists. This might be a general feature in different brain areas since a similar nAChR-mediated bolstering of NMDA-induced ([3H]D-Asp) overflow was also observed in hippocampal synaptosomes.

Glutathionylation of the Aquaporin-2 Water Channel: A Novel Post-Translational Modification Modulated by the Oxidative Stress

Aquaporin-2 (AQP2) is the vasopressin-regulated water channel that controls renal water reabsorption and urine concentration. AQP2 undergoes different regulated post-translational modifications, including phosphorylation and ubiquitylation, which are fundamental for controlling AQP2 cellular localization, stability, and function. The relationship between AQP2 and S-glutathionylation is of potential interest because reactive oxygen species (ROS), produced under renal failure or nephrotoxic drugs, may influence renal function as well as the expression and the activity of different transporters and channels, including aquaporins. Here, we show for the first time that AQP2 is subjected to S-glutathionylation in kidney and in HEK-293 cells stably expressing AQP2. S-Glutathionylation is a redox-dependent post-translational modification controlling several signal transduction pathways and displaying an acute effect on free cytosolic calcium concentration. Interestingly, we found that in fresh kidney slices, the increased AQP2 S-glutathionylation correlated with tert-butyl hydroperoxide-induced ROS generation. Moreover, we also found that cells expressing wild-type human calcium-sensing receptor (hCaSR-wt) and its gain of function (hCaSR-R990G; hCaSR-N124K) had a significant decrease in AQP2 S-glutathionylation secondary to reduced ROS levels and reduced basal intracellular calcium concentration compared with mock cells. Together, these new findings provide fundamental insight into cell biological aspects of AQP2 function and may be relevant to better understand and explain pathological states characterized by an oxidative stress and AQP2-dependent water reabsorption disturbs.

Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guard cells are of an oscillatory nature.

Changes in cytosolic free calcium ([Ca(2+)]cyt) are an early and essential element of signalling networks activated by the perception of pathogen-associated molecular patterns (PAMPs), such as flg22. The flg22-induced calcium signal has been described on whole-plant, but not on single-cell scale so far. Also, the Ca(2+) sources and channels contributing to its generation are still obscure. Ratiometric fluorescence imaging employing the calcium reporter Yellow Cameleon 3.6 was performed to analyse the flg22-induced calcium signature in single guard cells of Arabidopsis thaliana. Calcium stores and channel types involved in its generation were determined by a pharmacological approach. In contrast to the calcium signal determined on whole-plant level, the signature on single-cell level is not characterized by one sustained response, but by oscillations in [Ca(2+)]cyt. These oscillations were abolished by EGTA and lanthanum, as well as by U73122, neomycin and TMB-8, but only partially or not at all affected by inhibitors of glutamate receptor-like channels and cyclic nucleotide-gated channels. Our analyses suggest that the response observed on whole-plant level is the summary of oscillations occurring in single cells. Parallel to external calcium, influx via channels located at internal stores contributes to the signal.

OSCA1 mediates osmotic-stress-evoked Ca2+ increases vital for osmosensing in Arabidopsis.

Water is crucial to plant growth and development. Environmental water deficiency triggers an osmotic stress signalling cascade, which induces short-term cellular responses to reduce water loss and long-term responses to remodel the transcriptional network and physiological and developmental processes. Several signalling components that have been identified by extensive genetic screens for altered sensitivities to osmotic stress seem to function downstream of the perception of osmotic stress. It is known that hyperosmolality and various other stimuli trigger increases in cytosolic free calcium concentration ([Ca(2+)]i). Considering that in bacteria and animals osmosensing Ca(2+) channels serve as osmosensors, hyperosmolality-induced [Ca(2+)]i increases have been widely speculated to be involved in osmosensing in plants. However, the molecular nature of corresponding Ca(2+) channels remain unclear. Here we describe a hyperosmolality-gated calcium-permeable channel and its function in osmosensing in plants. Using calcium-imaging-based unbiased forward genetic screens we isolated Arabidopsis mutants that exhibit low hyperosmolality-induced [Ca(2+)]i increases. These mutants were rescreened for their cellular, physiological and developmental responses to osmotic stress, and those with clear combined phenotypes were selected for further physical mapping. One of the mutants, reduced hyperosmolality-induced [Ca(2+)]i increase 1 (osca1), displays impaired osmotic Ca(2+) signalling in guard cells and root cells, and attenuated water transpiration regulation and root growth in response to osmotic stress. OSCA1 is identified as a previously unknown plasma membrane protein and forms hyperosmolality-gated calcium-permeable channels, revealing that OSCA1 may be an osmosensor. OSCA1 represents a channel responsible for [Ca(2+)]i increases induced by a stimulus in plants, opening up new avenues for studying Ca(2+) machineries for other stimuli and providing potential molecular genetic targets for engineering drought-resistant crops.

Male-female communication triggers calcium signatures during fertilization in Arabidopsis.

Cell–cell communication and interaction is critical during fertilization and triggers free cytosolic calcium ([Ca2+]cyto) as a key signal for egg activation and a polyspermy block in animal oocytes. Fertilization in flowering plants is more complex, involving interaction of a pollen tube with egg adjoining synergid cells, culminating in release of two sperm cells and their fusion with the egg and central cell, respectively. Here, we report the occurrence and role of [Ca2+]cyto signals during the entire double fertilization process in Arabidopsis. [Ca2+]cyto oscillations are initiated in synergid cells after physical contact with the pollen tube apex. In egg and central cells, a short [Ca2+]cyto transient is associated with pollen tube burst and sperm cell arrival. A second extended [Ca2+]cyto transient solely in the egg cell is correlated with successful fertilization. Thus, each female cell type involved in double fertilization displays a characteristic [Ca2+]cyto signature differing by timing and behaviour from [Ca2+]cyto waves reported in mammals.

Arabidopsis thaliana calmodulin-like protein CML24 regulates pollen tube growth by modulating the actin cytoskeleton and controlling the cytosolic Ca(2+) concentration.

Cytosolic free calcium ([Ca(2+)]cyt), which is essential during pollen germination and pollen tube growth, can be sensed by calmodulin-like proteins (CMLs). The Arabidopsis thaliana genome encodes over 50 CMLs, the physiological role(s) of most of which are unknown. Here we show that the gene AtCML24 acts as a regulator of pollen germination and pollen tube extension, since the pollen produced by loss-of-function mutants germinated less rapidly than that of wild-type (WT) plants, the rate of pollen tube extension was slower, and the final length of the pollen tube was shorter. The [Ca(2+)]cyt within germinated pollen and extending pollen tubes produced by the cml24 mutant were higher than their equivalents in WT plants, and pollen tube extension was less sensitive to changes in external [K(+)] and [Ca(2+)]. The pollen and pollen tubes produced by cml24 mutants were characterized by a disorganized actin cytoskeleton and lowered sensitivity to the action of latrunculin B. The observations support an interaction between CML24 and [Ca(2+)]cyt and an involvement of CML24 in actin organization, thereby affecting pollen germination and pollen tube elongation.

Local membrane deformation and micro-injury lead to qualitatively different responses in osteoblasts

Micro-damage of bone tissue is known to regulate bone turnover. However, it is unknown if individual bone cells can differentiate between membrane deformation and micro-injury. We generated osteoblasts from mouse bone marrow or bone morphogenetic protein 2-transfected C2C12 cells. Single cells were mechanically stimulated by indentation with the atomic force microscopy probe with variable force load either resulting in membrane deformation only, or leading to membrane penetration and micro-injury. Changes in the cytosolic free calcium concentration ([Ca 2+] i) in fluo4-AM loaded cells were analyzed. When deformation only was induced, it resulted in an immediate elevation of [Ca 2+] i which was localized to the probe periphery. Multiple consecutive local Ca 2+ responses were induced by sequential application of low level forces, with characteristic recovery time of ~2 s. The duration of [Ca 2+] i elevations was directly proportional to the tip-cell contact time. In contrast, cell micro-injury resulted in transient global elevations of [Ca 2+] i, the magnitude of which was independent of the tip-cell contact time. Sequential micro-injury of the same cell did not induce Ca 2+ response within 30 s of the first stimulation. Both local and global Ca 2+elevations were blocked in Ca 2+-free media or in the presence of stretch-activated channel blocker Gd 3+. In addition, amount of Ca 2+ released during global responses was significantly reduced in the presence of PLC inhibitor Et-18-OCH 3. Thus, we found qualitative differences in calcium responses to mechanical forces inducing only membrane deformation or deformation leading to micro-injury.