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1.
Spermine (SPM) and spermidine (SPD) activate isolated phosphatidylinositol-4-phosphate 5-kinases (PI(4)P5K), enzymes that convert phosphatidylinositol-4-phosphate to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). PI(4,5)P2 formation is known to be involved in cellular actin reorganization and motility, functions that are also influenced by polyamines. It has not been proven that endogenous polyamines can control inositol phospholipid metabolism. We evoked large decreases in SPD and putrescine (PUT) contents in HL60 cells, using the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine (DFMO), which resulted in decreases in PI(4,5)P2 content per cell and inositol phosphate formation to 76.9 +/- 3.5% and 81.5 +/- 4.0% of control, respectively. Accurately reversing DFMO-evoked decreases in SPD content by incubating cells with exogenous SPD for 20 min rescued these decreases. DFMO treatment and SPD rescues also changed the ratio of total cellular PI(4,5)P2 to PIP suggesting involvement of a SPD-sensitive PI(4)P5K. PUT and SPM were not involved in DFMO-evoked changes in cellular PI(4,5)P2 contents. In DFMO-treated HL60 cells, the percent of total actin content that was filamentous was decreased to 59.1 +/- 5.8% of that measured in paired control HL60 cells, a finding that was rescued following reversal of DFMO-evoked decreases in SPD and PI(4,5)P2 contents. In slowly proliferating DMSO-differentiated HL60 cells, inositol phospholipid metabolism was uncoupled from SPD control. We conclude: in rapidly proliferating HL60 cells, but not in slowly proliferating differentiated HL60 cells, there are endogenous SPD-sensitive PI(4,5)P2 pools, probably formed via SPD-sensitive PI(4)P5K, that likely control actin polymerization.  相似文献   

2.
Phosphatidylinositol‐specific phospholipase C (PI‐PLC) is involved in stress signalling but its signalling function remains largely unknown in crop plants. Here, we report that the PI‐PLC4 from rice (Oryza sativa cv), OsPLC4, plays a positive role in osmotic stress response. Two independent knockout mutants, plc4‐1 and plc4‐2, exhibited decreased seedling growth and survival rate whereas overexpression of OsPLC4 improved survival rate under high salinity and water deficiency, compared with wild type (WT). OsPLC4 hydrolyses PI, phosphatidylinositol 4‐phosphate (PI4P), and phosphatidylinositol‐4,5‐bisphosphate (PIP2) to generate diacylglycerol (DAG) in vitro. Knockout of OsPLC4 attenuated salt‐induced increase of phosphatidic acid (PA) whereas overexpression of OsPLC4 decreased the level of PI4P and PIP2 under salt treatment. Applications of DAG or PA restored the growth defect of plc4‐1 to WT but DAG kinase inhibitor 1 blocked the complementary effect of DAG in plc4‐1 under salt stress. In addition, the loss of OsPLC4 compromised the increase of inositol triphosphate and free cytoplasmic Ca2+ ([Ca2+]cyt) and inhibited the induction of genes involved in Ca2+ sensor and osmotic stress response to salt stress. The results indicate that OsPLC4 modulates the activity of two signalling pathways, PA and Ca2+, to affect rice seedling response to osmotic stress.  相似文献   

3.
Oscillations in cytoplasmic Ca2+ concentration are a universal mode of signaling following physiological levels of stimulation with agonists that engage the phospholipase C pathway. Sustained cytoplasmic Ca2+ oscillations require replenishment of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2), the source of the Ca2+-releasing second messenger inositol trisphosphate. Here we show that cytoplasmic Ca2+ oscillations induced by cysteinyl leukotriene type I receptor activation run down when cells are pretreated with Li+, an inhibitor of inositol monophosphatases that prevents PIP2 resynthesis. In Li+-treated cells, cytoplasmic Ca2+ signals evoked by an agonist were rescued by addition of exogenous inositol or phosphatidylinositol 4-phosphate (PI4P). Knockdown of the phosphatidylinositol 4-phosphate 5 (PIP5) kinases α and γ resulted in rapid loss of the intracellular Ca2+ oscillations and also prevented rescue by PI4P. Knockdown of talin1, a protein that helps regulate PIP5 kinases, accelerated rundown of cytoplasmic Ca2+ oscillations, and these could not be rescued by inositol or PI4P. In Li+-treated cells, recovery of the cytoplasmic Ca2+ oscillations in the presence of inositol or PI4P was suppressed when Ca2+ influx through store-operated Ca2+ channels was inhibited. After rundown of the Ca2+ signals following leukotriene receptor activation, stimulation of P2Y receptors evoked prominent inositol trisphosphate-dependent Ca2+ release. Therefore, leukotriene and P2Y receptors utilize distinct membrane PIP2 pools. Our findings show that store-operated Ca2+ entry is needed to sustain cytoplasmic Ca2+ signaling following leukotriene receptor activation both by refilling the Ca2+ stores and by helping to replenish the PIP2 pool accessible to leukotriene receptors, ostensibly through control of PIP5 kinase activity.  相似文献   

4.
We reported recently that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release. IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P2 to IP3 and diacylglycerol. We show that Sprouty2 (Spry2), which binds PI(4,5)P2 and PLCγ, interfered with PI(4,5)P2 in a manner similar to that of U73122, an inhibitor of PI(4,5)P2 hydrolysis, suggesting that Spry2 negatively regulates IP3R by preventing formation of its activating ligand, IP3. Mutation to Asp of R252, a crucial determinant of PI(4,5)P2 binding in the C-terminal domain of Spry2, prevented the interference, indicating that binding to the phospholipid is required. By contrast, deletion of the PLCγ binding region or mutation of a critical Tyr residue in the region did not prevent the interference but Spry2-PI(4,5)P2 colocalization was not detected, suggesting that PLC binding is required for their stable association. Like U73122, Spry2 over-expression inhibited wild type Gag release as virus-like particles. Disrupting either binding determinant relieved the inhibition. IP3R-mediated Ca2+signaling, in turn, was found to influence Spry2 subcellular distribution and ERK, a Spry2 regulator. Our findings suggest that Spry2 influences IP3R function through control of PI(4,5)P2 and IP3R influences Spry2 function by controlling its distribution and ERK activation.  相似文献   

5.
Activation of muscarinic cholinergic receptors was studied by measuring agonist-stimulated inositol lipid turnover and changes in [Ca2+]i in dissociated salt gland secretory cells. Carbachol stimulation of quin2-loaded cells results in a sustained 4-fold increase in [Ca2+]i, while incorporation of [32P]Pi into phosphatidylinositol (PI) and phosphatidate are similarly increased. [3H]Inositol phosphates, measured in the presence of Li+, increased 13-fold. The stimulated increment in [Ca2+]i required extracellular Ca2+, whereas [3H]inositol phosphate accumulation was independent of external Ca2+. Dose-response curves for carbachol-induced increments in [Ca2+]i, PI labeling, and labeled inositol phosphate release are similar, with EC50 values of 6, 4.5 and 8 μM, respectively. Dissociation constants for atropine vs. the quin2 and phospholipid responses are 0.59 ± 0.3 nM and 0.48 ± 0.28 nM, respectively. These cells thus provide a model system for the study of non-exocytotic secretion as a consequence of stimulated inositol lipid turnover.  相似文献   

6.
Metabolism of the putative messenger molecule d-myo-inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] in plant cells has been studied using a soluble fraction from pea (Pisum sativum) roots as enzyme source and [5-32P]Ins(1,4,5)P3 and [2-3H]Ins(1,4,5)P3 as tracers. Ins(1,4,5)P3 was rapidly converted into both lower and higher inositol phosphates. The major dephosphorylation product was inositol(4,5)bisphosphate [Ins(4,5)P2] whereas inositol(1,4)bisphosphate [Ins(1,4)P2] was only present in very small quantities throughout a 15 minute incubation period. In addition to these compounds, small amounts of nine other metabolites were produced including inositol and inositol(1,4,5,X)P4. Dephosphorylation of Ins(1,4,5)P3 to Ins(4,5)P2 was dependent on Ins(1,4,5)P3 concentration and was partially inhibited by the phosphohydrolase inhibitors 2,3-diphosphoglycerate, glucose 6-phosphate, and p-nitrophenylphosphate. Conversion of Ins(1,4,5)P3 to Ins(4,5)P2 and Ins(1,4,5,X)P4 was inhibited by 55 micromolar Ca2+. This study demonstrates that enzymes are present in plant tissues which are capable of rapidly converting Ins(1,4,5)P3 and that pathways of inositol phosphate metabolism exist which may prove to be unique to the plant kingdom.  相似文献   

7.
Depletion of intracellular Ca2+ stores evokes store‐operated Ca2+ entry through the Ca2+ release‐activated Ca2+ (CRAC) channels. In this study, we found that the store‐operated Ca2+ entry was inhibited by neomycin, an aminoglycoside that strongly binds phosphatidylinositol 4,5‐bisphosphate (PtdIns(4,5)P2). Patch clamp recordings revealed that neomycin blocked the CRAC currents reconstituted by co‐expression of Orai1 and Stim1 in HEK293 cells. Using a rapamycin‐inducible PtdIns(4,5)P2‐specific phosphatase (Inp54p) system to manipulate the PtdIns(4,5)P2 in the plasma membrane, we found that the CRAC current was not altered by PtdIns(4,5)P2 depletion. This result suggests that PtdIns(4,5)P2 is not required for CRAC channel activity, and thereby, neomycin inhibits CRAC channels in a manner that is independent of neomycin–PtdIns(4,5)P2 binding. Copyright © 2015 John Wiley & Sons, Ltd.  相似文献   

8.
Calcium has been shown to induce clustering of PI(4,5)P2 at high and non-physiological concentrations of both the divalent ion and the phosphatidylinositol, or on supported lipid monolayers. In lipid bilayers at physiological conditions, clusters are not detected through microscopic techniques. Here, we aimed to determine through spectroscopic methodologies if calcium plays a role in PI(4,5)P2 lateral distribution on lipid bilayers under physiological conditions. Using several different approaches which included information on fluorescence quantum yield, polarization, spectra and diffusion properties of a fluorescent derivative of PI(4,5)P2 (TopFluor(TF)-PI(4,5)P2), we show that Ca2 + promotes PI(4,5)P2 clustering in lipid bilayers at physiological concentrations of both Ca2 + and PI(4,5)P2. Fluorescence depolarization data of TF-PI(4,5)P2 in the presence of calcium suggests that under physiological concentrations of PI(4,5)P2 and calcium, the average cluster size comprises ~ 15 PI(4,5)P2 molecules. The presence of Ca2 +-induced PI(4,5)P2 clusters is supported by FCS data. Additionally, calcium mediated PI(4,5)P2 clustering was more pronounced in liquid ordered (lo) membranes, and the PI(4,5)P2-Ca2 + clusters presented an increased affinity for lo domains. In this way, PI(4,5)P2 could function as a lipid calcium sensor and the increased efficiency of calcium-mediated PI(4,5)P2 clustering on lo domains might provide targeted nucleation sites for PI(4,5)P2 clusters upon calcium stimulus.  相似文献   

9.
Human immunodeficiency virus type 1 (HIV-1) release efficiency is directed by late (L) domain motifs in the viral structural precursor polyprotein Gag, which serve as links to the ESCRT (endosomal sorting complex required for transport) machinery. Linkage is normally through binding of Tsg101, an ESCRT-1 component, to the P7TAP motif in the p6 region of Gag. In its absence, budding is directed by binding of Alix, an ESCRT adaptor protein, to the LY36PXnL motif in Gag. We recently showed that budding requires activation of the inositol 1,4,5-triphosphate receptor (IP3R), a protein that “gates” Ca2+ release from intracellular stores, triggers Ca2+ cell influx and thereby functions as a major regulator of Ca2+ signaling. In the present study, we determined whether the L domain links Gag to Ca2+ signaling machinery. Depletion of IP3R and inactivation of phospholipase C (PLC) inhibited budding whether or not Tsg101 was bound to Gag. PLC hydrolysis of phosphatidylinositol-(4,5)-bisphosphate generates inositol (1,4,5)-triphosphate, the ligand that activates IP3R. However, with Tsg101 bound, Gag release was independent of Gq-mediated activation of PLC, and budding was readily enhanced by pharmacological stimulation of PLC. Moreover, IP3R was redistributed to the cell periphery and cytosolic Ca2+ was elevated, events indicative of induction of Ca2+ signaling. The results suggest that L domain function, ESCRT machinery and Ca2+ signaling are linked events in Gag release.  相似文献   

10.
Double C2 domain protein B (DOC2B) is a high‐affinity Ca2+ sensor that translocates from the cytosol to the plasma membrane (PM) and promotes vesicle priming and fusion. However, the molecular mechanism underlying its translocation and targeting to the PM in living cells is not completely understood. DOC2B interacts in vitro with the PM components phosphatidylserine, phosphatidylinositol (4, 5)‐bisphosphate [PI(4, 5)P2] and target SNAREs (t‐SNAREs). Here, we show that PI(4, 5)P2 hydrolysis at the PM of living cells abolishes DOC2B translocation, whereas manipulations of t‐SNAREs and other phosphoinositides have no effect. Moreover, we were able to redirect DOC2B to intracellular membranes by synthesizing PI(4, 5)P2 in those membranes. Molecular dynamics simulations and mutagenesis in the calcium and PI(4, 5)P2‐binding sites strengthened our findings, demonstrating that both calcium and PI(4, 5)P2 are required for the DOC2B–PM association and revealing multiple PI(4, 5)P2–C2B interactions. In addition, we show that DOC2B translocation to the PM is ATP‐independent and occurs in a diffusion‐like manner. Our data suggest that the Ca2+‐triggered translocation of DOC2B is diffusion‐driven and aimed at PI(4, 5)P2‐containing membranes.   相似文献   

11.
Eisosomes are multiprotein structures that generate linear invaginations at the plasma membrane of yeast cells. The core component of eisosomes, the BAR domain protein Pil1, generates these invaginations through direct binding to lipids including phosphoinositides. Eisosomes promote hydrolysis of phosphatidylinositol 4,5 bisphosphate (PI(4,5)P2) by functioning with synaptojanin, but the cellular processes regulated by this pathway have been unknown. Here, we found that PI(4,5)P2 regulation by eisosomes inhibits the cell integrity pathway, a conserved MAPK signal transduction cascade. This pathway is activated by multiple environmental conditions including osmotic stress in the fission yeast Schizosaccharomyces pombe. Activation of the MAPK Pmk1 was impaired by mutations in the phosphatidylinositol (PI) 5-kinase Its3, but this defect was suppressed by removal of eisosomes. Using fluorescent biosensors, we found that osmotic stress induced the formation of PI(4,5)P2 clusters that were spatially organized by eisosomes in both fission yeast and budding yeast cells. These cortical clusters contained the PI 5-kinase Its3 and did not assemble in the its3-1 mutant. The GTPase Rho2, an upstream activator of Pmk1, also co-localized with PI(4,5)P2 clusters under osmotic stress, providing a molecular link between these novel clusters and MAPK activation. Our findings have revealed that eisosomes regulate activation of MAPK signal transduction through the organization of cortical lipid-based microdomains.  相似文献   

12.
Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCβ1 enzymatically reduce plasma membrane PI(4,5)P2 levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P2 abundance by these enzymes releases the PI(4,5)P2‐binding protein cofilin from its inactive membrane‐associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid‐dependent sequestration of an actin‐remodeling factor.  相似文献   

13.
Abstract

The activation of Ca2+-mobilising receptors on hepatocytes and many other cells leads to a prompt reduction in the cellular content of inositol phospholipids. The primary event which underlies these changes is, most probably, a phospholipase C-catalysed attack upon phosphatidylinositol 4,5 bisphosphate. The receptor-mediated breakdown of this lipid in stimulated cells is: (i) not mediated by an increase in cytosol [Ca2+] and (ii) closely coupled to receptor occupation. Phosphatidylinositol 4,5 bisphosphate degradation may be studied by measuring the appearance of the water-soluble product, inositol trisphosphate (and its metabolites: inositol bisphosphate and inositol monophosphate), in stimulated cells. Recent evidence indicates that inositol trisphosphate and the lipid soluble product of phosphatidylinositol 4,5 bisphosphate breakdown, 1,2 diacylglycerol, may act as ‘second messengers’ which mediate the effects of many extracellular signals in stimulated cells.  相似文献   

14.
Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the two‐pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH‐dependent manner. Under controlled Mg2+, TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP‐mediated Ca2+ release in intact cells is regulated by Mg2+, PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP‐mediated Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer.  相似文献   

15.
The generation of phosphoinositides (PIs) with spatial and temporal control is a key mechanism in cellular organization and signaling. The synthesis of PIs is mediated by PI kinases, proteins that are able to phosphorylate unique substrates at specific positions on the inositol headgroup to generate signaling molecules. Phosphatidylinositol 5 phosphate 4 kinase (PIP4K) is one such lipid kinase that is able to specifically phosphorylate phosphatidylinositol 5 phosphate, the most recently discovered PI to generate the well-known and abundant PI, phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2]. PIP4K appears to be encoded only in metazoan genomes, and several genetic studies indicate important physiological functions for these enzymes in metabolism, immune function, and growth control. PIP4K has recently been reported to localize to multiple cellular compartments, including the nucleus, plasma membrane, endosomal systems, and autophagosome. However, the biochemical activity of these enzymes that is relevant to these physiological functions remains elusive. We review recent developments in this area and highlight emerging roles for these enzymes in cellular organization.  相似文献   

16.
The role of second messengers in the diversion of cellular processes by pathogens remains poorly studied despite their importance. Among these, Ca2+ virtually regulates all known cell processes, including cytoskeletal reorganization, inflammation, or cell death pathways. Under physiological conditions, cytosolic Ca2+ increases are transient and oscillatory, defining the so‐called Ca2+ code that links cell responses to specific Ca2+ oscillatory patterns. During cell invasion, Shigella induces atypical local and global Ca2+ signals. Here, we show that by hydrolyzing phosphatidylinositol‐(4,5)bisphosphate, the Shigella type III effector IpgD dampens inositol‐(1,4,5)trisphosphate (InsP3) levels. By modifying InsP3 dynamics and diffusion, IpgD favors the elicitation of long‐lasting local Ca2+ signals at Shigella invasion sites and converts Shigella‐induced global oscillatory responses into erratic responses with atypical dynamics and amplitude. Furthermore, IpgD eventually inhibits InsP3‐dependent responses during prolonged infection kinetics. IpgD thus acts as a pathogen regulator of the Ca2+ code implicated in a versatility of cell functions. Consistent with this function, IpgD prevents the Ca2+‐dependent activation of calpain, thereby preserving the integrity of cell adhesion structures during the early stages of infection.  相似文献   

17.
Abstract: Extracellular ATP has neurotransmitter-like properties in the CNS and PNS that are mediated by a cell-surface P2 purinergic receptor. In the present study, we have extensively characterized the signal transduction pathways that are associated with activation of a P2U receptor in a cultured neuroblastoma × glioma hybrid cell line (NG108-15 cells). The addition of ≥1 μM ATP to NG108-15 cells caused a transient increase in [Ca2+]i that was inhibited by 40% when extracellular calcium was chelated by EGTA. ATP concentrations ≥500 μM also elicited a sustained increase in [Ca2+]i that was inhibited when extracellular calcium was chelated by EGTA. The increase in [Ca2+]i elicited by ATP occurred concomitantly with the hydrolysis off [32P]-phosphatidylinositol 4,5-bisphosphates and an increase in the level of inositol 1,4,5-trisphosphate. ATP also caused a time- and dose-dependent increase in levels of [3H]inositol monophosphates in lithium-treated cells. Separation of the inositol monophosphate isomers by ion chromatography revealed a specific increase in the level of inositol 4-monophosphate. The magnitude of the increase in [Ca2+]i elicited by ATP correlated with the concentration of the fully ionized form of ATP (ATP4-) in the medium and not with the concentration of magnesium-ATP (MgATP2-). Similar to ATP, UTP also induced polyphosphoinositide breakdown, inositol phosphate formation, and an increase in [Ca2+]i. ADP, ITP, TTP, GTP, ATP-γS, 2-methylthio ATP, β,γ-imidoATP or 3′-O-(4-benzoyl)benzoylATP, but not CTP, AMP, β,γ-methylene ATP, or adenosine, also caused an increase in [Ca2+]i. In cells labeled with [32P]Pi or [14C]-arachidonic acid, ATP caused a transient increase in levels of labeled phosphatidic acids, but had no effect on levels of arachidonic acid. The increase in phosphatidic acid levels elicited by ATP apparently was not due to activation of a phospholipase D because ATP did not induce the formation of phosphatidylethanol in [14C]myristic acid-labeled cells incubated in the presence of ethanol. These findings support the hypothesis that a P2 nucleotide receptor in NG108-15 cells is coupled to a signal transduction pathway involving the activation of a phospholipase C and a plasma membrane calcium channel, but not the activation of phospholipases A2 and D.  相似文献   

18.
Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca2+ and Fe2+ release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1–3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P2-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca2+, Mn2+, and Fe2+, but not Fe3+. The TRPML currents were inhibited by trivalent cations Fe3+, La3+, and Gd3+. These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.  相似文献   

19.
The transport of Ca2+ across membranes precedes the fusion and fission of various lipid bilayers. Yeast vacuoles under hyperosmotic stress become fragmented through fission events that requires the release of Ca2+ stores through the TRP channel Yvc1. This requires the phosphorylation of phosphatidylinositol‐3‐phosphate (PI3P) by the PI3P‐5‐kinase Fab1 to produce transient PI(3,5)P2 pools. Ca2+ is also released during vacuole fusion upon trans‐SNARE complex assembly, however, its role remains unclear. The effect of PI(3,5)P2 on Ca2+ flux during fusion was independent of Yvc1. Here, we show that while low levels of PI(3,5)P2 were required for Ca2+ uptake into the vacuole, increased concentrations abolished Ca2+ efflux. This was as shown by the addition of exogenous dioctanoyl PI(3,5)P2 or increased endogenous production of by the hyperactive fab1T2250A mutant. In contrast, the lack of PI(3,5)P2 on vacuoles from the kinase dead fab1EEE mutant showed delayed and decreased Ca2+ uptake. The effects of PI(3,5)P2 were linked to the Ca2+ pump Pmc1, as its deletion rendered vacuoles resistant to the effects of excess PI(3,5)P2. Experiments with Verapamil inhibited Ca2+ uptake when added at the start of the assay, while adding it after Ca2+ had been taken up resulted in the rapid expulsion of Ca2+. Vacuoles lacking both Pmc1 and the H+/Ca2+ exchanger Vcx1 lacked the ability to take up Ca2+ and instead expelled it upon the addition of ATP. Together these data suggest that a balance of efflux and uptake compete during the fusion pathway and that the levels of PI(3,5)P2 can modulate which path predominates.  相似文献   

20.
Adipocytes of white adipose tissue are the cells maintaining glucose homeostasis in an organism, which is controlled by insulin. Insulin stimulates the translocation of glucose transporter GLUT4 from the cytosol into the cell membrane, as well as glucose transport and utilization in these cells. Here we show that insulin-induced [Ca2+]i oscillations are supported by the two signaling pathways involving: (1) phosphoinositide 3-kinase (PI3K), protein kinase B (Akt/PKB), endothelial NO synthase (eNOS), nitric oxide (NO), and ryanodine receptor (RyR) and (2) phospholipase C (PLC) and inositol 3-phosphate receptor (IP3R). Thus, the PI3K Akt/PKB signaling pathway initiates not only metabolic but also Ca2+-signaling pathways in response to insulin.  相似文献   

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