首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Chemicals of various origins: chlorambucil, maitotoxin, sigmoidines, caulerpenyne, tributyltin, thapsigargin, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) and retinoid CD 367 were assayed on the cleavage of sea urchin eggs, their embryonic development and mechanisms regulating Ca2+ homeostasis. Compounds were used at therapeutic doses or at concentrations which were previously shown to be cytotoxic. These molecules did not affect the fertilization of Paracentrorus lividus eggs but all of them delayed the first cleavage. Only chlorambucil and CD 367 retarded hatching. All compounds provoked embryonic abnormalities if development was followed up to the pluteus stage, 72 hr after fertilization. Chemicals inhibited the ability of ATP-driven Ca2+ accumulation by the eggs in non-mitochondrial intracellular stores. Chlorambucil, maitotoxin and sigmoidines provoked a release of the Ca2+ sequestered with kinetics comparable to those provoked by the Ca2+ ionophore A23187. Ca2+ permeability of the plasma membrane was greatly increased by maitotoxin and 2,4,5-T whereas the other compounds were without effect. A drug-induced change in the Ca2+ storage capacity of sea urchin eggs resulting in retardation of cleaving stages and in further developmental defects is discussed in view to the possibility of relating changes in Ca2+-homeostasis with teratogenicity.  相似文献   

2.
S100a is a heterodimeric, acidic calcium-binding protein that interacts with calmodulin antagonists in a Ca2+-dependent manner. In order to study the behavior of the hydrophobic domain on S100a when bound to Ca2+, its interaction with trifluoperazine (TFP) was investigated using16F nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The dissociation constant (K d) values of TFP, as estimated from the chemical shifts of19F NMR, were 191 and 29 μm in the absence and presence of Ca2+, respectively, and were similar to those previously reported for S100b. However, the TFP linewidth in the presence of Ca2+-bound S100a was 65 Hz greater than in the presence of Ca2+-bound S100b. This suggests a slower TFP exchange rate for S100a than for S100b. Thus, the TFP linewidths observed for each isoform may reflect differences in structural and modulatory properties of the Ca2+-dependent hydrophobic domains on S100a and S100b. Additionally, the presence of magnesium had no effect on the observed Ca2+-induced TFP spectral changes in S100a solutions. Circular dichroism studies indicate that Ca2+ induces a small transition from α-helix to random coil in S100a; in contrast, the opposite transition is reported for calmodulin (Hennesseyet al., 1987). However, TFP did not significantly alter the secondary structure of Ca2+-bound S100a; this observation is similar to the effect of TFP on Ca2+-bound calmodulin and troponin C (Shimizu and Hatano, 1984; Gariépy and Hodges, 1983). It is, therefore, proposed that TFP binds to a hydrophobic domain on S100a in a fashion similar to other calcium-modulated proteins.  相似文献   

3.
The motility of spermatozoa of both Lytechinus pictus and Strongylocentrotus purpuratus sea urchin species is modulated by the egg-derived decapeptide speract via an oscillatory [Ca2+]-dependent signaling pathway. Comprehension of this pathway is hence directly related to the understanding of regulated sperm swimming. Niflumic acid (NFA), a nonsteroidal anti-inflammatory drug alters several ion channels. Though unspecific, NFA profoundly affects how sea urchin sperm respond to speract, increasing the [Ca2+]i oscillation period, amplitude, peak and average level values of the responses in immobilized and swimming cells. A previous logical network model we developed for the [Ca2+] dynamics of speract signaling cascade in sea urchin sperm allows integrated dissection of individual and multiple actions of NFA. Among the channels affected by NFA are: hyperpolarization-activated and cyclic nucleotide gated Na+ channels (HCN), [Ca2+]-dependent Cl channels (CaCC) and [Ca2+]-dependent K+ channels (CaKC), all present in the sea urchin genome. Here, using our model we investigated the effect of blocking in silico HCN and CaCC channels suggested by experiments. Regarding CaKC channels, arguments can be provided for either their blockage or activation by NFA. Our study yielded two scenarios compliant with experimental observations: i) under CaKC inhibition, this [Ca2+]-dependent K+ channel should be different from the Slo1 channel and ii) under activation of the CaKC channel, another [Ca2+] channel not considered previously in the network is required, such as the pH-dependent CatSper channel. Additionally, our findings predict cause-effect relations resulting from a selective inhibition of those channels. Knowledge of these relations may be of consequence for a variety of electrophysiological studies and have an impact on drug related investigations. Our study contributes to a better grasp of the network dynamics and suggests further experimental work.  相似文献   

4.
The extracellular matrix is now recognized as a dynamic structure which influences cellular properties. Many matrix metalloproteinase activities have been identified and characterized in vertebrates and constitute important agents in controlling the composition of the extracellular matrix. We have begun a study of matrix metalloproteinase activities in the developing sea urchin embryo. Using sea urchin peristome collagen or gelatin as physiological substrates we have determined the kinetic parameters, Km and Vmax, for an 87 kDa gelatinase activity expressed in late stage sea urchin embryos. We also determined the kinetic parameters Km, Vmax and kcat, for a 41 kDa species, expressed in the early sea urchin embryo, which possesses both collagenase and gelatinase activities. All values determined were similar to those reported in the literature for vertebrate collagenases and gelatinases and Km values in the micromolar range suggest that both species possess physiologically relevant activities. Both activities have previously been shown to require Ca2+ for activity. Using an assay for quantitating the cleavage of gelatin into trichloroacetic acid soluble peptides we report here markedly different effects of Ca2+ on the thermal denaturation profiles of the gelatinases. This latter finding may be indicative of different modes of action for this activating cation. Collectively, these results demonstrate both similarities and differences between vertebrate and invertebrate sea urchin gelatinases.  相似文献   

5.
The stimulation of the (Ca2+ + Mg2+)ATPase of erythrocyte ghosts by calmodulin was observed not only in intact ghosts, but also in the solubilized (Triton X-100) and partially purified, reconstituted (phosphatidylserine liposomes) forms. Since the solubilized form of the enzyme migrated on Sepharose 6B at a position corresponding to a molecular weight of about 150,000, these results show that calmodulin stimulates by direct interaction with the ATPase complex. Additionally, the effects of calmodulin on erythrocyte ghosts prepared by the Dodge-EDTA method (hypotonic ghosts) and by the method of Ronner et al. (involving lysis followed by an isotonic wash repeated several times) were compared (P. Ronner, P. Gazzotti, and E. Carafoli, 1977, Arch. Biochem. Biophys. 179, 578–583). The (Ca2+ + Mg2+)ATPase of the hypotonic ghosts was low and was stimulated by added calmodulin while that of the isotonic ghosts was high and changed only slightly upon calmodulin addition; this difference in response to calmodulin persisted in the solubilized and reconstituted forms. Hypotonic ghosts bound 125I-labeled calmodulin, while isotonic ghosts did not. This comparison of two types of ghosts showed that isotonic ghosts possess an intact calmodulin-(Ca2+ + Mg2+)ATPase complex, and that the calmodulin remained with the ATPase during solubilization and reconstitution. The isotonic preparation is a particularly useful method of preparing ghosts with an intact calmodulin-ATPase complex, since it requires no special equipment and produces an enzyme activity which is stable to freezing.  相似文献   

6.
The structure of actin bundles from internodal cells of Chara australis, an algal plant, was studied by electron microscopy of negatively stained specimens and optical diffraction. Gently prepared bundles revealed paracrystalline structures resembling the Mg2+-induced paracrystals of rabbit skeletal muscle actin (Hanson, 1968). In addition, the algal actin bundles sometimes had transverse striations at intervals of about 130 Å, as has been observed in actin bundles from sea urchin eggs (DeRosier et al., 1977; Spudich & Amos, 1979) and sea urchin coelomocytes (De Rosier & Edds, 1980; Otto & Bryan, 1981). This finding suggests that a common mechanism might be working in a variety of cells to organize actin filaments into functional bundles.  相似文献   

7.
The (Ca2+ + Mg2+-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tej?ka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81–88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two Km values of 3.5 and 120 μM. The enzyme is phosphorylated by ATP in the presence of Ca2+ only.  相似文献   

8.
  • 1.1. About 0.3–0.4% of total water-soluble protein extracted from sea urchin embryos at the two-cell and early-gastrula stages was Ca2+-dependently bound to immobilized calmodulin.
  • 2.2. SDS-PAGE of calmodulin-binding proteins revealed at least 20 polypeptides ranging from 200 to 15.5 kDa, and 70–80% of the protein belonged to a dozen major polypeptides. Polypeptides of 70, 55, 50, 45 and 18 kDa seemed to be the same as those that were detected earlier (Iwasa and Mohri, J. Biochem.94, 575–587, 1983).
  • 3.3. The polypeptide spectrum of calmodulin target proteins changed significantly, e.g. the major polypeptides of 70 and 41 kDa increased, and the 200 and 43 kDa polypeptides decreased sharply during development from the two-cell to the early-gastrula stage.
  • 4.4. According to our estimates, the molar concentrations of the calmodulin and targets were close enough, and therefore the Ca2+ signal should depend on the spatial-temporal distribution of free calmodulin in the cells.
  相似文献   

9.
Evidence for the involvement of Ca2+ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-14C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-14C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca2+, whereas lipolytic acyl hydrolase proved to be insensitive to Ca2+. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca2+. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC50 values ranging from 10 to 15 micromolar. Thus the Ca2+-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca2+ on phospholipase D is independent of calmodulin. The role of Ca2+ as a second messenger in the initiation of membrane lipid degradation is discussed.  相似文献   

10.
Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca2+ by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K+ leading to an enhancement of the Ca2+ transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca2+ at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K+ but not by Li+ and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K+. These data suggest that the regulation of the SERCA pump by K+ and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.  相似文献   

11.
1. The quality of unfertilized eggs of the sea urchin Strongylocentrotus intermedius, kept for a long time (50 days) in a sea water containing water soluble hydrocarbons of diesel fuel in sublethal concentrations (0.3–0.04 mg/l), was assessed through observation of embryogenesis and the intensity of 45Ca2+ uptake.2. It has been shown that such treatment led to delay and asynchronism of embryonal and larval development and to appearance of a greater number of abnormalities compared to the control.3. Unfertilized eggs of sea urchins exposed to the hydrocarbons in sublethal concentrations accumu- lated 30–60% more 45Ca2+ than those of control animals. Short-term incubation (2 hr) of eggs at the same hydrocarbon concentrations did not change 45Ca2+ uptake by unfertilized eggs of control animals.4. The increase of hydrocarbon concentration up to 1 mg/l (i.e. to a concentration causing disturbance of embryogenesis in acute experiments) in short-term experiments caused a small elevation in the 45Ca2+ uptake by unfertilized eggs of control animals (30% more than in untreated eggs).5. Ionomycin-induced (concentration 10−8−10−9) increase of 45Ca2+ uptake by unfertilized eggs (50–100% more than the untreated eggs) caused the same disturbance of embryogenesis as under hydrocarbon exposure.6. It is suggested that one of the mechanisms inducing the deleterious effect of hydrocarbons in sea urchin gametes is related to the increase of membrane permeability to calcium ions.  相似文献   

12.
Fertilized eggs of the mollusk Ilyanassa obsoleta (Nassarius obsoletus) form large blebs resembling polar lobes within 12 min of exposure to solutions of isotonic CaCl2, whereas control eggs in sea water remain spherical. Under identical conditions, fertilized eggs of the sea urchin, Strongylocentrotus purpuratus, an organism which normally does not form polar lobes, do not form blebs upon exposure to solutions of isotonic CaCl2. The calcium-induced blebbing in Ilyanassa still occurs if other cations such as Na+, Mg2+, or Mn2+ are present in addition to Ca2+, but not if comparable concentrations of K+ are present. Cytochalasin B prevents the calcium-induced blebbing, whereas colchicine does not. Cytokinesis in both Ilyanassa and Strongylocentrotus and normal polar lobe formation in Ilyanassa appear to require exogenous K+ but not exogenous Ca2+. Preliminary electron microscopy of Ilyanassa eggs exposed to isotonic solutions of CaCl2 has shown microfilaments in the cortical cytoplasm in the region of the bleb constriction but no microfilaments in spherical control eggs in sea water. These data suggest that high concentrations of exogenous Ca2+ trigger the polymerization and contraction of a ring of microfilaments in the cortical cytoplasm of the Ilyanassa egg which results in the formation of a lobelike bleb of cytoplasm. The observation that K+ antagonizes this Ca2+-induced blebbing has led to the formulation of a theory which postulates that the ratio of intracellular Ca2+ to intracellular K+ is critical in the control of polar lobe formation and cytokinesis.  相似文献   

13.
Marine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca2+-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca2+ increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca2+ fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca2+ fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca2+ fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca2+ fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general.  相似文献   

14.
Sea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca2+ concentration ([Ca2+]i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na+/Ca2+ exchange inhibitor) increases [Ca2+]i with at least two differing profiles. These increases depend on the presence of extracellular Ca2+, which indicates they involve Ca2+ uptake and not only mitochondrial Ca2+ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn2+. Store-operated Ca2+ channel (SOC) blockers (Ni2+, SKF96365 and Gd2+) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca2+ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca2+ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca2+]i, apparently oligomycin also has an extra mitochondrial target.  相似文献   

15.
A method for monitoring exocytotic membrane fusion by using a fluorescent membrane probe is presented. The method is based on the relief from concentration-dependent self-quenching (dequenching) of fluorescence of 5-N-(octadecanoyl)aminofluorescein (AF18), an amphiphilic derivative of fluorescein. The validity and usefulness of this method were shown by the following results: 1) self-quenching of AF18 fluorescence occurred in the plasma membrane of unfertilized eggs of a sea urchin, Pseudocentrotus depressus, which were heavily stained with the fluorescent dye; 2) dequenching of AF18 fluorescence occurred upon fertilization in normal eggs but not in EGTA-injected eggs; 3) Ca2+ induced both AF18 fluorescence dequenching and cortical granule disappearance in the isolated sea urchin egg cortex; and 4) simultaneous measurements of the intracellular Ca2+ concentration ([Ca2+]i) and dequenching of AF18 fluorescence by using a simple one-excitation and two-emission wavelength system.  相似文献   

16.
(1) The effects of calmodulin binding on the rates of Ca2+-dependent phosphorylation and dephosphorylation of the red-cell Ca2+ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg2+-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg2+-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca2+ affinity for sustaining ATP hydrolysis is raised at least 20-fold, Km(Ca) ? 10 μM (—calmodulin) and Km(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca2+ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (Km(Ca) ? 3–4 · 10-8 M). (Ca2+ + Mg2+)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca2+ + Mg2+)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca2+ + Mg2+)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (Km(MgATP) ? 360 μM), and CaATP is an inhibitor (Ki(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca2+- pump active site, producing a high (Ca2+ + Mg2+)-ATPase activity, high Ca2+ affinity and regulation of activity by MgATP.  相似文献   

17.
Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca2+]cyt) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca2+]cyt were observed in response to glutamic acid (Glu), ATP, and aluminum (Al3+). Each chemical induced a [Ca2+]cyt signature that differed among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggered patterns of [Ca2+]cyt increases that were similar among the different root zones, whereas Al3+ evoked [Ca2+]cyt transients that had monophasic and biphasic shapes, most notably in the root transition zone. The Al3+-induced [Ca2+]cyt increases generally started in the maturation zone and propagated toward the cap, while the earliest [Ca2+]cyt response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca2+]cyt signature resulting from Al3+ treatment originated mostly from cortical cells located at 300 to 500 μ m from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca2+ channel blockers, elicited [Ca2+]cyt responses similar to those induced by Al3+. The trivalent ion-induced [Ca2+]cyt signatures in roots of an Al3+-resistant and an Al3+-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca2+]cyt changes we report here may not be tightly linked to Al3+ toxicity but rather to a general response to trivalent cations.The role of calcium ions (Ca2+) as a ubiquitous cellular messenger in animal and plant cells is well established (Berridge et al., 2000; Sanders et al., 2002; Ng and McAinsh, 2003). Cellular signal transduction pathways are elicited as a result of fluctuations of free Ca2+ in the cytoplasm ([Ca2+]cyt) in response to external and intracellular signals. These changes in [Ca2+]cyt influence numerous cellular processes, including vesicle trafficking, cell metabolism, cell proliferation and elongation, stomatal opening and closure, seed and pollen grain germination, fertilization, ion transport, and cytoskeletal organization (Hepler, 2005). [Ca2+]cyt fluctuations occur because cells have a Ca2+ signaling “toolkit” (Berridge et al., 2000) composed of on/off switches and a multitude of Ca2+-binding proteins. The on switches depend on membrane-localized Ca2+ channels that control the entry of Ca2+ into the cytosol (Piñeros and Tester, 1995, 1997; Thion et al., 1998; Kiegle et al., 2000a; White et al., 2000; Demidchik et al., 2002; Miedema et al., 2008). On the other hand, the off switches consist of a family of Ca2+-ATPases and Ca2+/H+ exchangers in the plasma membrane or endomembrane that remove Ca2+ from the cytosol, bringing the [Ca2+]cyt down to the initial resting level (Lee et al., 2007; Li et al., 2008).The numerous cellular processes regulated by Ca2+ have led investigators to ask how specificity in Ca2+ signaling is maintained. It has been proposed that specificity in Ca2+ signaling is achieved because a particular stimulus elicits a distinct Ca2+ signature, which is defined by the timing, magnitude, and frequency of [Ca2+]cyt changes. For instance, tip-growing plant cells such as root hairs and pollen tubes exhibit oscillatory elevations in [Ca2+]cyt that partly mirror the oscillatory nature of growth in these cell types (Cárdenas et al., 2008; Monshausen et al., 2008). Another example is nuclear Ca2+ spiking in root hairs of legumes exposed to NOD factors (Oldroyd and Downie, 2006; Peiter et al., 2007). Recently, it was shown that mechanical forces applied to an Arabidopsis (Arabidopsis thaliana) root can trigger a stimulus-specific [Ca2+]cyt response (Monshausen et al., 2009). Translating the Ca2+ signature into a defined cellular response is governed by a number of Ca2+-binding proteins such as calreticulin that act as [Ca2+]cyt buffers, which shape both the amplitude and duration of the Ca2+ signal or Ca2+ sensors such as calmodulin that impact other downstream cellular effectors (Berridge et al., 2000; White and Broadley, 2003; Hepler, 2005).A deeper understanding of Ca2+ signaling mechanisms in plants has been driven in large part by our ability to monitor dynamic changes in [Ca2+]cyt in the cell. Such measurements have been conducted using Ca2+-sensitive fluorescent indicator dyes (e.g. Indo and Fura), the luminescent protein aequorin (Knight et al., 1991, 1996; Legué et al., 1997; Wymer et al., 1997; Cárdenas et al., 2008), and more recently the yellow cameleon (YC) Ca2+ sensor, a chimeric protein that relies on fluorescence resonance energy transfer (FRET) as an indicator of [Ca2+]cyt changes in the cell (Allen et al., 1999; Miwa et al., 2006; Qi et al., 2006; Tang et al., 2007; Haruta et al., 2008). The YC reporter is composed of cyan fluorescent protein (CFP), the C terminus of calmodulin (CaM), a Gly-Gly linker, the CaM-binding domain of myosin light chain kinase (M13), and a yellow fluorescent protein (YFP; Miyawaki et al., 1997, 1999). The increased interaction between M13 and CaM upon binding of Ca2+ to CaM triggers a conformational change in the protein that brings the CFP and YFP in close proximity, resulting in enhanced FRET efficiency between the two fluorophores (Miyawaki, 2003). Thus, changes in FRET efficiency between CFP and YFP in the cameleon reporter are correlated with changes in [Ca2+]cyt.Since it was first introduced, improved versions of the cameleon reporter have been selected to more accurately report [Ca2+]cyt levels in the cell. For instance, the YC3.60 version was selected because of its resistance to cytoplasmic acidification and its higher dynamic range compared with the earlier cameleons. The higher dynamic range of YC3.60 is due to the use of a circularly permutated YFP called Venus (cpVenus) that is capable of absorbing a greater amount of energy from CFP (Nagai et al., 2004). Recently, the utility of YC3.60 for monitoring [Ca2+]cyt was demonstrated in Arabidopsis roots and pollen tubes using ratiometric imaging approaches (Monshausen et al., 2007, 2008, 2009; Haruta et al., 2008; Iwano et al., 2009). Here, we further evaluated YC3.60 as a [Ca2+]cyt sensor in plants using confocal microscopy and FRET-sensitized emission imaging. Unlike the direct ratiometric measurement of cpVenus and CFP reported in previous studies using YC3.60-expressing plants (Monshausen et al., 2008, 2009), the sensitized FRET approach we describe here involves the use of donor-only (CFP) and acceptor-only (YFP) controls, allowing us to correct for bleed-through and background signals from the FRET specimen (van Rheenen et al., 2004; Feige et al., 2005).For this study, we focused on monitoring [Ca2+]cyt changes in Arabidopsis seedling roots after aluminum (Al3+) exposure. Although Ca2+ signaling has long been implicated in mediating Al3+ responses in plants (Rengel and Zhang, 2003), the [Ca2+]cyt changes evoked by Al3+ reported in the literature have been inconsistent, and as such, the significance of these [Ca2+]cyt responses to mechanisms of Al3+ toxicity are not very clear. For instance, some studies reported that Al3+ caused a decrease in [Ca2+]cyt in plants (Jones et al., 1998b; Kawano et al., 2004), and others demonstrated elevated [Ca2+]cyt upon Al3+ treatment (Nichol and Oliveira, 1995; Lindberg and Strid, 1997; Jones et al., 1998a; Zhang and Rengel, 1999; Ma et al., 2002; Bhuja et al., 2004).Here, we report that Arabidopsis roots expressing the YC3.60 reporter exhibited transient elevations in [Ca2+]cyt within seconds of Al3+ exposure. The general pattern of [Ca2+]cyt changes observed after Al3+ treatment were distinct from those elicited by ATP or Glu, reinforcing the concept of specificity in [Ca2+]cyt signaling. We also observed root zone-dependent variations in the [Ca2+]cyt signatures evoked by Al3+ in regard to the shape, duration, and timing of the [Ca2+]cyt response. Other trivalent ions such as lanthanum (La3+) and gadolinium (Ga3+), which have been widely used as Ca2+ channel blockers (Monshausen et al., 2009), also induced a rapid rise in [Ca2+]cyt in root cells that were similar to those elicited by Al3+. Al3+, La3+, and Gd3+ elicited similar [Ca2+]cyt signatures in the Al3+-tolerant mutant alr104 (Larsen et al., 1998) and the Al3+-sensitive mutant als3-1 (Larsen et al., 2005), indicating that the early [Ca2+]cyt increases we report here may not be tightly linked to mechanisms of Al3+ toxicity but rather to a general trivalent cation response. Our study further shows that FRET-sensitized emission imaging of Arabidopsis roots expressing YC3.60 provides a robust method for documenting [Ca2+]cyt signatures in different root developmental zones that should be useful for future studies on Ca2+ signaling mechanisms in plants.  相似文献   

18.
Summary The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca2+-activated K+ channels due to the oscillatory increase in the intracellular Ca2+ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 m, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca2+, Sr2+, Mn2+ and Ni2+, elicited slow hyperpolarizations, whereas Mg2+ and Ba2+, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca2+-activated K+ channels, abolished both Ca2+-and calmodulin-induced hyperpolarizations. TFP prevented Ca2+-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca2+-activated K+ channels in fibroblasts.  相似文献   

19.
Summary Pulses of some Ca2+ channel blockers (dantrolene, Co2+, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca2+, or of low Ca2+, a Ca2+ ionophore (A23187) together with Ca2+, and other Ca2+ channel blockers (La3+, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca2+ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca2+ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with35S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca2+ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca2+-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca2+ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.  相似文献   

20.
Symmetry/asymmetry conversion of eukaryotic flagellar waveform is caused by the changes in intracellular Ca2+. Animal sperm flagella show symmetric or asymmetric waveform at lower or higher concentration of intracellular Ca2+, respectively. In Chlamydomonas, high Ca2+ induces conversion of flagellar waveform from asymmetric to symmetry, resulting in the backward movement. This mirror image relationship between animal sperm and Chlamydomonas could be explained by the distinct calcium sensors used to regulate the outer arm dyneins (Inaba 2015). Here we analyze the flagellar Ca2+-response of the prasinophyte Pterosperma cristatum, which shows backward movement by undulating four flagella, the appearance similar to animal sperm. The moving path of Pterosperma shows relatively straight in artificial seawater (ASW) or ASW in the presence of a Ca2+ ionophore A23187, whereas it becomes circular in a low Ca2+ solution. Analysis of flagellar waveform reveals symmetric or asymmetric waveform propagation in ASW or a low Ca2+ solution, respectively. These patterns of flagellar responses are completely opposite to those in sperm flagella of the sea urchin Anthocidaris crassispina, supporting the idea previously proposed that the difference in flagellar response to Ca2+ attributes to the evolutional innovation of calcium sensors of outer arm dynein in opisthokont or bikont lineage.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号