增强UV-B辐射对小麦根尖细胞游离钙离子分布的影响

以小麦根尖为材料,利用低温装载法在根尖细胞中成功地装载了酯化形式的钙离子荧光指示剂fluo-3/AM,利用激光共聚焦显微技术检测了增强UV-B辐射后小麦根尖细胞内游离钙离子荧光强度的分布,并对胞质内游离钙离子浓度进行了测定.结果表明:(1)对照组小麦根尖细胞内钙离子荧光主要分布于细胞胞质周缘;而经UV-B辐射处理后,细胞内钙离子荧光不仅分布在胞质周缘,且在细胞壁与胞间隙可观察到大量钙离子荧光.(2)对单个细胞内钙离子荧光强度进行测定,发现UV-B处理使细胞胞质内游离钙离子浓度明显升高.     

外源钙调素对粟酒裂殖酵母细胞增殖的影响

外源钙调素（CaM）对粟酒裂殖酵母（Schizosaccharomycespombe）细胞增殖的影响。实验结果表明外源CaM能明显抑制粟酒裂殖酵母细胞的增殖,其作用方式是延长了粟酒裂殖酵母细胞生长的延滞期。抗粟酒裂殖酵母CaM抗体、TFP及Phenyl-SepharoseCL－4B能降低CaM对细胞生长的抑制作用,而Ca2+及Ca2+螫合剂EGTA对CaM的抑制作用均无影响。以上结果提示,外源CaM对粟酒裂殖酵母细胞增殖的抑制作用可能是由于胞外CaM激活了细胞膜上的Ca2+泵,使胞内Ca2+浓度降低所致。     

拟南芥叶细胞游离钙离子的测定

低温(4℃)条件下将钙离子荧光探针Fluo-3／AM导入拟南芥叶细胞,利用激光共聚焦显微技术检测了胞内钙离子荧光强度的分布。实验证明,低温导入Fluo-3／AM法测定拟南芥叶细胞中钙离子荧光强度的变化切实可行。茉莉酸(JA)处理能够诱导胞内游离钙离子浓度的升高。     

17β-雌二醇快速诱导静止状态小鼠囊胚细胞胞内钙离子增加

用钙离子荧光探针fluo-3/AM标记囊胚细胞内钙离子,用激光共聚焦扫描显微镜连续检测17β-雌二醇(17β-E2)作用所致囊胚胞内钙离子浓度的动态变化,用荧光显微镜检测偶联牛血清白蛋白的17β-雌二醇(E2-BSA)所致囊胚胞内钙离子浓度的改变,以及在去钙镁M2液、传统雌激素受体阻断剂tamoxifen和磷脂酶C特异抑制剂U73122作用下17β-E2所致囊胚细胞内钙离子浓度的改变。结果显示:17β-E2和E2-BSA均可引起静止状态囊胚细胞内[Ca2 ]的快速升高;17β-E2诱导的囊胚细胞内[Ca2 ]的快速升高不依赖于胞外钙离子的内流,且不被传统雌激素受体阻断剂所阻断,而磷脂酶C特异性抑制剂可明显抑制该效应。     

细胞转化过程中胞内钙离子浓度的变化

使用钙离子荧光探针Ｆｌｕｏ－３ＡＭ和ＤＮＡ荧光染料Ｈｏｅｃｈｓｔ３３３４２联染细胞的方法,利用显微分光光度计ＭＰＶⅡ测量了两种温度敏感细胞—６Ｍ２和ｔｓＲＳＶＮＩＨ３Ｔ３－ＬＡ９０细胞及Ｃ３Ｈ１０Ｔ１／２和转化Ｃ３Ｈ１０Ｔ１／２细胞在不同细胞周期时相内钙的浓度,发现从Ｇ１期到Ｓ期到Ｇ２期,６ｍ２细胞当在３３℃培养时（转化状态）胞内钙的浓度〔Ｃａ＾２＋〕ｉ分别为８５．０,１３８．４２３９．０ｎｍｏｌ     

百合花粉细胞中钙离子的荧光测定法1

以川百合花粉为材料,利用孵育法在花粉原生质体中,特别是利用低温装载法在完整花粉粒中,成功地载入酯化形式的钙离子荧光探针fluo-3AM.利用激光共聚焦显微技术对花粉细胞质中游离钙离子的分布特点进行研究的结果表明,花粉萌发初期细胞质内游离钙离子呈极性分布,萌发沟附近的钙离子浓度明显高于其他部位,萌发孔附近最高,花粉细胞核中较低.文中对低温装载法的可行性进行了讨论.     

百合花粉细胞中钙离子的荧光测定法

以川百合花粉为材料,利用孵育法在花粉原生质体中,特别是利用低温装载法在完整花粉粒中,成功地载入酯化形成的钙离子荧光探针fluo-3AM,利用激光共聚焦显微技术对花粉细胞质中游离钙离子的分布特点进行了研究的结果表明,花粉萌发初期细胞质内游离钙离子呈极性分布,萌发沟附近的钙离子浓度明显高于其他部位,萌发孔附近最高,花粉细胞核中较低,文中对低温装载法的可行性进行了讨论。     

小鼠巨噬细胞内游离钙离子变化介导的吞噬作用

为评估小鼠巨噬细胞吞噬死亡细胞时胞质内游离钙离子的变化。实验使用F luo-3标记巨噬细胞内钙离子和碘化丙碇对死亡细胞核染色,观察吞噬过程中细胞内钙离子的变化和显示巨噬细胞的吞噬功能,检测含死亡细胞的巨噬细胞内荧光密度图像。利用激光扫描共聚焦显微镜检测钙离子的释放。在缺钙的溶液中,可见巨噬细胞接触死细胞时细胞内钙离子快速地聚集和增高。在吞噬体形成时,巨噬细胞内钙离子上升到较高的水平。快速上升后,当吞噬小泡消化时,细胞内游离钙下降,随后钙离子恢复到低水平。研究显示伴随着吞噬小泡中红色荧光的死细胞的出现和消失,巨噬细胞内出现一系列钙离子变化的图像。提示巨噬细胞内钙离子改变在细胞吞噬作用中具有一定的作用。     

花粉蛋白诱导胞内钙离子信号波动的研究

钙离子是细胞生命活动中的一个重要的调节因子,在细胞对外界刺激产生反应以及细胞死亡过程中,它扮演着重要的角色。天花粉蛋白是一种抗肿瘤药物,对绒毛膜上皮癌细胞的杀伤力特别强。通过对绒毛膜上皮癌细胞内钙离子进行ｆｌｕｏ－３／ＡＭ荧光染色发现,天花粉蛋白的加入能诱导绒毛膜上皮癌细胞内钙离子浓度的增加。经天花粉蛋白作用２４小时后,被天花粉蛋白损伤的绒毛膜上皮癌细胞内钙离子的浓度比正常细胞要高得多。在开花粉蛋     

钙离子浓度对暗适应时的中华绒螯蟹光感受器超微结构的影响

用透射电镜研究了暗适应时中华绒螯蟹的光感受器超微结构与外界钙离子浓度的关系,结果显示出与培育在生理溶液中的光感受器相比,细胞外钙离子浓度升高,使得感杆束的直径急剧缩小,感杆束周围胞质增厚,胞饮泡增加,膜下猪泡囊极度减小。胞质中多囊体的数量和直径减小,而板模体和溶酶体的数量增加,同时细胞内的色素颗粒增多。分布在小网膜细胞的远端。细胞的结构表现为类似光适应状态,与之相反,细胞外钙离子浓度降低时小眼的感     

Direct measurement of Ca2+ concentration in the SR of living cardiac myocytes

Although abnormal sarcoplasmic reticulum (SR) Ca(2+) handling may cause heart failure, there has been no method to directly measure Ca(2+) concentration in SR ([Ca(2+)](SR)) of living cardiomyocytes. We have measured [Ca(2+)](SR) by expressing novel fluorescent Ca(2+) indicators yellow cameleon (YC) 2.1, YC3er, and YC4er in cultured neonatal rat cardiomyocytes. The distribution of YC2.1 was uniform in the cytoplasm, while that of YC3er/YC4er, containing the signal sequence which recruits them to SR, showed reticular pattern and was co-localized with SERCA2a. The treatment with caffeine reversibly decreased the emission ratio (R) in YC3er/YC4er-expressing myocytes, and the treatment with ryanodine and thapsigargin decreased R irreversibly. During the contraction-relaxation cycle, R was changed periodically in the YC2.1- and YC3er-expressing myocytes, but its direction of the change was opposite. These results suggest that YC3er/YC4er were specifically localized and functioned in SR as a [Ca(2+)](SR) indicator. This technique would be useful to understand the function of SR in failing myocardium.     

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca2+]cyt dynamics to be measured with a high level of reproducibility in Arabidopsis cells. This technical advance in combination with cell biological and molecular genetic approaches will become an invaluable tool in the dissection of plant cell signal transduction pathways.     

双重荧光染色监测听毛细胞游离钙

Calcium distribution and mobilization during mechanical stimulation in outer hair cells of the guinea pig were monitored using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes. Spatial calcium gradients were revealed among various subcellular areas. The ratios of the fluorescence intensity of fluo-3 and fura-red were 1.71 +/- 0.85, 1.61 +/- 0.75, 1.47 +/- 0.65 and 1.39 +/- 0.66 for the cytoplasm, the cytoplasmic membrane, the cuticular plate and the nucleus respectively, indicating that free calcium ion concentrations are the highest in the cytoplasm and the lowest in the nucleus. While the calcium concentration remained relatively constant under resting conditions, it increased during mechanical stimulation. The results show that confocal ratio imaging of fluo-3 and fura-red enables us to determine more accurately the subcellular calcium distribution and that the calcium ions make a contribution to the mechanic-electrical transduction in hair cells.     

High concentration of calcium ions in Golgi apparatus

The interphase NIH3T3 cells were vitally fluorescentstained with calcium indicator fluo-3 and Glogi probe C6-NBD-ceramide,and then the single cells were examined by laser scanning confocal microscopy(LSCFM) for subcellular distributions of Ca^2 and the location of Golgi apparatus.In these cells,the intracellular Ca^2 were found to be highly concentrated in the Golgi apparatus.The changes of distribution of cytosolic high Ca^2 region and the Golgi apparatus coincided with the cell cycle phase.In calcium free medium,when the plasma membrane of the cells which had been loaded with fluo-3/AM were permeated by digitonin,the fluorescence of the Golgi region decreased far less than that of the cytosol.Our results indicated that the Glogi lumen retained significantly high concentration of free calcium.     

Fluctuations in the pollen tube tip-focused calcium gradient are not reflected in nuclear calcium level: a comparative analysis using recombinant yellow cameleon calcium reporter

For the first time in pollen tubes, both cytoplasmic and nuclear calcium have been imaged to allow comparative analysis of calcium dynamics in these two compartments with high spatial and temporal dynamics. An improved cameleon (YC2.1) calcium reporter was expressed cytoplasmically in both Lilium longiflorum and Nicotiana tabacum pollen tubes and the periodically fluctuating tip-focused calcium gradient typical of normal growth was recorded by ratio image analysis. The nucleoplasmin targeting sequence was then used to localise expressed YC2.1 to the vegetative nucleus of N. tabacum pollen tubes to permit imaging of nuclear location, shape and calcium dynamics. Nuclear-targeted YC2.1 (NupYC2.1) showed an absence of any obvious regular fluctuations in nuclear calcium levels during tube extension in vitro with typical growth rate fluctuation. The use of targeted cameleons to study subcellular calcium dynamics in pollen tubes is discussed.     

A comparative assessment of fluo Ca2+ indicators in rat ventricular myocytes

The fluo family of indicators is frequently used in studying Ca(2+) physiology; however, choosing which fluo indicator to use is not obvious. Indicator properties are typically determined in well-defined aqueous solutions. Inside cells, however, the properties can change markedly. We have characterized each of three fluo variants (fluo-2MA, fluo-3 and fluo-4) in two forms-the acetoxymethyl (AM) ester and the K(+) salt. We loaded indicators into rat ventricular myocytes and used confocal microscopy to monitor depolarization-induced fluorescence changes and fractional shortening. Myocytes loaded with the indicator AM esters showed significantly different Ca(2+) transients and fractional shortening kinetics. Loading the K(+) salts via whole-cell patch-pipette eliminated differences between fluo-3 and fluo-4, but not fluo-2MA. Cells loaded with different indicator AM esters showed different staining patterns-suggesting differential loading into organelles. Ca(2+) dissociation constants (K(d,Ca)), measured in protein-rich buffers mimicking the cytosol were significantly higher than values determined in simple buffers. This increase in K(d,Ca) (decrease in Ca(2+) affinity) was greatest for fluo-3 and fluo-4, and least for fluo-2MA. We conclude that the structurally-similar fluo variants differ with respect to cellular loading, subcellular compartmentalization, and intracellular Ca(2+) affinity. Therefore, judicious choice of fluo indicator and loading procedure is advisable when designing experiments.     

Localization of calcium during somatic embryogenesis of carrot (Daucus carota L.)

Summary The distribution of free cytosolic Ca²⁺ was studied during somatic embryogenesis of carrot using confocal scanning laser microscopy with fluo-3 as a fluorescent Ca²⁺ indicator. Chlorotetracycline fluorescence, antimonate precipitation and proton induced X-ray emission analysis were used as additional methods to confirm the results obtained with fluo-3. The process of embryogenesis was found to coincide with a rise in the level of free cytosolic Ca²⁺. The level of Ca²⁺ was low in proembryogenic masses and relatively high in later stages of embryogenesis. The highest signal was found in the protoderm of embryos from the late globular to the torpedo-shaped stage. A gradient in fluorescence intensity was often observed along the longitudinal axis of the embryos. The most conspicuous intracellular signal was found in the nucleus. Other organelles did not take up the dye and were always without fluorescence. The changes in [Ca²⁺]_c are discussed in relation to physiological processes which are known to be important during somatic embryogenesis.This article is dedicated to the memory of the late Dr. Hans-Dieter Reiss.     

Increase in IP3 and intracellular Ca2+ induced by phosphate depletion in LLC-PK 1 cells

The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.     

FRET-based Ca2+ measurement in B lymphocyte by flow cytometry and confocal microscopy

Upon the B cell antigen receptor (BCR) ligation Ca²⁺ mobilization is induced, which is essential for activation of downstream signaling molecules such as MAP kinase. Although synthetic fluorescent chelators such as Fluo-4 and Indo-1 are widely used for Ca²⁺ measurement upon BCR ligation, they are leaked or unfavorably localized into some organelles with time post loading. To solve these problems, we introduce a genetically encoded fluorescent indicator cameleon which is a fluorescence resonance energy transfer (FRET)-based indicator comprising two fluorescent proteins (CFP and YFP) and two Ca²⁺-responsive elements (a variant of calmodulin (CaM) and a CaM-binding peptide). Here, we demonstrate that cameleon as well as a conventional synthetic Ca²⁺ indicator enables Ca²⁺ measurement by flow cytometry clearly upon BCR ligation. In addition, confocal microscopy analysis allows us to detect cameleon-based Ca²⁺ mobilization in a single cell upon BCR ligation.     

Cytosolic and intranuclear calcium signals in rat basophilic leukemia cells as revealed by a confocal fluorescence microscope.

A confocal fluorescence microscope with an argon-ion laser (488 nm) and a He-Cd laser (325 nm) was used to study spatial heterogeneity of the calcium signals in rat basophilic leukemia 2H3 cloned cell line (RBL-2H3). After stimulation with antigen (2,4-dinitrophenol-conjugated bovine serum albumin), fluo-3-fluorescence intensities increased in individual RBL-2H3 cells with different lag times. Time-dependent profiles of the fluo-3-fluorescence intensities resembled closely the patterns of the sequential fluorescence-ratio images of fura-2, which were used to measure the intracellular free-calcium concentration ([Ca2+]i) in individual RBL-2H3 cells using a conventional fluorescence microscope. The present results obtained using the confocal fluorescence microscope showed spatial heterogeneities of fluo-3-fluorescence intensities, suggesting the existence of spatial heterogeneity of [Ca2+]i in RBL-2H3 cells. That is, the results showed that calcium signals first occurred transiently at pseudopodia in RBL-2H3 cells, then the signals transferred to the central parts of the cells. In addition, from the fluorescence images of co-loaded Hoechst 33342 (bisbenzimide H 33342, a DNA-specific probe) which were produced by excitation with a He-Cd laser, it was found that the fluorescence images of the nucleus were quite similar to those of the calcium signals mentioned above. This suggested that the receptor-mediated calcium signals were transferred not only to the cytoplasm but also to the nucleus.