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.     

SIMS ion microscopy in cancer research: single cell isotopic imaging for chemical composition, cytotoxicity and cell cycle recognition.

A secondary ion mass spectrometry (SIMS) based isotopic imaging technique was used for studies of i/ total calcium stored in cancerous and normal cell lines and ii/ intracellular chemical composition (total K, Na, and Ca) in relation to DNA staining patterns in taxol-treated breast cancer cells. A Cameca IMS-3f ion microscope with 0.5 microm spatial resolution was used. Observations were made on frozen freeze-dried cells. In MCF-10A non-tumorigenic breast epithelial cells, the nucleus contained 0.6 +/- 0.10 mM and the cytoplasm 1.1 +/- 0.30 mM total calcium per unit volume (mean +/- S.D.). MCF-7 tumorigenic breast epithelial cells revealed an abnormal total calcium distribution. Their nuclei and cytoplasm were not significantly different in stored calcium concentrations (0.5 +/- 0.08 mM total calcium in the nucleus and 0.6 +/- 0.07 mM in the cytoplasm). Furthermore, in MCF-7 cells the cytoplasmic total calcium is significantly less than in MCF-10A cells. Both cell lines contained approximately 150 mM intracellular potassium and 13 mM sodium. As 80% of the cytoplasmic total calcium pool in MCF-10A cells could be released with thapsigargin, it is plausible that the calcium storage capacity of the endoplasmic reticulum in tumorigenic MCF-7 cells is compromised. Correlative SIMS and confocal laser scanning microscopy (CLSM) revealed an increase in intracellular sodium and a redistribution of calcium in taxol-arrested M-phase cells prior to any noticeable DNA fragmentation. This novel correlative approach opens new avenues of research for understanding intracellular ionic composition in relation to therapeutic cytotoxicity. Other valuable features of SIMS for cancer research shown in this study include subcellular imaging of calcium influx using 44Ca, 127I from iododeoxyuridine for S-phase recognition, and 19F from fluorinated deoxyglucose.     

Antigen receptor-mediated calcium signals in B cells as revealed by confocal fluorescence microscopy

A confocal fluorescence microscope was used to study the antigen receptor-mediated calcium signals in B cells. Anti-IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo-3-loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non-homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.     

Receptor-mediated calcium signal playing a nuclear third messenger in the activation of antigen-specific B cells.

We have studied receptor-mediated calcium signals in antigen-specific B cells (trinitrophenol-specific B cell clone, TP67.21) using a confocal fluorescence microscope with an argon ion laser (488 nm) and a He-Cd laser (325 nm). Confocal fluorescence images of fluo-3 loaded B cells, excited by an argon ion laser, became much brighter and more nonhomogeneous than those before antigen stimulation. Time-dependent fluorescence changes in intensities were abrupt and quite similar to the patterns of the intracellular calcium ion concentration [Ca2+]i observed by a conventional fluorescence microscope using fura-2. From the morphological patterns of the calcium images, the parts of the bright fluorescence seemed to belong to the nucleus in B cells. To confirm the above events we measured the confocal fluorescence images of the nucleus. From the fluorescence images of co-loaded Hoechst 33342 (a DNA-specific fluorescent probe), which excited by a He-Cd laser, the brighter parts of the fluo-3 fluorescence intensities were identified to the nucleus in B cells. This suggested the possibility that the increased intranuclear calcium ions may play a nuclear third messenger in B 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.     

Free intracellular calcium ([CA2+]i) in opioid sensitive cells of the porcine anterior pituitary.

A new experimental model was utilized to study calcium involvement in the mechanism of opioid influence on cultured porcine pituitary cells. The in vitro model involved interactive argon laser cytometry of pituitary cells pre-loaded by three dyes (fluo-3AM, fura-red and naloxone-conjugated to fluorescein). We compared: 1) the kinetics of free intracellular calcium ([Ca2+]i) in anterior pituitary cells of pregnant pigs (day 25-30) treated in vitro with naloxone (NAL) or gonadotrophin-releasing hormone (GnRH) and 2) the distribution of the opioid-sensitive cells by image analysis of doubly loaded cells. In experiment 1, the changes in [Ca2+]i of pituitary cells pre-loaded with fluo-3 AM (488(ex)/520(em) nm) in response to NAL (10(-6) M) or to GnRH (10(-8) M) were compared to a control cell group. Repetitive line scans across cells were performed and the fluorescence emission from individually selected cells was measured in a time-dependent manner (in 0.5 seconds intervals during periods of 50 seconds). Analysis of data indicated significant increases of [Ca2+]i in NAL- (P<0.001) and GnRH-treated cells (P<0.05) in comparison to the control group. In experiment 2, the fluorescence intensity of doubly-loaded pituitary cells (fura-red, 488(ex)/605(em) nm, as principal calcium indicator and NAL-conjugated with fluorescein, 488(ex)/520(em) nm, to distinguish opioid-sensitive cells) were measured using dual detector image analysis. We found that only approximately 8% of the entire population of anterior pituitary cells exhibited sensitivity to the opioid antagonist treatment. This paper demonstrates calcium involvement in the opioid action on anterior pituitary cells from pregnant pigs and provides a useful model for studies at the individual pituitary cell level and in time-dependent manner.     

Properties of intra- and intercellular Ca(2+)-wave propagation elicited by mechanical stimulation in cultured RPE cells

Membrane deformation induced by a mechanical stimulus increases the [Ca2+]i in cultured retinal pigment epithelial (RPE) cells, and in many other cell types. In this study, confocal microscopy and Ca(2+)-measurements using the fluorescent dye fluo-3 were used to measure the spatiotemporal characteristics of the Ca(2+)-wave propagation during a mechanical stimulation in Long Evans (LE) RPE cells or dystrophic Royal College of Surgeons (RCS) RPE cells. Ca2+ signals were recorded in the mechanically stimulated cell and in the neighboring cells. A regenerative Ca(2+)-wave with a decreasing rate of propagation was found in the stimulated cells. The rate of propagation was significantly slower in RCS-RPE cells compared to LE-RPE cells. Incubation with thapsigargin significantly lowered the propagation rate in both LE- and RCS-RPE cells. The amplitude of the [Ca2+]i-rise in the nucleus and cytoplasm was differentially modulated by protein kinase C in RCS-RPE cells, but not in LE-RPE cells. It is concluded that RCS-RPE cells have intracellular Ca(2+)-regulating properties which are different from those of LE-RPE cells.     

应用重组表达钙离子敏感蛋白YC2.1研究粟酒裂殖酵母细胞内钙离子浓度的分布

把重组表达钙离子敏感蛋白的YC2．1基因（yellow cameleon 2．1）导入了粟酒裂殖酵母中,观察了粟酒裂殖酵母细胞内钙离子浓度的分布。结果发现,钙离子敏感蛋白所指示的钙离子呈细胞周缘胞质较高浓度分布,而在细胞胞质中部的钙离子浓度相对低一些。通过DAPI染色实验证实这是由于胞质中部细胞核的填充而形成。fluo-3染色的裂殖酵母细胞,由于fluo-3进入到细胞器（房室化现象）,所以出现胞质的内部区域高的荧光信号,而在周缘的胞质区相对弱,不能真实反应胞质钙离子的分布。因此重组表达钙离子敏感蛋白测定钙离子的方法优于fluo-3荧光探针的方法,对于裂殖酵母细胞胞内钙离子的研究具有良好的应用前景。     

Calcium uptake and membrane trafficking in response to PTH or 25(OH)D3 in polarized intestinal epithelial cells

Cell culture techniques providing retention of the polarized enterocyte morphology has allowed, for the first time, comparison of parathyroid hormone (PTH)- and 25-hydroxyvitamin D(3) [25(OH)D(3)]-induced (45)Ca uptake with membrane trafficking events discerned using confocal microscopy. Treatment of cells with 65 pM bPTH(1-34) promoted enhanced (45)Ca uptake between 1 and 10 min after peptide. The protein kinase A (PKA) antagonist, RpcAMP inhibited hormone-mediated uptake. At the microscopic level, cells labeled with the endocytic tracking dye FM1-43 revealed increased punctate staining 50-550s after hormone. Pretreatment of cells with RpcAMP abolished this pattern of staining. The calcium indicator dye fluo-3 AM revealed faint punctate labeling in controls, with increased bands of punctate labeling in the apical region of the cells after peptide hormone, and ultimately the basal region. Parallel studies conducted with the metabolite 25(OH)D(3) resulted in a slower stimulation of (45)Ca uptake 5-10 min after steroid, which was also inhibited by preincubation with RpcAMP. Cells labeled with FM1-43 and then treated with steroid showed no change in distribution of fluorescence during the 10 min incubation period. Confocal microscopy with fluo-3 revealed intense apical fluorescence--that after steroid --streamed to a perinuclear position, and ultimately the basal area. Uniformly diffuse staining, which would indicate cytoplasmic calcium transport, was observed only in controls. Membrane trafficking and compartmentalized calcium appear to be integral to agonist mediated cation transport.     

RBPMS不同剪接体的真核表达和亚细胞定位

目的：构建具有多种剪接形式的RNA结合蛋白（RBPMS）基因的真核表达载体,并在真核细胞中表达,确定不同形式的RBPMS在细胞中的定位。方法：采用PCR技术从人卵巢cDNA文库中扩增RBPMS基因的几种完整编码序列（命名为RBPl～RBP4）,克隆到带绿色荧光蛋白标签的pEGFP-C1表达载体上,转染人胚肾细胞293T,Western印迹鉴定RBPMS的表达,并利用激光共聚焦显微镜观察RBPMS不同剪接体在细胞中的定位。结果：限制性内切酶分析和DNA序列测定表明构建的重组表达载体正确,Western印迹实验证明RBP1～RBP4表达成功。通过激光共聚焦显微镜观察,RBP1／4围绕胞核在核膜的周围呈聚集状分布;RBP2则在细胞质和细胞核中均有分布,但会出现斑点状聚集;RBP3呈半月状紧密分布在细胞核周围;RBPMS中的RNA识别基序缺失后,这种现象消失,与空载体对照类似,在细胞核和细胞质中均有分布。结论：构建并表达了RBPMS基因的真核表达载体,RBPMS不同剪接体及RNA识别基序缺失后具有不同的亚细胞分布模式,提示具有不同的功能。