Selective subcellular localization of cations with variants of the potassium (pyro)antimonate technique.

Fixation of rat parotid with an unbuffered osmium tetroxide solution containing nearly saturated potassium (pyro)antimonate resulted in abundant deposition of cation-antimonate precipatates in acinar cells. Altering the antimonate concentration, including buffers or chelators in the solution or changing the primary fixative resulted in an altered intensity and distribution of the precipitates formed in the tissue, apparently reflecting a degree of selectivity in ion localization. Decreasing the concentration of pyroantimonate to about half-saturation preserved predominantly the less soluble antimonate salts (e.g., Na+, Ca++) and resulted in preferential retention of deposits along the plasmalemma and in mitochondrial "dense bodies," with loss of most cytoplasmic and nuclear precipitates. A similar pattern was seen if fixation with the high concentration antimonate-osmium procedure was followed by a prolonged rinse. Adding phosphate or collidine buffers markedly decreased precipitates in the nuclei and on granular reticulum as well. Phosphate buffer or ehtyleneglycoltetraacetate inhibited in vitro precipitation of calcium and sodium and decreased or abolished plasmalemmal deposits. Glutaraldehyde fixation, either in the presence of antimonate or prior to antimonate-containing osmium tetroxide, abolished heterochromatin deposits. Mitochondrial dense bodies were of two types, one containing precipitate and the other inherently osmiophilic. The latter were also observed in pyrophosphate-osmium controls. Results from in vitro titrations of cations with the various antimonate methods and from neutron activation analyses of fixed tissues supported conclusions drawn from fine structural distribution patterns and were interpreted as follows. In rat parotid acinar cells, deposits in heterochromatin and on granular reticulum probably arose from precipitation in sites of high K+ and H+ as well as--NH3+-rich histones. Plasmalemmal antimonate deposits demonstrated sites of sodium and/or calcium accumulation. Some mitochondrial dense bodies contained Ca++ whereas others were inherently osmiophilic. Large, extracellular deposits were probably predominantly sodium precipitates.     

Ca++ localization in boar spermatozoa by the pyroantimonate technique and X-ray microanalysis

Intracellular, loosely bound Ca++ has been localized electron microscopically in freshly ejaculated boar spermatozoa by in situ precipitation with potassium antimonate. Ca++ was identified as the cation precipitated by testing the EGTA-sensitivity of the precipitates and by X-ray microprobe analysis. The data obtained revealed that the outer acrosomal membrane is the preferential site for Ca++ precipitation in the sperm head.     

Localization of submembranous cations to the leading end of human neutrophils during chemotaxis

Potassium pyroantimonate was used to localize sites of bound cations in human neutrophils under conditions of random migration, stimulated random migration (chemokinesis), and directed migration (chemotaxis). The cells were placed in a standard chamber in which 0.45-micron micropore filters separated the cells from the stimulus (buffer, Escherichia coli endotoxin-activated serum or the synthetic chemotactic peptide N-formyl-Met-Leu-Phe). The small pore filters permitted pseudopod formation but impeded cell imgration through the filter. Cells examined under all conditions had electron-dense precipitates of antimonate salts in some granules. However, antimonate deposits were localized in the condensed chromatin of the nucleus during random migration and associated to a large extent with the uncondensed nuclear chromatin during chemokinesis and chemotaxis. Under conditions of chemokinesis deposition of antimonate procipitates appeared on the cytoplasmic side of the plasma membrane of neutrophils whereas under conditions of chemotaxis cation deposits beneath the cell membrane were localized to the pseudopods which were directed toward the chemoattractant. In addition to endotoxin-activated serum, concentrations of N-formyl-Met-Leu-Phe which caused neutrophil chemotaxis (10(-8) M) also caused cation deposition beneath the cell membrane at the leading end of the cell regardless of whether albumin was present in the incubation media. However, with higher concentrations of the synthetic peptide (10(-5) M) which caused granule release and were not chemotactic, submembranous cation deposition was not seen. EDTA (10 mM) and EGTA (10 mM) removed nuclear, granular, and submembranous cation deposits from neutrophils examined under conditions of chemotaxis. X-ray microprobe analysis of antimonate deposits revealed the possible presence of calcium but did not detect sodium or magnesium. The data indicate that chemotactic factors induce submembranous deposition of cations, most likely Ca++, which localize to the leading edge of cells exposed to a gradient of chemoattractant.     

Calcium in the synergid cells and other regions of pearl millet ovaries

Summary The synergids and other cells of mature, unpollinated pearl millet ovaries were investigated using: (1) freeze-substitution fixation in conjunction with scanning electron microscope observations and energy-dispersive X-ray microanalysis to localize total calcium (Ca) and other elements, and (2) antimonate precipitation to selectively localize loosely sequestered, exchangeable calcium (Ca⁺⁺). In freeze-fixed ovaries, the synergid cells, ovary wall, nucellus, and other regions of the ovary displayed, respectively and relatively, extremely high, high, moderate, and low levels of Ca. In antimonate-fixed ovaries, Ca-containing antimonate precipitates exhibited similar distribution patterns. In ovaries fixed using the conventional 2% (w/v) antimonate in fixatives, the synergids were disrupted due to precipitate overload. In the ovary wall, precipitates were mainly located in the intercellular spaces. Some precipitates were observed at the micropyle and along the outer ovule integument, associated with diffuse extracellular material, and in the cell walls of nucellar cells proximal to the micropyle. Examination of precipitate distribution inside the synergids was possible in ovaries fixed using 0.5% (w/v) antimonate in the fixatives. Cytoplasmic organelles of all synergids examined exhibited variable states of disintegration. The amount of precipitates associated with the degenerated organelles appeared to be proportional to the degree of their degeneration. Distinct precipitates were localized in contiguous regions of the nucellar cells fused with the embryo sac, the micropylar half of the embryo sac wall, and the filiform apparatus. The results are discussed in relation to the involvement of Ca⁺⁺ in mediating the functions of synergid cells during fertilization in angiosperms.On Specific Cooperative Agreement 58-43YK-8-0026 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

A calsequestrin-like protein in the endoplasmic reticulum of the sea urchin: localization and dynamics in the egg and first cell cycle embryo

Using an antiserum produced against a purified calsequestrin-like (CSL) protein from a microsomal fraction of sea urchin eggs, we performed light and electron microscopic immunocytochemical localizations on sea urchin eggs and embryos in the first cell cycle. The sea urchin CSL protein has been found to bind Ca++ similarly to calsequestrin, the well-characterized Ca++ storage protein in the sarcoplasmic reticulum of muscle cells. In semi-thin frozen sections of unfertilized eggs, immunofluorescent staining revealed a tubuloreticular network throughout the cytoplasm. Staining of isolated egg cortices with the CSL protein antiserum showed the presence of a submembranous polygonal, tubular network similar to ER network patterns seen in other cells and in egg cortices treated with the membrane staining dye DiIC16[3]. In frozen sections of embryos during interphase of the first cell cycle, a cytoplasmic network similar to that of the unfertilized egg was present. During mitosis, we observed a dramatic concentration of the antibody staining within the asters of the mitotic apparatus where ER is known to aggregate. Electron microscopic localization on unfertilized eggs using peroxidase-labeled secondary antibody demonstrated the presence of the CSL protein within the luminal compartment of ER-like tubules. Finally, in frozen sections of centrifugally stratified eggs, the immunofluorescent staining concentrated in the clear zone: a layer highly enriched in ER and thought to be the site of calcium release upon fertilization. This localization of a CSL protein within the ER of the egg provides evidence for the ability of this organelle to serve a Ca++ storage role in the regulation of intracellular Ca++ in nonmuscle cells in general, and in the regulation of fertilization and cell division in sea urchin eggs in particular.     

黄瓜种子萌发过程中Ca~(2 )分布的变化

采用细胞化学方法 ,研究了黄瓜种子中贮藏Ca2 的分布特点及其在萌发过程中的变化动态。干种子的子叶细胞中贮藏有大量的蛋白体、油脂体 ,Ca2 沉淀颗粒大量分布于胞质、胞间隙以及细胞质膜上。大多数蛋白体中有 1至数个圆球形或椭圆体形含Ca2 的球状晶体。相比之下 ,胚芽和胚根细胞中Ca2 较少。种子萌发早期 ,子叶中的贮藏钙及晶体溶解释放出的Ca2 部分转运到生长发育中的胚芽和胚根中。随着萌发的继续 ,胚根和胚芽细胞中的Ca2 不会持续增多 ,反而下降     

Effects of EGF and calcium on adult parenchymal hepatocyte proliferation

Adult rat hepatocytes were grown in serum-free medium containing 0.05-4 mM Ca++ and 40 ng/ml EGF. After 48 hours of cultivation the mitotic index and the percentage of second division metaphases were determined. The results demonstrated a maximum proliferation response to EGF at a Ca++ concentration of 0.4 mM. With lower and higher external Ca++ concentrations the fraction of cells undergoing more than one cell division decreased. At lower Ca++ concentrations this decrease appears to result from a reduced viability. In contrast, the low response to EGF at higher Ca++ concentrations--especially in the physiological range--may reflect the influence of Ca++ on the state of hepatocyte differentiation.     

淹水玉米幼苗根尖分生细胞内Ca2+超微细胞化学定位

采用焦锑酸钾沉淀法,对遭受淹水胁迫的玉米幼苗初生根根尖分生细胞内钙离子分布变化情况进行了电镜细胞化学观察。在正常状态下,根尖分生细胞内Ca^2＋沉淀颗粒的分布较少．主要位于细胞核和细胞质中。在淹水1h后,根尖分生细胞内呈现有大量Ca^2＋沉淀颗粒分布,细胞核和细胞质中分布的Ca^2＋沉淀颗粒密度,远大于正常细胞。随着淹水时间的延长,根尖分生细胞的细胞核和细胞质中分布的Ca^2＋沉淀颗粒呈现不断增多的趋势,而液泡中分布的Ca^2＋沉淀颗粒则逐步明显减少。根据实验结果本文对受淹根尖分生细胞的死亡与Ca^2＋分布变化的关系进行了研究。     

低温胁迫下董棕（Garyota urens L.）幼苗叶肉细胞内Ca2+水平及细胞超微结构的变化

用焦锑酸钙沉淀的电镜细胞化学方法,研究了低温胁迫下董棕（Garyota urensL.） 幼苗叶肉细胞内Ca2+水平的变化。研究结果表明,未经低温处理的董棕幼苗叶肉细胞,焦锑 酸钙沉淀颗粒大量出现在液泡和细胞间隙中,细胞壁中也可见少量沉淀,而细胞基质中则看 不到焦锑酸钙沉淀;经2 ℃ 48 h低温处理后,细胞基质和细胞膜上焦锑酸钙沉淀增加,而液泡和细胞间隙中的焦锑酸钙沉淀则显著减少,并且超微结构已初步显示出寒害的特征,叶绿体外膜部分破损,类囊体片层稀疏且排列不规则,光合速率明显下降等;经2℃ 120 h低温处理后,细胞间隙内的焦锑酸钙沉淀极少,有的也紧贴在细胞外壁上,而细胞基质和细胞膜上则分布有非常多的焦锑酸钙沉淀,在核基质和液泡中也可见到少量的焦锑酸钙沉淀,并且超微结构遭到了显著破坏,叶绿体结构完全被破坏,核膜与液泡膜严重破损,内部结构模糊,细胞只表现为呼吸作用,不进行光合作用。表明Ca2+的区域性分布的变化与植物抗寒性存在一定关系。     

Studies on the contractile vacuole of Poterioochromonas malhamensis Peterfi. II. Antimonate stains the contractile vacuole of Poterioochromonas

Fixation of the chrysophycean flagellate Poterioochromonas malhamensis in the presence of potassium (pyro) antimonate have been carried out. Intense antimonate deposits are characteristic of the various elements of the contractile vacuole as well as a portion of the plasma membrane adjacent to the contractile vacuole. Neither pretreatment of the cells with EGTA or EDTA nor the treatment of sections with these chelators is successful in removing the antimonate precipitates. Despite this, X-ray microanalyses of these deposits indicate the presence of calcium.     

INORGANIC CATIONS IN RAT KIDNEY : Localization with Potassium Pyroantimonate-Perfusion Fixation

For localization of pyroantimonate-precipitable cations, rat kidney was fixed by perfusion with a saturated aqueous solution of potassium pyroantimonate (pH about 9.2, without addition of any conventional fixative). A remarkably good preservation of the tissue and cell morphology was obtained as well as a consistent and reproducible localization of the insoluble antimonate salts of magnesium, calcium, and sodium. All proximal and distal tubules and glomeruli were delimited by massive electron-opaque precipitates localized in the basement membrane and, to a lesser extent, in adjacent connective tissue. In the intraglomerular capillaries the antimonate precipitate was encountered in the basement membranes and also between the foot processes. In addition to a more or less uniform distribution in the cytoplasm and between the microvilli of the brush border, antimonate precipitates were found in all cell nuclei, mainly between the masses of condensed chromatin. The mitochondria usually contained a few large antimonate deposits which probably correspond to the so-called "dense granules" observed after conventional fixations.     

Identification of the small nuclear RNAs associated with the mitotic chromosomes of Amoeba proteus

Amebas contain 7 electrophoretically distinct species of small nuclear RNAs (snRNAs), some of which are known to associate in a striking manner with mitotic chromosomes. These RNAs can be divided into 2 classes, one consisting of 4 snRNA species that shuttle in a non-random way between nucleus and cytoplasm during interphase and one consisting of 3 snRNA species that do not leave the nucleus at all during interphase. In the work reported here we sought to determine which class is associated with mitotic chromosomes. Through a series of micromanipulative procedures we arranged for the shuttling snRNAs to be the only radioactive molecules in the cell. Such cells were allowed to enter mitosis, whereupon they were fixed and subjected to autoradiography. In those cells no radioactive snRNAs were found associated with mitotic chromosomes. It is concluded, therefore, that those snRNAs that do associate with mitotic chromosomes must be one or more of the non-shuttling species. — In the Discussion, how the non-shuttling snRNAs may function in cell activities is considered.     

Cell cycle-dependent regulation of structure of endoplasmic reticulum and inositol 1,4,5-trisphosphate-induced Ca2+ release in mouse oocytes and embryos

The organization of endoplasmic reticulum (ER) was examined in mouse eggs undergoing fertilization and in embryos during the first cell cycle. The ER in meiosis II (MII)-arrested mouse eggs is characterized by accumulations (clusters) that are restricted to the cortex of the vegetal hemisphere of the egg. Monitoring ER structure with DiI18 after egg activation has demonstrated that ER clusters disappear at the completion of meiosis II. The ER clusters can be maintained by inhibiting the decrease in cdk1-cyclin B activity by using the proteasome inhibitor MG132, or by microinjecting excess cyclin B. A role for cdk1-cyclin B in ER organization is further suggested by the finding that the cdk inhibitor roscovitine causes the loss of ER clusters in MII eggs. Cortical clusters are specific to meiosis as they do not return in the first mitotic division; rather, the ER aggregates around the mitotic spindle. Inositol 1,4,5-trisphosphate-induced Ca(2+) release is also regulated in a cell cycle-dependent manner where it is increased in MII and in the first mitosis. The cell cycle dependent effects on ER structure and inositol 1,4,5-trisphosphate-induced Ca(2+) release have implications for understanding meiotic and mitotic control of ER structure and inheritance, and of the mechanisms regulating mitotic Ca(2+) signaling.     

Localization of calcium in roots and microsomal membranes of corn by direct pyroantimonate precipitation

Many cell membrane systems, including microsomal vesicles of corn, are able to regulate calcium levels both in vivo and in vitro, often in an ATP-dependent, calmodulin-stimulated fashion. The purpose of this study was to determine calcium distribution in meristematic cells of intact tissue and microsomal vesicles from corn roots using direct pyroantimonate-osmium fixation. In root cells, precipitates were localized in mitochondria, plastids, the nucleus, endoplasmic reticulum, Golgi apparatus, and along the plasma membrane. Plasma membrane-enriched microsomal vesicles isolated from corn roots incubated in media to permit calcium transport before pyroantimonate-osmium fixation show internal precipitates associated with the membrane and in the lumen of the vesicles. De-staining of the sections with 1 mM EDTA or EGTA removed precipitate from the sections, confirming the presence of calcium in the antimonate precipitates. These data support biochemical data that this same membrane preparation exhibited ATP-dependent calcium sequestration that was stimulated by calmodulin, as measured by retention of 45Ca. This provides evidence that these membranes are responsible for ATP-requiring, calmodulin-stimulated calcium transport in the intact cell.     

Effect of heat shock on ultrastructure and calcium distribution in Lavandula pinnata L. glandular trichomes

The effects of heat shock (HS) on the ultrastructure and calcium distribution of Lavandula pinnata secretory trichomes are examined using transmission electron microscopy and potassium antimonate precipitation. After 48-h HS at 40°C, plastids become distorted and lack stroma and osmiophilic deposits, the cristae of the mitochondria become indistinct, the endoplasmic reticulum acquires a chain-like appearance with ribosomes prominently attached to the lamellae, and the plasma and organelle membranes become distorted. Heat shock is associated with a decrease in calcium precipitates in the trichomes, while the number of precipitates increases in the mesophyll cells. Prolonged exposure to elevated calcium levels may be toxic to the mesophyll cells, while the lack of calcium in the glands cell may deprive them of the normal protective advantages of elevated calcium levels. The inequality in calcium distribution may result not only from uptake from the transpiration stream, but also from redistribution of calcium from the trichomes to the mesophyll cells.     

光敏核不育水稻农垦58S发育过程中药壁超微结构和Ca2+分布的变化

利用焦锑酸钾沉淀法分析光敏感核不育水稻农垦58S药壁超微结构和Ca²⁺分布的变化,发现在长日照条件下,药壁绒粘层细胞和药壁中Ca²⁺从花粉母细胞时期开始出现异常;随着花药的发育,药壁中Ca²⁺沉淀增加,主要分布在绒粘层细胞和乌氏体表面,长日照条件下不能很好地形成乌氏体,且乌氏体表面Ca²⁺沉淀要比短日照条件下少。     

Changes of the Level of Ca2+ in Cells of Rice Seedlings Under Low Temperature Stress

The changes of Ca2+ localization in ceils of rice (Oryza sativa L. ) seedlings under chilling stress were investigated with calcium antimonate precipitate-electromicroscopic-cyto- chemical methods. When rice seedlings grew at the optimum temperature, it was shown that the deposits of calcium antimonate, being the indicator for Ca2+ localization, mainly concen-trated within the vacuoles and intercellular spaces, and that there was also some Caz+ deposits in plastid, mitochondria, cytoplasm and nucleus. This indicates that under the normal condition, the vacuoles are the main pool of Ca2+ in plant cells, and that there is quite an amount of Ca2+ in the intercellular spaces. On the contrary, the free Ca2+ in cytoplasm and nucleus is very low under the normal condition. When the rice seedlings were treated at the temperature of 1 ℃ for 24 h, there nearly appeared a ring of well arranged Ca2+ precipitates in the inner side of plasmalemma. Meanwhile, the level of Ca2+ in cytoplasm and nucleus increased considerably. When the chilling stress of 1 ℃ continued for 48 h, a great amount of Ca2+ distributed within the cytoplasm and nucleus, and there was also a large quantity of Ca2+ deposits on vacuolar membranes and envelope of plastid. However, the ultrastructures of the cells remained normal. Based on the above observations, the authors proposed that the increase of Ca2+ in cytoplasm and nucleus under chilling stress might be related to the ulterior changes of physiological-biochemical processes.     

Electron-dense endoplasmic reticulum-like profiles closely associated with mitochondria in glomus cells of the carotid body after fixation with oxalate

Fixation in the presence of oxalate was used to demonstrate the electron-dense Ca2+ precipitates in the endoplasmic reticulum in glomus cells of the carotid body. Glomus cells in intact carotid bodies or cells dissociated from the organ by treatment with collagenase were studied electron microscopically. In the intact organ as well as in dissociated glomus cells, electron-dense endoplasmic reticulum-like profiles were seen closely associated with mitochondria, while these lacked reaction product. The interspace between mitochondria was occupied by electron-dense, slightly distended ER, which appeared to contact the outer membrane of the mitochondria. Occasionally, a mitochondrion was in contact with several ER profiles or the ER formed an electron-dense 'cap' on the mitochondrion. The electron-dense precipitates could be removed from ultrathin sections with the calcium chelator ethyleneglycol-2(2-aminoethyl tetra-acetic acid) (EGTA). It is tentatively suggested that the endoplasmic reticulum could be involved in intracellular buffering of Ca2+ in the glomus cell, as has been previously suggested for neurons.     

Distribution of the antimonate precipitate in normal and segregated nucleoli after potassium pyroantimonate fixation

Fixation with a saturated aqueous solution of potassium pyroantimonate produces electron-opaque antimonate deposits in the nucleoli of lutheinic and hepatic cells. The comparative analysis between normal and actinomycin D segregated nucleoli fixed either with glutaraldehyde-osmium or potassium pyroantimonate facilitates location of the ring-shaped precipitates in the fibrillar centers and the fine dense ones in the pars fibrosa.     

Localization of calcium in the pericarp cells of tomato fruits during the development of blossom-end rot

Summary. Blossom-end rot (BER) of tomato (Lycopersicon esculentum) fruits is considered to be a physiological disorder caused by calcium deficiency. We attempted to clarify the localization of calcium in the pericarp cells and the ultrastructural changes during the development of BER. Calcium precipitates were observed as electron-dense deposits by an antimonate precipitation method. Some calcium precipitates were localized in the cytosol, nucleus, plastids, and vacuoles at an early developmental stage of normal fruits. Calcium precipitates were increased markedly on the plasma membrane during the rapid-fruit-growth stage compared with their level at the early stage. Cell collapse occurred in the water-soaked region at the rapid-fruit-growth stage in BER fruits. There were no visible calcium precipitates on the traces of plasma membrane near the cell wall of the collapsed cells. The amount of calcium precipitates on plasma membranes near collapsed cells was smaller than that in the cells of normal fruits and normal parts of BER fruits, and the amount on cells near collapsed cells was small. The amount of calcium precipitates on the plasma membranes increased as the distance from collapsed cells increased. On the other hand, calcium precipitates were visible normally in the cytosol, organelles, and vacuoles and even traces of them in collapsed cells. The distribution pattern of the calcium precipitates on the plasma membrane was thus considerably different between normal and BER fruits. On the basis of these observations, we concluded that calcium deficiency in plasma membranes caused cell collapses in BER tomato fruits.Correspondence and reprints: National Institute of Vegetable and Tea Science, National Agricultural Research Organization, Ano, Mie, 514-2392, Japan.