The intracellular localization of inorganic cations with potassium pyroantimonate. Electron microscope and microprobe analysis.
TL;DR: In both animal and plant tissues, the electron-opaque antimonate precipitates were found deposited in the nucleus—as well as within the nucleolus—and in the cytoplasm, largely at the site of the ribonucleoprotein particles; the condensed chromatin appeared relatively free of precipitates.
Abstract: Potassium pyroantimonate, when used as fixative (saturated or half-saturated, without addition of any conventional fixative) has been demonstrated to produce intracellular precipitates of the insoluble salts of calcium, magnesium, and sodium and to preserve the general cell morphology. In both animal and plant tissues, the electron-opaque antimonate precipitates were found deposited in the nucleus—as well as within the nucleolus—and in the cytoplasm, largely at the site of the ribonucleoprotein particles; the condensed chromatin appeared relatively free of precipitates. The inorganic cations are probably in a loosely bound state since they are not retained by conventional fixatives. The implications of this inorganic cation distribution in the intact cell are discussed in connection with their anionic counterparts, i.e., complexing of cations by fixed anionic charges and the coexistence of a large pool of inorganic orthophosphate anions in the nucleus and nucleolus.
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TL;DR: It is hoped that this cxercise provides evidence that it is possible to employ antimonate as a selective electron microscopic histoehemical stain for localization of exchangeable cellular Ca2 and that, in spite of inevitable limitations, it is a useful tool for exploring Ca2 regulation.
Abstract: Introduction As a result of concerted efforts by scientists from a wide range of disciplines, we are coming to realize the crucial role played by Ca2 in biological functions. In the realm of biochemistry, especially, there has been recently an explosive rise in our knowledge about Ca2 effects on numerous cellular reactions, processes, and structural components. One emerging concept is that Ca2 ‘ serves as regulator of many of these. In such wellexamined phenomena as muscle contraction, a high degree of compartmentalization of Ca2 , coupled with the cell’s ability to mobilize Ca2 among various compartments (and thus to locally alter levels of reactive Ca2 ), is the means by which this is achieved (2). Often, direct measurement of the dynamics of free Ca2 is technologically difficult. Many have chosen instead to cxamine intracellular Ca2 -binding or Ca2 -sequestration sites in the search for clues on regulatory processes. There exist several histochemical techniques for localization of these sites, among them being in situ precipitation ofCa2 with potassium antimonate. The ideal probe should retain the cell’s in vivo Ca2 distribution, maximize its detection, and minimize interferenee from other reacting species. Realistically speaking, few, if any, techniques in science match up to their ideal: limitations and dangers of artifact abound, and histoehemistry holds no exception. Use of antimonate has received its share of criticism, and, indeed, the variety of eations reported to precipitate with antimonate could easily lead one to conclude that specificity for Ca2 is not possible with the reaction. However, a closer look at the literature reveals that “antimonate precipitation” does not specify a unique procedure, but rather encompasses a bewildering array of variations. A survey of results obtained by others using different buffers, pH’s, antimonate concentrations, fixatives, and tissue pretreatments, as well as our own experience in handling the reagent, indicates that reaction parameters strongly influence retention of and precipitation of physiological cations relative to each other. Thus, while originally proposed and used as a means of loealizing Na (40), antimonate’s use recently has been almost exclusively in studies involving Ca2 localization. As elaborated in this review, careful choice of reaction conditions can make the antimonate technique highly selective for Ca2 in comparison to the other cations that are capable of precipitation. Also, other variations on the antimonate reaction, while not so specific for Ca2 , can be used in conjunction with analytical techniques such as X-ray analysis or chelator treatments to ascertain which of the deposits formed contain Ca2 . By means of several different antimonate procedures, coupled thus with deposit analysis, previous studies have localized Ca2 in a wide variety of tissue and cell types, and cumulatively have revealed Ca2 in nearly every type of membranous organelle, as well as in association with some nonmembranous cellular components (Table 1 ). We believe that a discussion of some parameters of antimonate precipitation is instructive for those considering its use, as well as for those trying to understand results obtained with it in the past. When comparing results obtained in various laboratories, it is often difficult to pinpoint the influence exerted by any single parameter of the technique, since even the most similar protocols usually differ from each other in several details. While we have tried to sort these out as much as possible, there are substantial areas ofunavoidable overlap with material discussed in other sections. In these eases, the reader is requested to cross-refer to appropriate sections for a more detailed analysis of other influencing factors. We hope this cxercise provides evidence that it is possible to employ antimonate as a selective electron microscopic histoehemical stain for localization of exchangeable cellular Ca2 and that, in spite of inevitable limitations, it is a useful tool for exploring Ca2 regulation.
187 citations
TL;DR: This chapter discusses the methods of measuring intracellular calcium, and the emphasis on the location of Ca 2+ may change from the assay of cytoplasmic Ca2 + toward determination of the source ofCa 2+ which causes alteration of cytopsized Ca2+ and toward the binding site of Ca 1+ which serves to produce the response.
Abstract: Publisher Summary This chapter discusses the methods of measuring intracellular calcium. The techniques described offer a wide range of approaches for assaying intracellular Ca 2+ . The variety of problems involved in monitoring in vivo Ca diffusion among compartments in the short period of the physiological event present peculiar technical challenges Techniques have kept pace with the problems, and at the same time the problems have stimulated development of the techniques. Thus, optical methods for following rapid Ca 2+ transients have been developed over the past few years, and the search continues for more sensitive and selective dyes. The emphasis on the location of Ca 2+ may change from the assay of cytoplasmic Ca2 + toward determination of the source of Ca 2+ which causes alteration of cytoplasmic Ca 2+ and toward the binding site of Ca 2+ which serves to produce the response. In some cells this development has already occurred. The technical problems in monitoring Ca 2+ fluxes within organelles of cells may be considerable, although some progress has already been made.
159 citations
TL;DR: Electron microscopy, freeze-etching, and optical diffraction show how the structure of the septate junction may provide the basis for the low-resistance pathway between the electrically coupled cells in mussel gill epithelia.
Abstract: Electron microscopy, freeze-etching, and optical diffraction show how the structure of the septate junction may provide the basis for the low-resistance pathway between the electrically coupled cells in mussel gill epithelia. Conventional electron microscopy suggests that the septa are pleated sheets that differentiate from and are structurally continuous with the junctional cell membranes. Freeze-etching exposes geometrically arranged rows of 85-A particles within the junctional cell membranes. Diffraction evidence shows that these membrane particles and the alternate vertices of the intercellular septal sheets are congruent and therefore superposable. Together, the membrane particles and septal sheets provide a channel that extends from the cytoplasm of one cell through the septate junction to the cytoplasm of the adjacent cell.
127 citations
TL;DR: 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.
Abstract: Fixation of rat parotid with an unbuffered osmium tetroxide solution containing nearly saturated potassium (pyro)antimonate resulted in abundant deposition of cation-antimonate precipitates 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 ethyleneglycoltetraacetate 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 precipi� * .�,..‘ �
124 citations
Cites background or methods from "The intracellular localization of i..."
..., Cat”, Mg and Na which have been demonstrated by other methods in nuclei) (26, 62, 69, 80)....
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...(68, 69), the fine structural morphology was not well preserved and the essential absence of precipitates within cells, except those having obviously disrupted membranes, indicated both inadequate cellular fixation and poor penetration of the antimonate molecule into cells....
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...Studies have been performed in which the nature of the cation(s) precipitated with selected pyroantimonate methods was evaluated by means of in vitro precipitation (5, 32, 53, 77), cation chelation (32, 37), emission and absorption spectrometry (15), electron-probe x-ray microanalysis (25, 36, 41, 69, 74), neutron activation analysis (60) and autoradiography (73)....
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...Antimonate fixation: Several variants of the Tandler method (30, 51, 68, 69) were tried....
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TL;DR: Fusion of plant gametes in vitro triggers calcium oscillations evident in both the zygote and primary endosperm during double fertilization that are similar to those in animals.
Abstract: Calcium has an essential signaling, physiological, and regulatory role during sexual reproduction in flowering plants; elevation of calcium amounts is an accurate predictor of plant fertility. Calcium is present in three forms: (1) covalently bound calcium, (2) loosely bound calcium typically associated with fixed and mobile anions (ionic bonding); and (3) cytosolic free calcium-an important secondary messenger in cell signaling. Pollen often requires calcium for germination. Pollen tube elongation typically relies on external calcium stores in the pistil. Calcium establishes polarity of the pollen tube and forms a basis for pulsatory growth. Applying calcium on the tip may alter the axis; thus calcium may have a role in determining the directionality of tube elongation. In the ovary and ovule, an abundance of calcium signals receptivity, provides essential mineral nutrition, and guides the pollen tube in some plants. Calcium patterns in the embryo sac also correspond to synergid receptivity, reflecting programmed cell death in one synergid cell that triggers degeneration and prepares this cell to receive the pollen tube. Male gametes are released in the synergid, and fusion of the gametes requires calcium, according to in vitro fertilization studies. Fusion of plant gametes in vitro triggers calcium oscillations evident in both the zygote and primary endosperm during double fertilization that are similar to those in animals.
108 citations
Cites background from "The intracellular localization of i..."
...Because they occur in an unbound soluble form, some of these calcium binding proteins can serve as transport molecules, acquiring calcium from storage and releasing it where calcium is required (Tandler et al., 1970)....
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References
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TL;DR: Experiments in which radioactive label is chased shows that there is no high molecular weight RNA permanently associated with the nucleus, and all of the early precursors to cytoplasmic ribosomal RNA are contained in the nuclear fraction.
1,086 citations
TL;DR: In the interphase nucleus, the distribution and fine structure of various components known to contain or should carry RNP have been investigated on ultrathin sections by means of a new preferential staining method and the use of various enzymatic and chemical extraction procedures.
617 citations
TL;DR: Tissue examined in the electron microscope showed a consistent and reproducible localization of the electron-opaque pyroantimonate salts of sodium and calcium to distinct sites in the tissue, suggesting that subcellular calcium is sequestered in specific sites in mammalian myocardium.
Abstract: This study was designed to investigate the proposition that subcellular calcium is sequestered in specific sites in mammalian myocardium. 29 functioning dog papillary muscles were fixed through the intact vascular supply by means of osmium tetroxide containing a 2% concentration of potassium pyroantimonate (K2H2Sb2O7·4H2O). Tissue examined in the electron microscope showed a consistent and reproducible localization of the electron-opaque pyroantimonate salts of sodium and calcium to distinct sites in the tissue. Sodium pyroantimonate was found exclusively in the extracellular space and clustered at the sarcolemmal membrane. Calcium pyroantimonate, on the other hand, identified primarily by its susceptibility to removal by chelation with EGTA and EDTA, was consistently found densely concentrated in the lateral sacs of the sarcoplasmic reticulum and over the sarcomeric I bands. M zones were virtually free of precipitate. The implications of these findings with respect to various parameters of muscle function are discussed.
165 citations
TL;DR: Ribonucleoprotein particles containing the precursors of ribosomal RNA were extracted from L cell nucleoli and analyzed under conditions comparable to those used in the characterization of cytoplasmic ribosomes, suggesting that the precursor particles have a relatively higher ratio of protein to RNA, and that ribosome maturation involves a progressive decrease in the proportion of associated protein.
Abstract: Ribonucleoprotein (RNP) particles containing the precursors of ribosomal RNA were extracted from L cell nucleoli and analyzed under conditions comparable to those used in the characterization of cytoplasmic ribosomes. Using nucleoli from cells suitably labeled with 3H-uridine, we detected three basic RNP components, sedimenting at approximately 62S, 78S, and 110S in sucrose gradients containing magnesium. A fourth particle, sedimenting at about 95S, appears to be a dimer of the 62S and 78S components. When centrifuged in gradients containing EDTA, the 62S, 78S, and 110S particles sediment at about 55S, 65S, and 80S, respectively. RNA was extracted from RNP particles which were prepared by two cycles of zonal centrifugation. The 62S particles yielded 32S RNA and a detectable amount of 28S RNA, the 78S structures, 32S RNA and possibly some 36S RNA, and the 110S particles, a mixture of 45S, 36S, and 32S RNA's. When cells were pulsed briefly and further incubated in the presence of actinomycin D, there was a gradual shift of radioactivity from heavier to lighter particles. This observation is consistent with the scheme of maturation: 110S → 78S → 62S. The principal buoyant densities in cesium chloride of the 110S, 78S, and 62S particles are 1.465, 1.490, and 1.545, respectively. These densities are all significantly lower than 1.570, which is characteristic of the mature large subunit of cytoplasmic ribosomes, suggesting that the precursor particles have a relatively higher ratio of protein to RNA, and that ribosome maturation involves, in addition to decrease in the size of the RNA molecules, a progressive decrease in the proportion of associated protein.
111 citations
TL;DR: The distribution and specificity of the resulting precipitate in rat kidney is described and it appeared that sodium ions could move after fixation but that sodium pyroantimonate precipitate could not.
Abstract: Potassium pyroantimonate added to fixative solutions has been used in tissue localization of sodium ions. The distribution and specificity of the resulting precipitate in rat kidney is described in this study. Two reproducible patterns of precipitate were obtained in control tissues. The first pattern, which occurred after fixation in solutions containing aldehyde, showed the precipitate to be mainly extracellular. The second pattern, showing the precipitate in both intracellular and extracellular locations, occurred after aldehyde fixation in those experimental situations favoring cellular swelling or after fixation with solutions containing osmium tetroxide. It appeared that sodium ions could move after fixation but that sodium pyroantimonate precipitate could not. Since model systems demonstrated that dense precipitate formed when potassium pyroantimonate was added to solutions containing certain biological amines or some divalent cations, it appeared likely that the reagent did not provide specific tissue localization for sodium ions.
77 citations