Mitochondrial calcium of intact and mechanically damaged bone and cartilage cells studied with K-pyroantimonate

Summary Cellular cation was localized with K-pyroantimonate osmium fixation in whole fetal mouse metatarsal bones and in deliberately mechanically damaged specimens. X-ray microprobe analysis of ultrathin sections showed a positive correlation between the concentration of Ca (and Sb) and the amount of electron-dense precipitate.In non-damaged osteoblasts and growth-plate chondrocytes dense precipitate had accumulated along the plasmalemma and the mitochondrial membranes, whereas damaged cells showed the precipitate on round granules in the mitochondrial matrix but not on membranes. Intermediate stages between these two patterns were also found.In a non-calcifying tissue such as liver no membrane-bound precipitate was found in intact cells. However, damaged liver cells showed precipitate-containing mitochondrial granules similar to those in damaged bone cells, but only after incubation of the damaged tissue for l h in a Ca-containing balanced salt solution.Freezing of fresh whole bones in liquid N₂ before fixation in K-pyroantimonate osmium did not change the precipitate pattern in the damaged cells, but in intact cells it produced a random distribution of precipitate unrelated to membranes.The results are compared with those obtained in other studies on the subcellular localization of calcium and in biochemical studies on membrane versus matrix loading in calcium-accumulating isolated mitochondria.     

Mitochondrial calcium of intact and mechanically damaged bone and cartilage cells studied with K-pyroantimonate.

Cellular cation was localized with K-pyroantimonate osmium fixation in whole fetal mouse metatarsal bones and in deliberately mechanically damaged specimens. X-ray microprobe analysis of ultrathin sections showed a positive correlation between the concentration of Ca (and Sb) and the amount of electron-dense precipitate. In non-damaged osteoblasts and growth-plate chondrocytes dense precipitate had accumulated along the plasmalemma and the mitochondrial membranes, whereas damaged cells showed the precipitate on round granules in the mitochondrial matrix but not on membranes. Intermediate stages between these two patterns were also found. In a non-calcifying tissue such as liver no membrane-bound precipitate was found in intact cells. However, damaged liver cells showed precipitate-containing mitochondrial granules similar to those in damaged bone cells, but only after incubation of the damaged tissue for 1 h in a Ca-containing balanced salt solution. Freezing of fresh whole bones in liquid N2 before fixation in K-pyroantimonate osmium did not change the precipitate pattern in the damaged cells, but in intact cells it produced a random distribution of precipitate unrelated to membranes. The results are compared with those obtained in other studies on the subcellular localization of calcium and in biochemical studies on membrane versus matrix loading in calcium-accumulating isolated mitochondria.     

Presence of calcium in polychaete cortical granules demonstrated by X-ray microanalysis on ultrathin cryo-sections of oocytes and eggs

Summary Cortical granules from fertilized eggs, oocytes and nurse cells of Ophryotrocha labronica have been analyzed for the presence of calcium using cryo-ultramicrotomy and X-ray microprobe analysis. All cortical granules showed a significant peak for calcium, but yolk granules were without calcium. These results support the hypothesis that the discharge of cortical granules shortly after fertilization is a self-propagating phenomenon involving the diffusion of Ca²⁺ from bursting granules.     

Development of brown fat cells in monolayer culture. II. Ultrastructural characterization of precursors, differentiating adipocytes and their mitochondria

The stroma of mature brown fat has been shown to contain cells which can proliferate and accumulate fat in monolayer cultures, and which have inherent characteristics distinct from those of white fat precursor cells. The purpose of the present investigation was to characterize by electron microscopic analysis these brown fat cells and their subsequent development when they were grown in vitro. By comparison with the existing ultrastructural data on brown fat in situ, it could thus be determined whether or not the precursor cells have the capacity to differentiate in culture. The stromal-vascular fraction isolated from the brown fat of weaned rats was identified as containing adipocyte stem cells, preadipocytes, endothelial cells and a few mature adipocytes. During the first week in culture (i.e., growth phase to confluence), when multilocular fat accumulation occurred, the mitochondria of the preadipocytes developed cristae and matrix granules, as they do in differentiating brown fat in situ. Such granules have been shown to be a sign of intense inner membrane synthetic activity. After confluence, the mitochondria regressed in internal structure and became morphologically more similar to white fat mitochondria. It was concluded that mature brown fat contains precursor cells which can differentiate in vitro. However, this differentiation was incomplete, and the necessity of specific factors for a full mitochondrial development in brown fat is discussed.     

Electron microscopic and microprobe analysis of calciuminduced differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum

Differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum leading to spherule formation can be induced by CaCl(2) if the concentration in the nutrient medium is increased by 5mM prior to the transfer to a non-nutrient salts medium. All stages previously reported for the typical (M(3)cVII) strain of Physarum polycephalum from microplasmodia to spherules are seen but the mutant lacks the synchrony that the replacement technique induces in the typical strain. X-ray microanalyses locate calcium and phosphorus in granules in mitochondria and in the cytoplasm of specimens fixed without osmium. Mitochondria accumulate calcium-containing granules during early differentiation and appear to be essentially without granules in mature spherules. Mobilization of mitochondrial calcium is implicated in the initiation of differentiation. A longitudinally striated cytoplasmic inclusion is abundant in microplasmodia grown in media that have not been supplemented with additional calcium and is seen more rarely during calcium-induced spherulation. Whether or not this inclusion represents cytoplasmic contractile elements is unknown. The calcium-treated mutant strain, previously considered non-differentiating, may prove to be a good alternate model for the study of factors influencing differentiation. It was employed earlier as a control in studies of strains that readily spherulate in response to routine procedures.     

Zinc sulfide in intestinal cell granules of Ancylostoma caninum adults

A source of confusion has existed since the turn of the century about the reddish brown, weakly birefringent "sphaerocrystals" located in the intestines of strongyle nematodes, Strongylus and Ancylostoma. X-ray diffraction and energy dispersive spectrometric analyses were used for accurate determination of the crystalline order and elemental composition of the granules in the canine hookworm Ancylostoma caninum. The composition of the intestinal pigmented granules was identified unequivocally as zinc sulfide. It seems most probable that the granules serve to detoxify high levels of metallic ions (specifically zinc) present due to the large intake of host blood.     

Calcium binding by subcellular fractions of bovine adrenal medulla

Significantly more calcium per gram protein was found in a relatively pure granule fraction isolated from fresh bovine adrenal medulla than in predominantly mitochondrial fractions isolated from the same tissue. Sixty-four and 55% of the calcium associated with chromaffin granule and mitochondrial fractions, respectively, was released into the supernatant upon lowering the tonicity of the medium. The per cent calcium released by this procedure was significantly greater for granules than for mitochondria (p < 0.05). The amount of calcium per gram protein released into the supernatant also was greater in granule fractions than in mitochondrial fractions (p < 0.05). These data, coupled with a previous report that 10^?3 M EDTA does not markedly decrease the calcium content of whole granules, indicate that the excess calcium of the granule fractions relative to the mitochondrial fractions is maintained within the particles of that fraction. The functional significance of the relatively large amount of calcium in chromaffin granules is not clear. The presence of 150 mM sodium chloride or potassium chloride decreases calcium binding by granule or mitochondrial fragments incubated in 2.2 mM calcium chloride in 0.2 M Tris, pH 7, by about 50%. EDTA, 10^?3 M, removes all but a small residual of the calcium associated with the granule or mitochondrial fragments whereas lowering the concentration of Tris increases calcium binding to about the same extent in both these subcellular fractions. The calcium-binding properties of granule and mitochondrial fragments therefore appear to be quantitatively and qualitatively similar. Inhibition of catecholamine release by relatively high concentrations of sodium may be explained by competitive inhibition of calcium binding. Calcium binding by granule fragments decreases with an increase in hydrogen ion concentration.     

Calcium containing lysosomes in the normal chick duodenum: a histochemical and analytical electron microscopic study.

Absorptive cells of the normal chick duodenum contain numerous supranuclear vesicular/vacuolar structures. By routine transmission electron microscopy, such structures are membrane bound and demonstrate a granular content. These vesicles appear to move laterally and eventually coalesce with the lateral plasma membrane (exocytosis). The granular contents are resistant to high temperature microincineration, thus revealing their mineral-containing nature. The granular vesicular matrix also stains intensely with osmium pyroantimonate EGTA chelation of pyroantimonate-stained vesicles selectively extracts the granules indicating a high concentration of calcium. X-ray microanalysis also demonstrates a significant intravesicular calcium localization. When tissues were incubated for the presence of acid phosphatase, the supranuclear vesicles were markedly positive for this lysosomal enzyme. A possible role for these calcium-containing lysosomes in the transcellular flux of calcium ions across the intestinal absorptive cell is discussed.     

ELECTRON MICROSCOPY AND ELECTRON PROBE ANALYSIS OF MITOCHONDRIAL CATION ACCUMULATION IN SMOOTH MUSCLE

The contractile responses to barium and the ultrastructure and ionic composition of mitochondria were studied in vascular smooth muscle. In normal rabbit portal anterior mesenteric vein (PAMV) and main pulmonary artery (MPA) smooth muscle mitochondria were frequently associated with the surface vesicles. The average distance between the outer mitochondrial and inner surface vesicle membrane was 4–5 nm. Ba contractures of MPA were tonic and of PAMV were phasic. Incubation of MPA and PAMV with Ba resulted in the accumulation of mitochondrial granules, followed in the MPA by massive mitochondrial swelling. Oligomycin and anoxia inhibited the appearance of mitochondrial electron-opaque granules and prevented the Ba-induced mitochondrial swelling in the MPA. Electron probe analysis of mitochondria in PAMV incubated with Ba and containing granules showed characteristic Ba signals over the mitochondria. Electron probe X-ray microanalysis also showed a highly significant (P < 0.001) correlation of P with mitochondrial Ba, in an estimated elemental ratio of approximately 3 Ba/4 P. Mitochondrial granules were still prominent after block staining of the osmium-fixed, Ba-loaded PAMV, but electron probe microanalysis showed no Ba, but only U, emissions. Tissues incubated with strontium had electron-opaque mitochondrial granules and deposits in the sarcoplasmic reticulum. X-ray microanalysis of mitochondria containing granules showed the presence of characteristic Sr and Ca emissions. The presence of Sr was similarly verified in the sarcoplasmic reticulum. These findings indicate the energy dependent uptake of divalent cations, in association with phosphate, by mitochondria in vascular smooth muscle in situ and the possibility that mitochondria may contribute to the regulation of intracellular divalent cation levels in smooth muscle.     

Cortical membrane-trafficking during the meiotic resumption of Xenopus laevis oocytes

Summary The giant mucous cells in the skin of the terrestrial banana slug Ariolimax columbianus secret intact granules containing mucins. Electron microscopy, after ultrarapid freezing and freeze-substitution in osmium, shows that the secreted granules are bounded by two distinct membranes, presumably derived from the Golgi apparatus and the plasmalemma. Relatively stable, intact granules can be obtained in great quantity in our in vitro system. Rapid lysis of the granules was induced by adenosine triphosphate. At much higher concentrations, adenosine diphosphate and 5-adenylimido-diphosphate also caused lysis. Other nucleotides and related compounds, as well as 1,4,5-inositol triphosphate and molluscan neurotransmitters and neuropeptides, had no effect on the granules. The stability of secreted granules varied with the ionic composition of the isosmotic medium in which they were suspended. When the predominant cation in the medium was potassium, and calcium was also present, granules lysed if exposed to shear stress (stirring of the suspension). This did not occur if sodium was the major cation present. None of the other ions in the suspension media had detectable effects on the stability of the granules.     

Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation

Mitochondria play an essential role in the ability of brown fat to generate heat, and the PGC-1 coactivators control several aspects of mitochondrial biogenesis. To investigate their specific roles in brown fat cells, we generated immortal preadipocyte lines from the brown adipose tissue of mice lacking PGC-1alpha. We could then efficiently knockdown PGC-1beta expression by shRNA expression. Loss of PGC-1alpha did not alter brown fat differentiation but severely reduced the induction of thermogenic genes. Cells deficient in either PGC-1alpha or PGC-1beta coactivators showed a small decrease in the differentiation-dependant program of mitochondrial biogenesis and respiration; however, this increase in mitochondrial number and function was totally abolished during brown fat differentiation when both PGC-1alpha and PGC-1beta were deficient. These data show that PGC-1alpha is essential for brown fat thermogenesis but not brown fat differentiation, and the PGC-1 coactivators play an absolutely essential but complementary function in differentiation-induced mitochondrial biogenesis.     

Autofluorescence and Ultrastructure in the Myxomycete Diachea leucopodia (Physarales)

Autofluorescence is reported for the first time in Myxomycete fruiting bodies. Ultrastructure of stalked sporangia of Diachea leucopodia (Didymiaceae, Physarales) was studied using scanning and transmission electron microscopy, energy-dispersive X-ray microanalysis, and fluorescence microscopy. External and internal properties of the peridium that surround the spores and capillitium exhibit autofluorescence. The stalk is composed of calcareous granules and energy-dispersive X-ray microanalysis demonstrates that the elemental composition of the peridium, capillitium, and stalk has varying concentrations of calcium.     

ELECTRON MICROSCOPE STUDIES ON THE ACTIVE ACCUMULATION OF SR++ BY RAT-LIVER MITOCHONDRIA

Electron-opaque granules are deposited in isolated rat-liver mitochondria concomitant with the energy-linked accumulation of Sr⁺⁺ by these organelles. High temperature microincineration (600°C) of thin sections of mitochondria containing different amounts of Sr⁺⁺ shows that a clear qualitative correlation exists between the number of inorganic residues remaining after incineration and the amount of Sr⁺⁺ translocated into the mitochondria. By loading the mitochondria with consecutive pulses of small amounts of Sr⁺⁺ ("multiple-pulse" loading), very early stages of granule formation can be detected; the first detectable deposits are seen closely associated with the cristae. The evidence presented supports the hypothesis that mineral deposition following or during the in vitro accumulation of ions by mitochondria occurs, at least initially, at sites on these membranes and not as nonspecific precipitates in the mitochondrial matrix. The large number of electron-opaque deposits (100 to 200) seen in single thin sections of individual mitochondria having accumulated intermediate levels of Sr⁺⁺ clearly exceeds the number of normal dense granules in rat-liver mitochondria, indicating that the normal matrix granules per se do not constitute sites essential for deposition. At the highest levels of Sr⁺⁺ uptake studied in the multiple-pulse loading experiments, needlelike deposits are seen, a result which suggests that the structural form ("crystallinity") of the mineral deposits may be determined by the rate of accumulation.     

Calcium binding sites in plasmodia of Physarum polycephalum as revealed by the pyroantimonate technique

Plasmodia of the acellular slime mold, Physarum polycephalum, were treated with an osmium tetroxide fixative containing potassium pyroantimonate to precipitate calcium and thereby localize calcium binding sites and sites of increased calcium concentration. Dense calcium pyroantimonate precipitates were detected within the nucleoli. The distribution of these precipitates during interphase and mitosis coincides with the distribution of the unique minichromosomes in Physarum, i.e., the numerous short pieces of extrachromosomal nucleolar chromatin containing segments of amplified DNA coding for ribosomal RNA. Calcium pyroantimonate precipitates were present as frequent dense granules in the mitochondrial matrix and as fine precipitates in the mitochondrial nucleoid. Large calcium-containing precipitates were seen within cytoplasmic vacuoles, confirming reports by others. In addition, we have identified calcium binding sites along the cytoplasmic surface of the plasma membrane. The distribution of calcium within the plasmodium is discussed in relation to the assembly of the mitotic spindle and the regulation of cell motility.     

Heterogeneity of the calcium-induced permeability transition in isolated non-synaptic brain mitochondria

Calcium overload of neural cell mitochondria plays a key role in excitotoxic and ischemic brain injury. This study tested the hypothesis that brain mitochondria consist of subpopulations with differential sensitivity to calcium-induced inner membrane permeability transition, and that this sensitivity is greatly reduced by physiological levels of adenine nucleotides. Isolated non-synaptosomal rat brain mitochondria were incubated in a potassium-based medium in the absence or presence of ATP or ADP. Measurements were made of medium and intramitochondrial free calcium, light scattering, mitochondrial ultrastructure, and the elemental composition of electron-opaque deposits within mitochondria treated with calcium. In the absence of adenine nucleotides, calcium induced a partial decrease in light scattering, accompanied by three distinct ultrastructural morphologies, including large-amplitude swelling, matrix vacuolization and a normal appearance. In the presence of ATP or ADP the mitochondrial calcium uptake capacity was greatly enhanced and calcium induced an increase rather than a decrease in mitochondrial light scattering. Approximately 10% of the mitochondria appeared damaged and the rest contained electron-dense precipitates that contained calcium, as determined by electron-energy loss spectroscopy. These results indicate that brain mitochondria are heterogeneous in their response to calcium. In the absence of adenine nucleotides, approximately 20% of the mitochondrial population exhibit morphological alterations consistent with activation of the permeability transition, but less than 10% exhibit evidence of osmotic swelling and membrane disruption in the presence of ATP or ADP.     

Mitochondrial biogenesis and thyroid status maturation in brown fat require CCAAT/enhancer-binding protein alpha

Brown fat differentiation in mice is fully achieved in fetuses at term and entails the acquisition of not only adipogenic but also thermogenic and oxidative mitochondrial capacities. The present study of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein alpha (C/EBPalpha-null mice) demonstrates that C/EBPalpha is essential for all of these processes. Developing brown fat from C/EBPalpha-null mice showed a lack of uncoupling protein-1 expression, impaired adipogenesis, and reduced size and number of mitochondria per cell when compared with wild-type mice. Furthermore, immature mitochondrial morphology was found in brown fat, but not in liver or heart, from C/EBPalpha-null mice. Concordantly, expression of both nuclear and mitochondrial genome-encoded genes for mitochondrial proteins was reduced in C/EBPalpha-null brown fat, although expression of mitochondrial rRNA and mitochondrial DNA content were unaltered. Expression of nuclear respiratory factor-2, thyroid hormone nuclear receptors, and peroxisome proliferator-activated receptor gamma coactivator-1, was delayed in C/EBPalpha-null brown fat. Iodothyronine 5'-deiodinase activity and thyroid hormone content were also reduced in brown fat from C/EBPalpha-null mice, indicating for the first time a crucial role for C/EBPalpha in controlling thyroid status in developing brown fat, which may contribute to impaired mitochondrial biogenesis and cell differentiation. When survival of C/EBPalpha-null mice was achieved by transgenically expressing C/EBPalpha only in the liver, a substantial recovery in brown fat differentiation was found by day 7 of postnatal age, which is associated with a compensatory overexpression of C/EBPdelta and C/EBPbeta.     

Sequential changes in brown adipose tissue composition, cytochrome oxidase activity and GDP binding throughout pregnancy and lactation in the rat

The sequential appearance of changes in interscapular brown adipose tissue composition, cytochrome oxidase activity and GDP binding was studied throughout pregnancy and lactation in the rat. Brown adipose tissue was hypertrophied during pregnancy because of progressive lipid accumulation, whereas its mitochondrial component and GDP binding to brown fat mitochondria were unchanged. In early lactation (day 5) there was a decrease in the overall GDP binding to brown fat only because of the lower mitochondrial protein content. In late stages of lactation (days 10 and 15), the amount of tissue and its mitochondrial protein content were minimal and the GDP binding per mitochondrial protein decreased substantially. Scatchard analysis in day-15-lactating rats indicated a large decrease in GDP binding sites without any changes in affinity. It is concluded that the diminished thermogenic activity of brown fat in lactation is attained through changes at different structural levels of the tissue occurring in a characteristic sequential trend; first a reduction in its mitochondrial component, and only later, at mid-lactation, a decrease in the specific mitochondrial proton conductance pathway activity.     

Elemental analysis of particles in fungal zoospores

The elemental composition of gamma particles in zoospores ofAllomyces macrogynus andBlastocladiella emersonii was determined by use of energy dispersive X-ray analysis of glutaraldehyde fixed, thin section zoospores. Isolated preparations of gamma particles were also examined. Gamma particles contained no detectable elements. Similar sized, globular, electron dense cytoplasmic inclusions contained phosphorus and calcium. We suggest that previous studies assigning calcium and phosphorus to gamma particles may have been based on confusion of these two types of cytoplasmic inclusions.     

Ca increase in secretory granules of stimulated mast cells

The elemental content of rat peritoneal mast-cell secretory granules has been measured by X-ray micro-analysis. Two distinct categories of granules were analyzed: intact granules, seen in control samples, and spumous granules, corresponding to exocytosed granule matrices. The average Ca content of intact granules was found to be approximately equal to cytosolic concentration, and to increase up to 40-fold in spumous granules. A significant increase was also observed for Na and Cl. These changes were not observed (for Ca) or weaker (for Na and Cl) if the cells had been challenged in the absence of nominal extracellular Ca; in this case, there was also a significant decrease in the sulphur content, suggesting a partial dispersion of the organic matrix components. In exocytosed granule matrices, in the presence but not in the absence of extracellular Ca, a slow and long-lasting increase of intragranular free Ca was monitored by changes in the fluorescence of the Ca-sensitive probes Fluo-3 and Calcium Green-5N, accumulated within rat mast-cell secretory granules. These findings are discussed along two lines: It is proposed that the calcium uptake by the exocytosed mast-cell granule matrices can have a physiological relevance for the surrounding tissue. Mast-cell granules do not disperse after exocytosis. The major uptake of Ca which is seen after opening of the exocytotic pore could be responsible for the exceptional stability of the externalized matrices.     

Elemental compositions and characteristics of aerobic granules cultivated at different substrate N/C ratios

The effects of the substrate N/C ratios on the formation, elemental compositions and characteristics of aerobic granules were investigated in four sequencing batch reactors. Results showed that aerobic granules could form at substrate N/C ratios ranging from 5/100 to 30/100 and the substrate N/C ratio had a direct and profound effect on the elemental compositions and characteristics of the aerobic granules. Nitrifying populations in aerobic granules were enriched significantly with the increase in the substrate N/C ratio, while the respective ratio of cell oxygen, nitrogen and calcium to cell carbon were also determined by the substrate N/C ratio. It was found that cell hydrophobicity of aerobic granules was inversely related to the ratio of cell oxygen normalized to cell carbon. Since the cell calcium content in aerobic granules developed at different substrate N/C ratios was even lower than that in the seed sludge, it is reasonable to conclude that the cell calcium would not contribute to aerobic granulation. This study probably for the first time demonstrates that the elemental composition, microbial distribution and characteristics of aerobic granules are related to the substrate N/C ratio applied.