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.     

Ultrastructural changes in neurohaemal tissue of the stick insect,Carausius morosus,induced by the ionophores Br-X-537 A and A-23187

Summary The antibiotic ionophores Br-X-537A and A-23187 alter the ultrastructure of neurohaemal tissue on the transverse nerve of the stick insect, Carausius morosus. Br-X-537A induces dramatic changes in the ultrastructural appearance of all three types of neurosecretory fibres present in the neurohaemal tissue. The neurosecretory granules become more electron-lucent and the mitochondria become more electron-opaque. The bounding membrane of the granules is frequently ruptured. A-23187, on the other hand, has no effect on two of the three types of fibres, but does produce an increase in the number of exocytotic profiles in the third.The two ionophores therefore have different effects on the same tissue. The results are discussed in the light of previous work with the use of these ionophores.We wish to thank Mrs. J. Birch for assistance with the electron micrographs, and Roche Products Ltd. and Lilly Research Centre Ltd. for gifts of the ionophores Br-X-537A and A-23187. The work was supported by the Science Research Council     

Calcium microdomains in mitochondria and nucleus

Endomembranes modify the progression of the cytosolic Ca(2+) wave and contribute to generate Ca(2+) microdomains, both in the cytosol and inside the own organella. The concentration of Ca(2+) in the cytosol ([Ca(2+)](C)), the mitochondria ([Ca(2+)](M)) and the nucleus ([Ca(2+)](N)) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca(2+) from the high [Ca(2+)](C) microdomains generated during cell activation near Ca(2+) channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca(2+) signal to the bulk cytosol. This shaping of [Ca(2+)](C) signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca(2+)](M) signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca(2+)](C) signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca(2+)](N) signals could be generated by direct release of stored Ca(2+) into the nucleoplasm. Ca(2+) release could even be restricted to subnuclear domains. Putative Ca(2+) stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca(2+) into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain.     

Calcium containing particles in mitochondria of heart muscle cells as shown by cryo-ultramicrotomy and X-ray microanalysis

Summary Mitochondria of normal myocardial cells of the sand rat and the mouse as well as of the left ventricle of man, have been examined for their content of calcium. Ultrahistochemistry and X-ray microanalysis revealed two basically different inclusions: Osmiophilic mitochondrial granules and Spherical mitochondrial particles. Osmiophilic mitochondrial granules were found in conventionally fixed and plastic embedded tissues as well as in cryosections of chemically fixed and sucrose infused tissues. Such granules lacked inert electron density and probably consisted mainly of unsaturated lipids. X-ray spectra obtained from these tissues revealed no peaks for calcium. Spherical mitochondrial particles were present in dry-cut cryo-sections of N₂-frozen tissues not treated by fixatives and/or cryoprotectants. These particles were deeply electron dense in unstained, freeze-dried cryo-sections. They usually measured from 600Å–900Å in diameter in the normal myocardium of the sand rat and the mouse and from 250 Å–400Å in diameter in the left ventricular myocardium of man. Significant calcium peaks could be identified in the X-ray spectra of these particles, whereas none occurred in the analyses of other tissue regions. Potassium was detected with about equal frequency in the particles and in other parts of the tissue. On the basis of the inert electron density of the particles and their absence in chemically fixed tissues as well as of the results of the X-ray analysis, it is concluded that they contain precipitates of extremely labile ions of mitochondrial calcium.We should like to thank Mrs. Trine Jensen for skillful technical assistance. This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from the Norwegian Research Council for Science and the Humanities     

Calcium content and distribution in egg vesicles ofGalleria mellonella (Lepidoptera) as determined by X-ray microanalysis

Summary In egg vesicles ofGalleria mellonella (Lepidoptera) electron microprobe analysis reveals calcium in concentrations of 9 and 3 mmoles per 1,000 g tissue wet weight in oocytes and accompanying trophic cells, respectively. This high average level of calcium characterizes both pre- and postvitellogenic oocytes, but the distribution of calcium is not uniform. In postvitellogenic vesicles the central area of the ooplasm shows a higher content of Ca than peripheral one, what may be correlated with the distribution of mature yolk platelets within the ooplasm.     

Protective role of transient pore openings in calcium handling by cardiac mitochondria

Long-lasting mitochondrial permeability transition pore (mPTP) openings damage mitochondria, but transient mPTP openings protect against chronic cardiac stress. To probe the mechanism, we subjected isolated cardiac mitochondria to gradual Ca(2+) loading, which, in the absence of BSA, induced long-lasting mPTP opening, causing matrix depolarization. However, with BSA present to mimic cytoplasmic fatty acid-binding proteins, the mitochondrial population remained polarized and functional, even after matrix Ca(2+) release caused an extramitochondrial free [Ca(2+)] increase to >10 μM, unless mPTP openings were inhibited. These findings could be explained by asynchronous transient mPTP openings allowing individual mitochondria to depolarize long enough to flush accumulated matrix Ca(2+) and then to repolarize rapidly after pore closure. Because subsequent matrix Ca(2+) reuptake via the Ca(2+) uniporter is estimated to be >100-fold slower than matrix Ca(2+) release via mPTP, only a tiny fraction of mitochondria (<1%) are depolarized at any given time. Our results show that transient mPTP openings allow cardiac mitochondria to defend themselves collectively against elevated cytoplasmic Ca(2+) levels as long as respiratory chain activity is able to balance proton influx with proton pumping. We found that transient mPTP openings also stimulated reactive oxygen species production, which may engage reactive oxygen species-dependent cardioprotective signaling.     

Ultrastructural location of calcium and magnesium during mineralisation of the cuticle of the shore crab,as determined by the K-pyroantimonate method and X-ray microanalysis

We have investigated the distribution of Ca²⁺ and Mg²⁺ in the new cuticle of moulting shore crabs (Carcinus maenas), using the K-pyroantimonate method in combination with X-ray microanalysis in order to identify antimony precipitates. During the premoult period, Ca²⁺ and Mg²⁺ accumulate in well-defined sites of the new pigmented layer. After moulting, mineralisation appears to begin preferntially at these sites. These form a honeycomb-like structure that quickly increases the rigidity of the new cuticle, with a small recruitment of material from extraneous sources. Mineralisation of the principal layer, on the other hand, immediately follows deposition of the organic matrix. Our experiments also provide evidence that the epidermal cell extensions associated with the pore canals are the means by which Ca²⁺ and Mg²⁺ are transferred from the epidermis into the mineralising cuticular layers. The plasma membrane of these cell extensions appears densely lined by particles of antimony precipitate that probably mark the location of the transporting sites. Shortly after moulting, the distribution of mineral deposits is such that the cell extensions cross the mineralised lamellae of the principal layer and constitute preferential access routes to the pigmented layer, where mineralisation is still in progress.     

The demonstration of calcium in the axonal granules of the peripheral nervous system of male Schistosoma mansoni by X-ray microanalysis

Summary A conspicuous feature of freeze-dried cryosections of male Schistosoma mansoni is the presence of groups of electron-dense granules. X-ray analysis in the TEM of these granules shows them, in comparison with the adjacent cytoplasm, to contain significantly greater amounts of sodium and calcium. In glutaraldehyde-fixed material, these granules have been correlated with the membrane-bounded, electron-dense granules found within the axons of the peripheral nervous system.deceased     

Calcium localization in oat aleurone cells using chlorotetracycline and X-ray microanalysis

Calcium distribution was studied in oat caryopses. Using the chlorotetracycline method it was found that membrane-associated Ca²⁺ was present in the aleurone layer. X-ray microanalysis confirmed the presence of calcium in aleurone cells; it also demonstrated the presence of considerable amounts of calcium in the cell wall surrounding these cells.Abbreviation CTC chlorotetracycline     

Calcium and magnesium levels in isolated cardiac mitochondria from mice injected with isoproterenol

Summary Calcium (Ca) and Magnesium (Mg) are determined by atomic absorption flame spectrometry in isolated cardiac mitochondria from mice receiving subcutaneous injections of DL-isoproterenol HC1 (ISO), and in mitochondria of untreated controls. In the controls, mitochondria were isolated in the presence or absence of ruthenium red. On the absence of ruthenium red in the isolation medium, mitochondrial Ca levels increase by about 300%, while levels of Mg remain unchanged. Focal myocardial necrosis following a single ISO-injection is shown by electron microscopy. Ca and Mg levels are largely unaffected by a single dose of ISO until 24 h after the injection. A slight increase in Ca occurs in the 48 h samples. When multiple injections of ISO are given every 12th hour for 48 h, 72 h and 96 h, respectively, endogenous Ca and Mg increase significantly. It is suggested that this increase might be associated with ISO-induced cardiac hypertrophy rather than with the pharmacological effects of ISO per se.This work was supported by grants from The Norwegian Council on Cardiovascular Disease and from The Norwegian Research Council for Science and the Humanities     

The smooth endoplasmic reticulum in neurohypophysial axons of the rat: Possible involvement in transport,storage and release of neurosecretory material

Summary The intra-axonal organization of the smooth endoplasmic reticulum was studied in the neurohypophysis of rats during and after water deprivation. Parallel to conventional electron microscopy, the material was treated with a double impregnation staining technique specifically designed to contrast the intracellular membranous system. In conventionally stained ultrathin sections from severely dehydrated rats most axons appeared to be free of membranous organelles, whereas corresponding axons treated with the double-impregnation technique generally exhibited a highly developed system of smooth endoplasmic reticulum. In axonal endings, both techniques revealed a profusion of microvesicles in intimate relationship with tubular elements of the smooth endoplasmic reticulum. In short-term (12 h) rehydrated rats, a similarly developed system of smooth endoplasmic reticulum was still observed at all axonal levels with both procedures. After 24 to 48 h of rehydration the tubules of the smooth endoplasmic reticulum exhibited, in double impregnated material, numerous dilatations which resembled the adjacent neurosecretory granules. In conventionally stained ultrathin sections, an accumulation of electron dense material occurred within tubules of the smooth endoplasmic reticulum in the more proximal axonal segments, while in the more terminal segments, which contained numerous elongated granules, membrane continuity was frequently observed between newly formed granules and the smooth endoplasmic reticulum. After 7 days of rehydration the general pattern of the axonal smooth endoplasmic reticulum was comparable to that in untreated rats. These results are discussed in the light of a suggested involvement of the axonal smooth endoplasmic reticulum in the non-granular transport of neurosecretory material in connection with (1) storage in distally formed granules, and (2) release via microvesicles. Acknowledgements: The authors wish to express their gratitude to Mrs. M. Balmefrézol for her skillful technical assistance     

Calcium localisation by X-ray microanalysis and fluorescence microscopy in larvae of zooxanthellate and azooxanthellate corals

X-ray microanalysis and fluorescence microscopy (Calcium Orangetrade mark) was used to determine the distribution of intracellular calcium (I(Ca)), in the form of total and ionic calcium respectively, in planulae and settled larvae of a zooxanthellate coral. The distribution of total calcium only was determined in larvae of an azooxanthellate coral. In azooxanthellate planulae and settled larvae, total I(Ca) concentration in the oral ectoderm was high and similar to that in seawater (SW). Calcium concentration did not vary (P > 0.05) between planulae and settled larvae. However, settled larvae accumulated large amounts of calcium in gastrodermal lipid-containing cells. In contrast, zooxanthellate planulae possessed significantly (P < 0.01) lower concentrations of total I(Ca) within ectodermal cells in comparison to settled larvae. In addition, in settled zooxanthellate larvae total calcium concentration in the mesogloea and coelenteron was significantly (P < 0.05) higher than in the oral ectodermal and gastrodermal cells, respectively. Total I(Ca) concentrations in the oral ectoderm of settled larvae were also significantly (P < 0.01) lower than that of the calicoblastic ectoderm. In zooxanthellate settled larvae, ionic I(Ca) levels in the aboral epithelium surrounding rapidly growing septa were high. These levels increased significantly (P < 0.05) within the tissue surrounding growing septa after incubation in high-calcium SW.     

Demonstration of sulphur in secretory granules of anterior and posterior pituitary by X-ray microanalysis (author's transl)]

The sulphur content of secretory granules and other subcellular structures in the cells of the anterior pituitary and of neurosecrotory granules in the nerve fibers of the neural lobe have been studied by energy dispersive microanalysis. The highest level has been found in the secretory granules of the neural lobe.     

Electron microscope studies of the intracellular origin and formation of calcifying granules and calcium spherites in the hepatopancreas of the snail,Helix pomatia L.

Summary It has been previously demonstrated that the hepatopancreas of the snail, Helix pomatia, produce proteinaceous particles (termed b-granules), which are involved in the process of calcification. In calcium cells of this organ calcium spherites arise from these granules. The present study retraces the intracellular development of b-granules from Golgi vesicles and from Golgi saccules encircling the cytoplasmic areas up to the formation of calcium spherites. It is suggested that the mature b-granules are well-defined cytoplasmic units with calcifying capacity. The fine structure of b-granules and calcium spherites is described. The possible function of various cell organelles in the formation of the b-granules and spherites is discussed.This investigation was supported by grants from the Swedish Natural Science Research Council. The assistance of Mrs. I. Rehnberg is gratefully acknowledged.     

Ultrastructural localization of calcium, by electron-probe X-ray microanalysis, in the small intestine of suckling rats

The subcellular distribution of calcium has been investigated in samples, from the intestinal mucosa of 10-day rats, prepared for X-ray microanalysis by various techniques designed to minimize the loss of this element. Calcium retention and its threshold of detection was most satisfactory in freeze-dried frozen thin sections. In resin-embedded samples the best retention of calcium was found in specimens fixed in absolute ethanol, embedded without osmication, and sectioned onto glycerol. The results of this investigation indicate the presence of calcium in the supranuclear vacuole of enterocytes in the distal intestine of the neonatal rat. This calcium is probably taken up during the endocytosis of material from the intestinal lumen. The same mechanism may also be important in the uptake of other metals by suckling animals.     

X-ray microanalysis and chlorotetracycline staining of calcium vesicles in the green alga Mougeotia

Calcium ions have been proposed to play a key role in the sensory transduction of phytochrome-governed chloroplast movement in the green alga Mougeotia. To test this hypothesis, the intracellular pattern of calcium distribution was studied in this alga by two independent techniques, namely, X-ray microanalysis of fixed and of unfixed frozen-hydrated cells, as well as in vivo fluorescence by chlorotetracycline. Both methods of detection reveal a significant compartmentation of calcium in vesicles close to the chloroplast edge and, less frequently, in the cortical cytoplasm. Microfilaments, presumably actin, which could function in driving chloroplast movement, have been observed running between the chloroplast edge and the cortical cytoplasm (Wagner, G., Klein, K. (1978) Photochem. Photobiol. 27, 137). The vesicular calcium concentration is stable and decays only slowly in the absence of extracellular calcium much in the same way as the ability of the chloroplast to perform movements decreases. A functional relationship between vesicular calcium compartmentation and phytochrome-governed chloroplast movement in the green alga Mougeotia seems indicated.Abbreviation CTC chlorotetracycline     

Mitofusin 2 regulates STIM1 migration from the Ca2+ store to the plasma membrane in cells with depolarized mitochondria

Store-operated Ca2+ channels in the plasma membrane (PM) are activated by the depletion of Ca2+ from the endoplasmic reticulum (ER) and constitute a widespread and highly conserved Ca2+ influx pathway. After store emptying, the ER Ca2+ sensor STIM1 forms multimers, which then migrate to ER-PM junctions where they activate the Ca2+ release-activated Ca2+ channel Orai1. Movement of an intracellular protein to such specialized sites where it gates an ion channel is without precedence, but the fundamental question of how STIM1 migrates remains unresolved. Here, we show that trafficking of STIM1 to ER-PM junctions and subsequent Ca2+ release-activated Ca2+ channel activity is impaired following mitochondrial depolarization. We identify the dynamin-related mitochondrial protein mitofusin 2, mutations of which causes the inherited neurodegenerative disease Charcot-Marie-Tooth IIa in humans, as an important component of this mechanism. Our results reveal a molecular mechanism whereby a mitochondrial fusion protein regulates protein trafficking across the endoplasmic reticulum and reveals a homeostatic mechanism whereby mitochondrial depolarization can inhibit store-operated Ca2+ entry, thereby reducing cellular Ca2+ overload.     

Intracellular site of Sr2+ and Ba2+ accumulation in frog twitch muscle fibres as determined by electron probe X-ray microanalysis

Strontium and barium can substitute for calcium at different levels of the excitation-contraction-relaxation cycle. The problem of sequestration of these ions in cellular microcompartments may be settled only by direct evidence obtained with analytical methods. Isolated frog twitch muscle fibres were perfused with increasing concentrations of potassium in Ca-free solution supplemented with Sr2+ (10 mmol/l) or Ba2+ (5 mmol/l). After equilibration in a Ca-free Ringer with Sr2+ or Ba2+ for 30 to 60 min the fibres were frozen in liquid propane (at 80 K) to immobilise ions. Ultrathin (150 nm) cryosections were cut at 170 K, freeze-dried, carbon-coated and analysed in an electron microscope equipped with an X-ray spectrometer. The ultrastructure of the superficial layer of the fibres was satisfactorily preserved. The terminal cisternae (t.c.) of the sarcoplasmic reticulum (SR) were dark and contained various amounts of Sr or Ba in addition to Ca. In Sr loaded fibres the longitudinal SR occasionally showed electron dense content with significant amounts of Ca; no Sr was present. The results suggest that t.c. is the common sequestering compartment for Ca, Sr and Ba. Essentially the same distribution pattern of Sr was found following precipitation of Sr with a solution containing digitonin and Koxalate.