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.     

Electron microscopy of primary zoosporogenesis inPlasmodiophora brassicae

Primary zoosporogenesis in resting sporangia ofPlasmodiophora brassicae that had been incubated for 14 d in culture solution containing turnip seedlings was examined by transmission electron microscopy. A single zoospore differentiated within each sporangium, the differentiation being initiated by the emergence, of two flagella in the tight space formed by invagination of the plasma membrane within the sporangium. The differentiazing zoospore was similar in intracellular aspects to sporangia within clubroot galls. Then a deep groove formed on the zoospore cell body by further invagination of the plasma membrane. Two flagella appeared to coil around the zoospore cell body in parallel along this groove. Thereafter, the cell body lost the groove and became rounded following the protoplasmic condensation (contraction of cell body) during late development, and assumed an irregular shape at the stage of maturation. Intracellular features in, developing and mature zoospores were complicated, being characterized by electron-dense nuclei and mitochondria, microbodies, cored vesicles and various unidentified cytoplasmic vesicles and granules. A nucleolus-like region was observed only in the nucleus of the mature zoospore. A partially opened germ, pore was also seem in the sporangium containing the mature zoospore.     

Further Observations on the Fine Structure of Acanthamoeba palestinensis (Reich, 1933). The Golgi Complex,Microbodies, and Mitochondria1

The fine structure of the trophozoite of Acanthamoeba palestinensis with a special emphasis on the Golgi complex, microbodies, and mitochondria has been examined. Golgi complexes are distributed throughout the cytoplasm but are most abundant in the perinuclear region. Usually two Goigi complexes are found in the same plane on opposite sides of the nucleus. One of them appears to be in an intimate association with the nuclear membrane. The region of contact contains compact cisternae, vesicles of various sizes, as well as granular and amorphous electron-dense material. Structural changes in the nuclear envelope are also observed in this area. A structure consisting of a Golgi complex and electron-dense microtubule organizing center, comparable to the centrosphere of other Acanthamoeba species, has been observed. Microbodies, surrounded by a single unit membrane and containing a granular matrix and tubular inclusions, are scattered throughout the cytoplasm. These organelles, circular (～1 μm in diameter) or ovoidal (～1 μm in length and ～0.5 μm in width) in section, have often an irregular outline. These microbodies are probably the morphological equivalent of peroxisomes and glyoxysomes. Most mitochondria show a typical structure including tubular cristae and intracristal inclusions. Occasionally mitochondria with two apposed double membranes running through the midline are found. Such atypical cristae have never been reported in small amoebae before.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

Replacement of calcium by cadmium ions from Ca-affinity sites localized in different cytoplasmic compartments of Acanthamoeba cells

Summary In Acanthamoeba cells both Ca and Cd may be precipitated in different cytoplasmic compartments forming electron-opaque deposits, as shown in cells treated with glutaraldehyde supplied with either Ca or Cd respectively. It was found by semiquantitative X-ray microanalysis that the transfer of cells containing Cadeposits to glutaraldehyde supplied with Cd causes a considerable replacement of Ca by Cd: in deposits formed at cell membrane, in cytoplasm, and in mitochondria the total weight percentage of Cd amounted to over 90, only in deposits formed in vacuoles the value was about 80. The replacement was not prevented by the presence of Ca in the transfer medium. When cells containing Cd-deposits were transferred to Ca-supplied medium, Cd predominated as well, its total weight percentage also amounting to over 90 in all the examined deposits. The results suggest that calcium bound in different cell structures may be easily replaced by cadmium, but not conversely, which suggests that Cd is more firmly than calcium linked to many cell constituents well preserved by fixation.     

Electron spectroscopic imaging (ESI) applied to the detection of potential Ca2+-binding sites in plant cells

The applicability of the electron spectroscopic imaging technique for detection of the intracellular distribution of calcium in plant cells was tested with calyptra cells ofZea mays and with pollen tubes ofLilium longiflorum. After fixation in enhanced Ca²⁺ levels and embedding in resin, ultrathin sections were analyzed for the elemental distribution. Calcium and phosphorus were enriched in cell wall, plasma membrane, endoplasmic reticulum, mitochondria, and Golgi vesicles, mainly in granular or globular deposits appearing electron dense in transmission electron microscopy. The results demonstrated that the ESI-technique allows exact localization of calcium enrichment relative to specific cell organelles.     

Evaluation of prokaryotic and eukaryotic cells as food source for Balamuthia mandrillaris

Balamuthia mandrillaris is a recently identified free-living protozoan pathogen that can cause fatal granulomatous encephalitis in humans. Recent studies have shown that B. mandrillaris consumes eukaryotic cells such as mammalian cell cultures as food source. Here, we studied B. mandrillaris interactions with various eukaryotic cells including, monkey kidney fibroblast-like cells (COS-7), human brain microvascular endothelial cells (HBMEC) and Acanthamoeba (an opportunistic protozoan pathogen) as well as prokaryotes, Escherichia coli. B. mandrillaris exhibited optimal growth on HBMEC compared with Cos-7 cells. In contrast, B. mandrillaris did not grow on bacteria but remained in the trophozoite stage. When incubated with Acanthamoeba trophozoites, B. mandrillaris produced partial Acanthamoeba damage and the remaining Acanthamoeba trophozoites underwent encystment. However, B. mandrillaris were unable to consume Acanthamoeba cysts. Next, we observed that B. mandrillaris-mediated Acanthamoeba encystment is a contact-dependent process that requires viable B. mandrillaris. In support, conditioned medium of B. mandrillaris did not stimulate Acanthamoeba encystment nor did lysates of B. mandrillaris. Overall, these studies suggest that B. mandrillaris target Acanthamoeba in the trophozoite stage; however, Acanthamoeba possess the ability to defend themselves by forming cysts, which are resistant to B. mandrillaris. Further studies will examine the mechanisms associated with food selectivity in B. mandrillaris.     

Acrosome differentiation in the ascidians Clavelina lepadiformis and Ciona intestinalis

The spermatozoa of both Clavelina lepadiformis and Ciona intestinalis have architectural features characteristic of ascidian spermatozoa that have been previously described. They have an elongated head (6 microm and 3 microm long, respectively) and a single mitochondrion that is closely applied laterally to the nucleus; they lack a midpiece. The acrosome of Clavelina lepadiformis spermatozoa is a moderately electron-dense, pear-shaped flattened vesicle, approx. 300 nm x 200 nm x 40 nm in length, width, and height, respectively. The acrosome of Ciona intestinalis spermatozoa is a moderately electron-dense, round flattened vesicle with an electron-dense plate in its central region. It is approx. 200 nm x 200 nm x 50 nm in length, width, and height, respectively. During spermiogenesis in both ascidians, several proacrosomal vesicles (50-70 nm in diameter) appear in a blister at the future apex of the spermatids. These vesicles appear to be associated with the inner surface of the plasma membrane enclosing the blister. They come into contact with each other along the inner surface of the plasma membrane and fuse to form a horseshoe-shaped acrosomal vesicle, which becomes a round, flattened vesicle during further differentiation. Some speculations about the mechanism of acrosome differentiation, the possible role of the acrosome during fertilization, and in the speciation of ascidians are presented.     

Ultrastructural localization of calcium ions in ram spermatozoa before and after cold shock as demonstrated by a pyroantimonate technique

Ram spermatozoa were subjected to cold shock before fixation in pyroantimonate-osmium. Ultrathin sections revealed an electron-dense particulate precipitate in association with the cells. The precipitate was shown to be related to the presence of calcium by exposure of the material to EGTA which reduced or completely eliminated the deposits. In the acrosome region, very little precipitate was evident when the plasma membrane was intact. Cold shock resulted in the disruption of the plasma membrane. When the acrosome remained intact, precipitate was concentrated just anterior to the equatorial segment, but many cells also had acrosomal disruption and then a more even distribution of precipitate was seen on the outer acrosomal membrane. Precipitate was rarely visible within or beneath the acrosome. Post-acrosomally, calcium pyroantimonate deposits were frequently present in the dense lamina beneath the plasma membrane and these became more intense after cold shock. Midpiece sections revealed a few large granules beneath the plasma membrane and a fine particulate precipitate within mitochondria. Similarly, the fine precipitate was also associated with the outer dense fibres in midpieces and tails. Cold shock did not apparently increase the extent or intensity of precipitates in these sites.     

应用电镜X射线显微分析方法研究神经细胞内钙离子的分布

本文应用X射线能谱分析结合电镜技术研究了钙离子在青蛙交感神经节神经元内的分布及其在茶碱作用下分布的变化.实验结果表明在组织样品的电子致密沉积物EDD中含有钙离子成分.在青蛙交感神经节突触后神经元中,包含钙离子的EDD存在于质膜、亚表面池及线粒体中;在突触前神经末梢中,突触小泡的膜上也可观察到EDD.在茶碱作用下,交感神经节神经元的质膜、线粒体中的EDD大大地减少;在亚表面池中则没有或很少观察到EDD;突触前末梢中的突触小泡明显地趋向聚集,在突触小泡之间的连接处频繁地出现EDD.本文根据实验结果讨论了茶碱可能促使钙离子从交感神经元的上述部位中释放出来,并认为质膜、亚表面池和线粒体是细胞内钙离子的贮存部位,而亚表面池可能是主要的贮存释放部位.突触前神经末梢内形态上的变化可能与神经递质释放的机理有关.     

CYST FORMATION IN THE FRESHWATER DINOFLAGELLATE CERATIUM HIRUNDINELLA (DINOPHYCEAE)1

Cyst formation in Ceratium hirundinella (O. F. Müll.) Bergh was studied by light and electron microscopy, using material from several lakes and reservoirs and also laboratory cultures. Cells preparing to encyst build up large quantities of starch and lipid and at the same time reduce their other cell components. The cyst is released from the theca as a naked cell bounded by a double membrane. The most commonly found cyst deposits a layer of electron-dense granules containing silicon on the outer membrane and lays down a cellulose-like material between the two membranes. Cysts without the electron-dense granules are commonly formed in cultures but rarely found in lakes. These cysts appear less resistant to decay and do not show the reorganization of cell contents for dormancy. It is suggested that C. hirundinella has both a resting cyst, forming part of the life cycle, and a temporary cyst stage.     

Histochemical, ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calcium-repleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normally-fed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 micron or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30-100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. These results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Osmolarity is an independent trigger of Acanthamoeba castellanii differentiation

Like many yeasts, bacteria, and other sporulating microorganisms, Acanthamoeba castellanii (Neff), a free-living amoeba with pathogenic relatives, differentiates into a dormant form when deprived of nutrients. Acanthamoeba cysts redifferentiate into trophozoites when food is resupplied. We report here that Acanthamoeba encystment is also triggered by elevated osmolarity, and that osmolarity and cell surface receptor binding are synergistic in triggering differentiation. Additions of sodium chloride or glucose to rich growth media were used to produce specific osmolarity increases and similar encystment results were obtained with either additive. Although many organisms, including Acanthamoeba and mammalian cells, have been shown to adapt to hyperosmolar conditions, this is the first demonstration that hyperosmolarity can be a primary differentiation signal. © 1996 Wiley-Liss, Inc.     

Electron microscopic studies of coated membranes in two types of gill epithelial cells of lamprey

Summary Coated membranes in two types of gill epithelial cell of adult lamprey, Lampetra japonica, were studied by electron microscopy. The type 3 gill epithelial cells possess well-developed microvilli or microfolds, apical vesicles and abundant mitochondria. The cytoplasmic surface of the microvillous plasma membrane is covered by a coat of regularly spaced particles with a center-to-center distance of about 15 nm. Each particle consists of a bulbous free end, about 10 nm in diameter, and a connecting piece, about 5 nm long. Apical vesicles are covered by a surface coat which consists of fine filamentous material but lack any special coating on their cytoplasmic surface.The type 4 cells (chloride cells) are characterized by apical vesicles, abundant mitochondria and cytoplasmic tubules. These tubules possess a coat on their luminal surface which consists of spirally wound parallel rows of electron-dense materials. The rows are about 16 nm apart and wound at a pitch of about 45°. The cytoplasmic surface of these tubules does not display a special coat. These coated membranes are assumed to be the sites of active ion transport across the plasma membrane. In particular, particles in type 3 cells and linear coat materials in chloride cells may be either loci of transport enzymes or energy generating systems. Apical vesicles lack any coating on their cytoplasmic surface but a fine filamentous coat is present on their luminal surface. They contain intraluminal vesicles and are continuous with apical ends of cytoplasmic tubules.     

Fine Structure of Hepatozoon mocassini (Apicomplexa,Eucoccidiorida) Gamonts and Modifications of the Infected Erythrocyte Plasmalemma1

Transmission electron microscopy and scanning electron microscopy were used to investigate the fine structure of Hepatozoon mocassini gamonts and modifications of the infected erythrocyte plasmalemma. Intraerythrocytic gamonts were contained within a parasitophorous vacuole. An electron-lucid space observed between the gamont pellicle and the membrane of the vacuole corresponded to the unstained space described in light microscopy studies. Gamonts possessed a conoid, polar ring, subpellicular microtubules, four pairs of rhoptries, micronemes, ovoid granules, mitochondria with tubular cristae, and a pellicle composed of three individual unit membranes. The conoid had an anterior diameter of 320 nm, a posterior diameter of 360 nm, and a length of 150 nm. In contrast to a report on Hepatozoon aegypti, no micropore or “canopy-like structure” was observed. The plasmalemma of infected erythrocytes exhibited two types of modifications: gross membrane deformations and knobs with an electron-dense central mass. These knobs are structurally distinct from previously described membrane excrescences.     

Ultrastructural Distribution of Calcium in Cutaneous Electroreceptor Organs of Teleost Fish

The calcium distribution in the ampullary electroreceptor and the type B electrore-ceptor organ (gymnarchomast) of Gymnarchus niloticus (Gymnarchidae) and in the tuberous organ of Apteronotus leptorhynchus (gymnotidae) was studied. Endogenous calcium appeared as electron-dense precipitates when the cutaneous organs were pre-fixed with phosphate-buffered glutaraldehyde and postfixed with osmium tetroxide plus potassium bichromate. Calcium precipitates were localized in both intracellular compartments of sensory cells and afferent nerve fibers. In contrast to sensory cells, small amounts of calcium precipitates were found in the cytoplasm of accessory cells. In sensory cells, electron-dense deposits were apparent mainly in synaptic vesicles near synaptic ribbons, inside vacuoles of the endoplasmic reticulum, and between the layers of the nuclear membrane. Very few deposits were found in mitochondria. Precipitates were also observed within the axons of afferent nerves and between the layers of the myelin sheath. The synaptic cleft was devoid of calcium. Calcium deposits have a specific cellular distribution in electro-receptor organs of teleost fish.     

Histochemical,ultrastructural and X-ray microprobe analytical studies of localization of calcium in the mucous lining of the rat duodenum

Summary Localization of calcium in a rapid frozen and freeze substituted duodenum of normal, starved or calciumrepleted rat was examined using either of the glyoxal bis(2-hydroxyanil) (GBHA) staining method, a sensitive histochemical calcium stain or electron microscopy. In normallyfed rats, a majority of absorptive cells of the duodenum showed numerous discrete red granular GBHA reactions, approximately 1 m or less in diameter, located primarily along their lateral plasma membranes and within intercellular spaces. Electron microscopy also revealed electron-dense granules, 30–100 nm in diameter, showing a similar distribution as the GBHA granules in the respective absorptive cells, and confirmed their absence in mitochondria and other intracellular compartments. Some of the absorptive cells located exclusively at the tip of each villus contained highly GBHA-reactive tubulo-vesicular structures extending throughout the cytoplasm. However, they displayed virtually no granular GBHA reaction. In these cells, electron microscopy revealed numerous electron-dense granules in the nucleus, mitochondria and in other unidentified organelles. X-ray microprobe analyses of ultrathin sections confirmed the presence of calcium within electron-dense granules associated with both types of absorptive cells. The number and intensity of all GBHA reactions fluctuated according to luminal calcium concentration. In calcium-repleted rats, strong GBHA reactions appeared in a narrow zone of lamina propria at the tip of the villus, overlaid, predominantly, with absorptive cells showing tubulo-vesicular GBHA reactions. these results suggest the existence of distinct types of absorptive epithelial cells in the rat duodenum, with respect to patterns of calcium localization which they display.     

Role of mitochondrial membrane potential in the regulation of murine erythroleukemia cell differentiation

The level of cytoplasmic calcium ions appears to be important in the control of murine erythroleukemia (MEL) cell differentiation. Our interest in this study focuses on the relationship between the regulation of calcium concentration and differentiation. We used the fluorescent membrane probe DiOC₆ to examine the relationship between MEL cell mitochondria and changes in cytoplasmic calcium levels occurring at the initiation of commitment. Fluorescence microscopy reveals the selective association of DiOC₆ with MEL cell mitochondria, where an enhanced fluorescence is observed. Treatment of cells with dimethylsulfoxide (DMSO) or other inducers causes a decrease in mitochondria-associated fluorescence levels that occurs with the initiation of commitment. A decrease in DiOC₆ fluorescence is caused by agents that reduce mitochondrial membrane potential, but is only slightly affected by agents that alter plasma membrane potential. Amiloride and EGTA, agents that prevent commitment and inhibit calcium uptake, also prevent the decrease in DiOC₆ uptake caused by DMSO. The effect of DMSO on MEL cell mitochondria is mimicked by FCCP, a proton ionophore that dissipates mitochondrial membrane potential. FCCP also caused MEL cell mitochondria to release calcium into the cytoplasm. When MEL cells are treated with DMSO plus FCCP, commitment is initiated without the lag period observed when cells are treated with DMSO alone. These results are consistent with the hypothesis that mitochondrial transmembrane potential is important in the regulation of cytoplasmic calcium levels at the time of commitment of MEL cells to terminal differentiation.     

Electron-microscopic demonstration of the distribution of calcium deposits in the exocrine pancreas of the rat after application of carbachol,atropine, cholecystokinin,and procaine

Summary In an attempt to identify a cellular Ca²⁺-pool, from which calcium is released when secretagogues are applied, tissue fragments of the rat exocrine pancreas were incubated and fixed with glutaraldehyde in the presence of calcium. By means of this procedure electron-dense deposits were found on plasma membranes. X-ray microanalysis showed that these deposits contain calcium. Stimulation of tissue fragments with the use of the secretagogues carbachol or cholecystokinin reduced the number of deposits by about 80%. When the antagonist atropine was applied after carbachol stimulation, deposits reappeared on cell membranes, which then disappeared again after a second stimulation with cholecystokinin. In the presence of procaine, carbachol was inhibited and only slightly reduced the Ca²⁺-deposits on the plasma membranes.These results suggest that a calcium pool, from which calcium is released to induce enzyme secretion on stimulation, is located in the cell membrane     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane