DEVELOPMENT OF PERIPHERAL VACUOLES IN PLANT CELLS

The vacuolar apparatus of various plant cells consists of two distinct features: the large central vacuole and peripheral vacuoles which are derived from invaginations of the plasma membrane. Peripheral vacuoles are conspicuous structures in both living and fixed hair or filament cells of Tradescantia virginiana. They occur as spherical structures along the inner boundary of the peripheral cytoplasm and can be recognized as projections into the central vacuole. These structures are variable in size and number within a cell and can represent a significant proportion of the volume of the vacuole. Peripheral vacuoles most frequently are observed in motion with the streaming cytoplasm although their velocity is usually somewhat slower that that of the cytoplasmic organelles. Ultrastructural studies show two closely approximated membranes, one for each vacuole, in areas where a peripheral vacuole projects into the central vacuole. These are separated by an intermembrane zone continuous with the peripheral cytoplasm. The movement of organelles over the perimeter of the peripheral vacuole is presumed to occur along this intermembrane zone. The internal area of the peripheral vacuoles may appear empty although some contain a vesicular content of unknown origin and function.     

ULTRASTRUCTURAL STUDIES PLASMA MEMBRANE RELATED SECONDARY VACUOLES IN CULTURED CELLS

The plasma membrane of cultured cells of several plant species was observed to possess invaginations, or secondary vacuoles, of variable size in the adjacent cytoplasm. These structures, which occurred in cells at different phases in vacuolation, were very numerous in thin sections of some cells but fewer in others. In vacuolated cells enlarged secondary vacuoles protrude into the primary vacuole but are delimited from the tonoplast by an intermembrane zone of variable width. The plasma membrane at the orifice of an invagination may fuse and detach the secondary vacuole from the membrane to form in the cytoplasm a structure bounded by a single membrane. Complex accumulations of membranes consisting of spherical, tubular, and laminar structures, possibly containing cytoplasm, may develop within secondary vacuoles. Contents of many of these vacuoles arise from folds along its limiting membrane which pinch off into the interior of the secondary vacuole. A fibrous substance, possibly derived from the wall, is present in some secondary vacuoles. Observed folding of the plasma membrane and measurements of membrane width of various organelles and cytomembranes support an interpretation that endocytosis occurs in cultured cells.     

Timing of perialgal vacuole membrane differentiation from digestive vacuole membrane in infection of symbiotic algae Chlorella vulgaris of the ciliate Paramecium bursaria

Each symbiotic Chlorella of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole to protect from lysosomal fusion. To understand the timing of differentiation of the perialgal vacuole from the host digestive vacuole, algae-free P. bursaria cells were fed symbiotic C. vulgaris cells for 1.5min, washed, chased and fixed at various times after mixing. Acid phosphatase activity in the vacuoles enclosing the algae was detected by Gomori's staining. This activity appeared in 3-min-old vacuoles, and all algae-containing vacuoles demonstrated activity at 30min. Algal escape from these digestive vacuoles began at 30min by budding of the digestive vacuole membrane into the cytoplasm. In the budded membrane, each alga was surrounded by a Gomori's thin positive staining layer. The vacuoles containing a single algal cell moved quickly to and attached just beneath the host cell surface. Such vacuoles were Gomori's staining negative, indicating that the perialgal vacuole membrane differentiates soon after the algal escape from the host digestive vacuole. This is the first report demonstrating the timing of differentiation of the perialgal vacuole membrane during infection of P. bursaria with symbiotic Chlorella.     

Symbiotic Chlorella sp. of the ciliate Paramecium bursaria do not prevent acidification and lysosomal fusion of host digestive vacuoles during infection

Summary. Each symbiotic Chlorella sp. of the ciliate Paramecium bursaria is enclosed in a perialgal vacuole derived from the host digestive vacuole, and thereby the alga is protected from digestion by lysosomal fusion. Algae-free cells can be reinfected with algae isolated from algae-bearing cells by ingestion into digestive vacuoles. To examine the timing of acidification and lysosomal fusion of the digestive vacuoles and of algal escape from the digestive vacuole, algae-free cells were mixed with isolated algae or yeast cells stained with pH indicator dyes at 25 ± 1 °C for 1.5 min, washed, chased, and fixed at various time points. Acidification of the vacuoles and digestion of Chlorella sp. began at 0.5 and 2 min after mixing, respectively. All single green Chlorella sp. that had been present in the host cytoplasm before 0.5 h after mixing were digested by 0.5 h. At 1 h after mixing, however, single green algae reappeared in the host cytoplasm, arising from those digestive vacuoles containing both nondigested and partially digested algae, and the percentage of such cells increased to about 40% at 3 h. At 48 h, the single green algae began to multiply by cell division, indicating that these algae had succeeded in establishing endosymbiosis. In contrast to previously published studies, our data show that an alga can successfully escape from the host’s digestive vacuole after acidosomal and lysosomal fusion with the vacuole has occurred, in order to produce endosymbiosis. Correspondence and reprints: Biological Institute, Faculty of Science, Yamaguchi University, Yoshida 1677-1, Yamaguchi 753-8512, Japan.     

Involvement of smooth muscle cells in collagen degradation in the postpartum uterus

Ultrastructural study of gravid and postpartum involuting human uteri revealed a number of cells containing collagen fibrils in their cytoplasm. In gravid uteri these cells could be identified as macrophages and fibroblasts; in the postpartum uteri smooth muscle cells (SMC) were also found, containing cytoplasmic collagenous vacuoles. The morphology of intracellular collagen in SMC was similar to that observed in macrophages: fragments of banded collagen fibrils with a diameter corresponding to that of extracellular collagen were located within structures considered to be phagosomes. Limiting membranes were always smooth, most often in apposition to the fibrils that were single or packed in small groups; some cytoplasmic vacuoles contained banded elongated profiles barely discernable as collagen. The collagen fibrils within SMC of the involuting human uterus are regarded as a morphological manifestation of heterogenic enclosure of collagen fibrils and their intracellular degradation. It seems that in the postpartum uterus, where a substantial amount of collagen needs to be removed rapidly, both macrophages and SMC are involved in the process of collagen phagocytosis and degradation. These data suggest that SMC may be involved in the cellular mechanism for collagen breakdown in remodelling SMC-containing tissues like the uterus and the vascular wall.     

Comparative analysis of the structure of the vacuolar system of the bladder granular cells in the frog and of the contractile vacuole complex of protozoans

A comparative morphological study of the vacuolar system of the frog urinary bladder epithelial cells and of the contractile vacuole complex of Paramecium caudatum enabled us to reveal some common structural elements in these: spongial channels and general vacuole reservoir. The structural similarity of these organoids seems to be the base of their analogous functions in the cell. Detection of vacuoles of various forms in different areas of granular cells may point to a possible migration of the vacuoles around their cytoplasm. Localization of spheric vacuoles in the innermost contact with the plasma membrane, and dilution of the intercellular space in this epithelial part may suggest an expulsion of the vacuole content in the basolateral part of the cell. The "contractile" vacuoles of granular cells are related to other intracellular structure: the Golgi apparatus, coated vesicles, microtubules, microfilaments.     

Gibberellic acid and the fine structure of barley aleurone cells

Summary Ultrastructural changes in barley aleurone, cells treated with gibberellic acid (GA₃) for 24–36 hr are described. Many large vacuoles are seen in the ground cytoplasm; the coalasce to form one large central vacuole. Evidence is presented indicating that the vacuoles are formed from the aleurone grains. The dictyosomes of aleurone cells treated with GA₃ for 24 hr or longer proliferate many vesicles. This proliferation of dictyosome vesicles is associated with the phase of rapid ribonuclease release from the aleurone cell. Estimates indicate that microbodies are considerably reduced in number with GA₃ treatment from 24–36, hr while the number of mitochondria is not substantially affected relative to controls. P-Protein-like material is seen in the cytoplasm of these cells often in close proximity to endoplasmic reticulum and spiny vesicles.Supported by National Science Foundation Grant No. GB8332.     

Calcium and Hydrogen Ion Concentrations in the Parasitophorous Vacuoles of Epithelial Cells Infected with the Microsporidian Encephalitozoon hellem

ABSTRACT. Microsporidia of the genus Encephalitozoon undergo merogony and sporogony in a parasitophorous vacuole within the host cell. Cultured green monkey kidney cells infected with Encephalitozoon hellem were loaded with the fluorescent dyes fura-2 or BCECF in order to measure intracellular concentrations of calcium and hydrogen ions respectively. Both the parasitophorous vacuole calcium concentration and pH values resembled those of the host cell cytoplasm in infected cells. Calcein entered the parasitophorous vacuole but not other host cell vacuoles or parasite stages within the parasitophorous vacuole. The lack of a pH or calcium concentration gradient across the parasitophorous vacuole membrane and the permeability of this membrane to a large anion such as calcein suggest that the vacuole membrane surrounding E. hellem resembles that surrounding some other intracellular parasites such as Toxoplasma gondii. A potential role is discussed for the parasitophorous vacuole calcium concentration in germination in situ.     

Two functionally different vacuoles for static and dynamic purposes in one plant mesophyll leaf cell

It is a common belief that plant mesophyll cells are occupied up to 95% by a single multipurpose vacuole. The common ice plant, Mesembryanthemum crystallinum L., however, requires two contrasting functions of the vacuole under salt stress. Large amounts of NaCl have to be sequestered permanently for osmotic purpose and for protecting the cytoplasm from NaCl toxicity. A dynamic exchange with the cytoplasm is required because photosynthesis proceeds under these conditions via the metabolic cycle of crassulacean acid metabolism (CAM). Nocturnally acquired CO2 must be kept as malate in the vacuole and re-mobilized in the daytime. Here, we show that two large independent types of vacuoles with different transport properties meet the requirements for the contrasting functions within the same cell.     

FURTHER OBSERVATIONS ON THE PHENOMENON OF SECONDARY VACUOLATION IN LIVING CELLS

Invagination of the plasma membrane in plant cells forms peripheral or endocytic structures which often contain a complement of membrane-bound vesicles. These structures, or secondary vacuoles, move with the streaming cytoplasm although their velocities are somewhat slower than that for the various organelles within the cytoplasm. They glide over the nucleus or flow from the peripheral cytoplasm onto a transvacuolar strand and continue unabated along the length of a strand. These structures may detach from the plasma membrane as sacs to become positioned in the cytoplasm directly under the tonoplast and project into the primary vacuole. Some endocytic vacuoles may separate from the peripheral cytoplasm and remain free within the primary vacuole; subsequently they can re-associate with the cytoplasm. While the content and function of these vacuoles are yet to be determined, indirect evidence indicates that they are pinocytic in character since the content of an invagination is confined to the sac upon its detachment from the plasma membrane and is subsequently transported throughout the cell by cyclosis.     

Giant vacuoles observed in Dictyostelium discoideum

Large intracellular vacuoles, >4 microm in diameter and either round or oval-shaped, were observed infrequently in Dictyostelium discoideum amoebae of axenically-grown strain AX2 (only 1 in 10(6)-10(8)cells). These previously unreported single or multiple 'giant' vacuoles were more common, however, in newly germinated KAX3 cells (0.55% of the population) and AT-K(neg), a strain that lacks an esterase (0.47% of the population). A vacuolar H(+)-ATPase was enriched in their membranes of intracellular giant vacuoles, indicating that the vacuoles were related possibly to both endosomes and the contractile vacuole compartment. When monitored over time, giant vacuoles protruded from, and retracted back into cells under hyperosmotic conditions, suggesting an osmoregulatory role for these vacuoles. Some of the intracellular and protruded giant vacuoles harbored a fluid-phase marker, fluorescein-labeled dextran, implying a pinocytotic origin for the vacuoles.     

Ultrastructural characteristics of macrophage transformation in foam cells in in vitro experiments

Ultrastructural features of lipid vacuole formation in foam cells were studied in a mouse macrophage culture on the use of aggregated low density lipids (a-LDL) and fibroblast-modified LDL (fm-LDL). Modification of LDL properties is a factor which induces rapid transformation of macrophages to foam cells. Catabolism of a-LDL and fm-LDL and lipid vacuole formation is carried out in two ways: with and without involvement of lysosomes. In the former case membraneless lipid vacuoles are formed, in the latter one, lipid vacuoles surrounded by a membrane.     

Ultrastructural and cytochemical studies on the developing adhesive disc of Boston Ivy tendrils

Summary Ultrastructural observations of the immature adhesive disc from tendrils of Boston Ivy showed that the peripheral cells, which are the presumptive contact layer, contain vacuoles of varied sizes which are filled with electron-dense aggregates. In small vacuoles, these deposits were appressed to the tonoplast and fusion of these small vacuoles with the large vacuoles apparently occurs. Cells from the central zone were largely parenchymatous. The vacuoles of many of these parenchyma cells contained electron-dense spheres and hemispheres of material either appressed to the tonoplast or within the vacuole lumen. In these cells, the vacuole-cytoplasm interface was characterized by a filiform network of interconnected membranes. Positive reactions with reagents for the identification of polyphenols indicate that the vacuoles of nearly all the peripheral cells and scattered cells of the central zone contain tanniniferous substances. Insoluble carbohydrates also occur in the vacuoles of the peripheral cells, but they contain little or no protein or lipid.     

Establishment of Endoreticulatus N. G. for Pleistophora fidelis (Hostounský & Weiser, 1975) (Microsporida: Pleistophoridae) Based on the Ultrastructure of a Microsporidium in the Colorado Potato Beetle,Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)1,2

A new genus, Endoreticulatus n. g., is described for the microsporidium Pleistophora fidelis (Hostounský & Weiser, 1975) based on light and electron microscopic studies of a microsporidium in the Colorado potato beetle, Leptinotarsa decemlineata (Say). This latter microsporidium is considered to be conspecific with P. fidelis because both isolates have been shown to be infectious for L. decemlineata where infection is limited to the epithelial cells of the midgut; both are haplokaryotic and develop as groups of sporoblasts and spores in subpersistent vacuoles in the host cell. In addition, meronts, sporonts, and spores of each isolate often occur simultaneously in a common cell, and by light microscopy they both appear similar. Ultrastructural studies of the isolate from L. decemlineata revealed that all developmental stages occur in parasitophorous vacuoles derived from cisternae of rough endoplasmic reticulum of the host cell cytoplasm. Based on the unique nature of the parasitophorous vacuole, a new genus, Endoreticulatus, is proposed for P. fidelis. The genus is compared with the other genera whose species undergo multisporous sporogony in sporophorous vesicles. In addition, the nature of the parasitophorous vacuole of Endoreticulatus fidelis (Hostounský & Weiser, 1975) n. comb. is compared with the parasitophorous vacuole known to encase various developmental stages of several other microsporidian species.     

Characterization of cocaine-elicited cell vacuolation: the involvement of calcium/calmodulin in organelle deregulation

The sizes of organelles are tightly regulated in the cells. However, little is known on how cells maintain the homeostasis of these intracellular compartments. Using cocaine as a model compound, we have characterized the mechanism of deregulated vacuolation in cultured rat liver epithelial Clone 9 cells. The vacuoles were observed as early as 10 min following cocaine treatment. Removal of cocaine led to vacuole degeneration, indicating vacuolation is a reversible process. The vacuoles could devour intracellular materials and the vacuoles originated from late endosome/lysosome as indicated by immunofluorescence studies. Instant calcium influx and calmodulin were required for the initiation of vacuole formation. The unique properties of these late endosome/lysosome-derived vacuoles were further discussed. In summary, cocaine elicited a new type of deregulated vacuole and the involvement of calcium/calmodulin in vacuolation could shed light on prevention or treatment of cocaine-induced cytotoxicity.     

Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H⁺-ATPase inhibitors concanamycin A and bafilomycin A₁, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H⁺-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.     

Logistics in the life cycle of Photosystem II—lateral movement in the thylakoid membrane and activation of electron transfer

The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean ( Glycine max [L.] cv Williams 82) cell suspension cultures. [7–¹⁴C]SA was added to the cell cultures, the metabolites were extracted from the cells at various time points and analysed by TLC and HPLC. The [7–¹⁴C]SA was taken up rapidly from the culture media and converted primarily to SA 2- O -β- d -glucose (SAG). Lower levels of glucosylated 2,5-dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2- O -β- d -glucose were also formed. Examination of the intracellular localization of the glucose conjugates revealed that all of the conjugates associated with the protoplasts were found in the vacuoles. An SA glucosyltransferase (SAGT) that could catalyse the formation of SAG from SA and UDP-glucose could be extracted from soybean cells and assayed in vitro. Increasing concentrations of SA added to the culture media induced the SAGT activity. The highest levels of SAGT activity were observed in cells treated with 0.5 m M SA. The SAGT activity in these cells was 88-fold greater than the SAGT activity in the untreated cells. The intracellular localization of the SAGT activity was also examined and it was determined that the majority of the SAGT activity in the protoplasts was located outside the vacuole. Therefore, it appears as if SAG is formed from SA outside the vacuole, presumably in the cytoplasm, and then subsequently transported into the vacuole where it accumulates.     

An Ultrastructural Study of the Hemocytes of the Pericardial Body in the Ascidian Ciona intestinalis (L.)

Abstract The hemocytes of the pericardial body of Ciona intestinalis were studied by electron microscopy. Our findings showed that stem cells, clear vesicular granulocytes, microgranulocytes, unilocular granulocytes and globular granulocytes are present at the periphery of the smaller-sized pericardial bodies. The stem cells are small round cells with a large nucleus, with or without nucleolus, and homogeneous cytoplasm containing numerous ribosomes. The clear vesicular granulocytes are characterized by an ameboid shape and cytoplasm containing several large electron-lucent vacuoles and small electron-dense granules. The microgranulocytes are variable in shape and contain numerous large electron-dense granules. The unilocular granulocytes show a single large vacuole with an electron-dense or electron-lucent content and a thin layer of peripheral cytoplasm that contains the flattened nucleus. The globular granulocytes are characterized by the presence of large vacuoles containing either fibrogranular material or electron-dense aggregates.     

Two- and three-dimensional ultrastructural observations of angiogenesis in juvenile hemangioma

Two- and three-dimensional electron microscopic observations by a serial sectioning method revealed endothelial sprouts with intracytoplasmic vacuolization in rapidly growing human juvenile hemangioma. A large vacuole bounded by a single unit membrane was enclosed in the cytoplasm and on the inner aspect of the vacuolar membrane several short microvilli were demonstrated. These appearances have not been reported before. The presence of microvilli in the vacuole indicates that the endothelium has reached the point of differentiation when a vascular lumen forms. In the cytoplasm adjacent to the vacuolar membrane, a significant number of 7 to 10 nm microfilaments were identified. These intracytoplasmic microfilaments are assumed to play a mechanical role in the development of the cytoplasmic vacuole and/or the sprout. The formation of a vacuole observed in the endothelial sprout is similar to the findings of Sabin (1920) by light microscopy in endothelial sprouts in the blood island of the chick embryo. The active endothelial sprout in juvenile hemangiomas is considered to be at least partially responsible for the capillary proliferation and enlargement of the tumor.