Ultrastructure of haustoria oferysiphe graminis f. sp.hordei preserved by freeze-substitution

Summary The ultrastructure of freeze-substituted haustoria ofErysiphe graminis DC f. sp.hordei Marchal onHordeum vulgare L. cv. Villa is described. Freeze-substitution allows an improved visualization of thein vivo fine structure of haustoria of powdery mildews. The sheath membrane, as well as the profiles of the plasmalemma, nucleus, mitochondria, and vacuoles appear sharp and smoothly contoured. Invaginations are considered real features of the sheath membrane. Large vacuoles extending into the haustorial body and the haustorial lobes characterize older fungal structures. In the cytoplasm polyribosomes are homogeneously distributed whereas electron-dense glycogen-like inclusions are observed in the periphery of the cytoplasm. The rough endoplasmatic reticulum and the microtubules, primarily orientated with the longitudinal axis of the haustorium, are well resolved by means of the freeze-substitution technique. The method presented provides more detailed insight into the host-parasite interface under natural conditions.     

Fine structure of the erythrocytic stages of Plasmodium knowlesi

Summary The fine structure of erythrocytic stages of Plasmodium knowlesi was compared with that of the same parasite isolated from its host cell by a saponin technique. Rhesus monkeys experimentally infected with Plasmodium knowlesi were the source of parasitized red cells. The erythrocytic stages of this Plasmodium showed all the organelles described in other mammalian forms; the nucleus lacked a typical nucleolus but contained a cluster of granules. P. knowlesi did not have protozoan-type mitochondria as do the avian and reptilian forms, but had double-membrane-bounded bodies as observed in other mammalian malarial parasites.The isolation procedure caused a slight swelling of the parasite, but in general, the structure and structural relationships of the parasite were preserved. However, the isolation technique gave a new insight into the connection of the host cell cytoplasm with the large, so-called food vacuoles of the parasite. The parasite freed from its host cell showed clear spaces where the large vacuoles had been. The content of these vacuoles had been removed together with the red cell cytoplasm. As the nature of the isolation procedure precluded any disruption of the parasite itself, these findings support our view that the vacuoles are not true food vacuoles. If these were true food vacuoles, they would be completely enclosed by a parasite membrane within the parasite cytoplasm. However, we have demonstrated that they represent extensions of host cell cytoplasm in direct communication with the rest of the red cell. The outer membrane surrounding the intra-erythrocytic parasites disappeared after isolation of the parasite from the host cell. This strongly suggested that the outer membrane is of host cell origin. The budding process of the merozoites from a schizont was also described and discussed.This paper is contribution No. 558 from the Army Research Program on Malaria and was supported in part by Research Grant AI 08970-01 from the United States Public Health Service.     

Gas Vacuoles and Flotation in the Testate Amoeba Arcella discoides

The natural formation of gas vacuoles as a method of movement is described for the testate amoeba Arcella discoides. These vacuoles are used to float the organism from the substrate to the surface in an inverted position.     

An ultrastructural study of microsporogenesis in tobacco line SR1

This article provides an ultrastructural atlas of microsporogenesis in the tobacco model line SR1. The stages of cell-wall remodeling and reorganization of the intercellular channels, accompanying this process, are reported for the microspore mother cells. The meiotic changes in the cell nucleus and cytoplasm are traced. The appearance of single-, double-, or multi-membrane nuclear vacuoles in microspore mother cells and their further elimination from the nucleus are for the first time described for the genus Nicotiana as well as deviations from a normal course for this process. Intercellular chromatin migration (cytomixis) was observed in the microsporogenesis of the line SR1 and behavior of the nuclear vacuoles within the cytomictic nucleus was described for the first time. The enzymatic activity of spherosome-like vesicles in the tobacco microsporogenesis is discussed. The features of microsporogenesis in the tobacco line SR1 are compared with those of other plant species and its association with the transition from a diploid to a haploid phase of the life cycle is discussed.     

The origin of vacuoles in young embryos of Pelargonium x Hortorum Bailey

Summary The different mechanisms of vacuole formation in embryonic tissues of Pelargonium are described. Some vacuoles are formed by mechanisms widely reported in a variety of plant species and plant tissues, but other vacuoles are initiated as differentiated zones of the cytoplasm around which the tonoplast is gradually built up form vesicles and small cisternae.     

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.     

Chromosome cycles of the basidiomycete Cyathus stercoreus (Schw.) De Toni

Summary Chromosome cycles of the basidiomycete Cyathus stercoreus (meiosis and mitosis) are described. The fusion of two nuclei of compatible mating type takes place in the developing basidium at the end of telophase of the presynaptic mitosis. Synapsis follows immediately after nuclear fusion. During synapsis the chromosomes elongate, facilitating pairing. Meiosis and mitosis are essentially similar to those processes in higher organisms. Details of divisional stages are described and illustrated with photomicrographs. The presence of centrioles and spindles is demonstrated. The presence of quadrivalents as well as secondary associations of like chromosomes suggests that Cyathus stercoreus may be a tetraploid species.     

Effect of mitotic inhibitors on the ultrastructure of root meristem cells

After 5 h in 10^-3M vinblastine the most obvious effects upon Vicia faba L. cells are seen in the spindle apparatus. These include the microtubules themselves as indicated by c-type metaphases and the pole regions of otherwise intact spindles, leading to multipolar anaphases and to telophases with more than two daughter nuclei. Vesicle transport may be undisturbed and new cell walls can be formed, although cases of disturbed cell plate and cell wall formation were noted occasionally, accompanied by myelinizations in phragmosomes. After 24 h in the same concentration of vinblastine, divisions are no longer observed and the plasma membranes are severely affected. They show extensive myelinizations, accumulations of lipids and dehiscence from the cell walls which are frequently thickened and irregularly formed. Of the other cellular membranes, the nuclear envelope and, more frequently, the tonoplast may be affected. Electron-dense deposits appear in the vacuoles. Comparable, though less severe, changes including multipolar anaphases and myelinizations result from treatment with lumicolchicine, but not with colchicine. Vinblastine leads to the appearance of filament bundles both in cytoplasm and karyoplasm, lumicolchicine to morphologically identical filaments in the cytoplasm alone. The nature of these filaments is unknown.Abbreviation VLB vincaleukoblastine     

Nonlysosomal vesicles (acidosomes) are involved in phagosome acidification in Paramecium

Although acidification of phagocytic vacuoles has received a broadened interest with the development of pH-sensitive fluorescent probes to follow the pH changes of vacuoles and acidic vesicles in living cells, the mechanism responsible for the acidification of such vacuoles still remains in doubt. In previous studies of the digestive vacuole system in the ciliate Paramecium caudatum we observed and described a unique population of apparently nonlysosomal vesicles that quickly fused with the newly released vacuole before the vacuole became acid and before lysosomes fused with the vacuole. In this paper we report the following: (a) these vesicles, named acidosomes, are devoid of acid phosphatase; (b) these vesicles accumulate neutral red as well as acridine orange, two observations that demonstrate their acid content; (c) cytochalasin B given 15 s after exposure of the cells to indicator dye-stained yeast will inhibit the acidification of yeast-containing vacuoles; and that (d) we observed using electron microscopy, that fusion of acidosomes with the vacuole is inhibited by cytochalasin B. We conclude that the mechanism for acidification of phagocytic vacuoles in Paramecium resides, at least partially if not entirely, in the acidosomes.     

Some properties of vacuoles isolated from Neurospora crassa slime variant

A method for the isolation of vacuoles based on polybase induced lysis of protoplasts of the cell wall deficient Neurospora crassa slime variant is described. Isolated vacuoles are characterized by 12 to 50 times increased specific activities of several hydrolases as compared with the total homogenate of protoplasts. Total -amino nitrogen, arginine, and polyphosphate are also greatly enriched in these vacuoles. Vacuoles are equipped with a permease for the transport of basic amino acids across the tonoplast.Non-Standard Abbreviation DEAE-dextran diethylaminoethyl-dextran     

Polygenic impact of morningness on the overnight dynamics of sleep spindle amplitude

Sleep spindles are thalamocortical oscillations that contribute to sleep maintenance and sleep‐related brain plasticity. The current study is an explorative study of the circadian dynamics of sleep spindles in relation to a polygenic score (PGS) for circadian preference towards morningness. The participants represent the 17‐year follow‐up of a birth cohort having both genome‐wide data and an ambulatory sleep electroencephalography measurement available ( N = 154, Mean age = 16.9, SD = 0.1 years, 57% girls). Based on a recent genome‐wide association study, we calculated a PGS for circadian preference towards morningness across the whole genome, including 354 single‐nucleotide polymorphisms. Stage 2 slow (9‐12.5 Hz, N = 186 739) and fast (12.5‐16 Hz, N = 135 504) sleep spindles were detected using an automated algorithm with individual time tags and amplitudes for each spindle. There was a significant interaction of PGS for morningness and timing of sleep spindles across the night. These growth curve models showed a curvilinear trajectory of spindle amplitudes: those with a higher PGS for morningness showed higher slow spindle amplitudes in frontal derivations, and a faster dissipation of spindle amplitude in central derivations. Overall, the findings provide new evidence on how individual sleep spindle trajectories are influenced by genetic factors associated with circadian type. The finding may lead to new hypotheses on the associations previously observed between circadian types, psychiatric problems and spindle activity.     

ULTRASTRUCTURAL INVESTIGATIONS ON THE TWO-NUCLEATE POLLEN GRAIN OF TILLANDSIA CAPUT-MEDUSAE MORR. (BROMELIACEAE)

The fine structure of the pollen grain of Tillandsia caput-medusae Morr. (Bromeliaceae) prior to germination has been studied. The development, after the first mitosis, is here schematized in three stages which are in accordance with the main steps described in angiosperms. The ultrastructural modifications occurring in the generative and vegetative cells are discussed in view of their different destiny. The results obtained are compared with the data known about tropical orchids and epiphyteic plants like Tillandsia. The following differences have been observed: a large vacuole in the vegetative cell; rapid thinning of the wall between the generative and vegetative cells; great quantity of ribosomes and rough endoplasmic reticulum surrounding the vacuoles in the generative cell. The above-listed ultrastructural features may have a meaning, considering the peculiar environmental conditions in which the epiphytism of Tillandsia is achieved.     

VACUOLE FORMATION IN THE ACTIVELY GROWING ROOT MERISTEM OF BARLEY (HORDEUM SATIVUM)

The subapical meristem of actively growing barley roots produces series of undifferentiated cells, some of which are devoid of vacuoles. At the beginning of their differentiation, the Golgi apparatus gives rise to vesicles and tubules which concentrate hydrolases, acid phosphatase being the typical representative of these enzymes. Some of these structures organize themselves as sequestration vacuoles. Then, the imprisoned fraction is destroyed by the process of autophagy after an alteration of the vacuolar internal membrane. These structures are identical to the “provacuolar apparatus” described by Marty in Euphorbia characias roots. Lytic processes which develop in autophagic vacuoles give rise to the first true meristematic vacuoles. Relations between dictyosomes, provacuoles and vacuoles, and their degree of exclusivity are discussed.     

Vacuolar dynamics in synchronously budding yeast

Summary A simple and rapid method for obtaining synchronously budding cultures of Saccharomyces cerevisiae is described. Synchronous cultures were started with homogeneous cell fractions isolated from exponentially growing cultures by isopycnic centrifugation in osmotically inactive media. The technique of fractionation is based on changes of cell density throughout the budding cycle. These changes are correlated with vacuolar changes observed in the light and electron microscope. During bud initiation the large vacuoles in late budding cells shrink and fragment into small vacuoles. Simultaneously the density of the cells increases. Later stages of the budding cycle are characterized by the distribution of the small vacuoles between mother and daughter cell, followed by their fusion and expansion, and by a decreasing density of the cells. The relative changes in cell density and dry weight and in the content of different macromolecules during the budding cycle suggest a cyclic change between utilization of endogenous and exogenous substrates. This is discussed in terms of a cyclic consumption and accumulation of vacuolar pools.     

Cellular changes associated with rest and quiescence in winter-dormant vascular cambium of<Emphasis Type="Italic"> Pinus contorta</Emphasis>

In winter, dormant cambial cells contain many small vacuoles interspersed throughout the cytoplasm. This differs dramatically from actively growing cambial cells whose structure is dominated by large central vacuoles. Structure reported in studies using conventional chemical fixation and transmission electron microscopy (TEM) conflicts with that described earlier for live cambial cells using light microscopy. In this study, cryofixation (high-pressure freezing/freeze substitution) was used to preserve dormant Pinus contorta fusiform cambial cells, revealing structure more consistent with that in early micrographs of live cambial cells. At the ultrastructural level, the plasmalemma was consistently smooth and tightly associated with the cell wall, contrary to the highly in-folded plasmalemma seen in chemically fixed cambial cells. In addition, both TEM and live-cell confocal microscopy demonstrated that, in some places, dormant cells were partitioned into more numerous, smaller vacuoles than were observed after chemical fixation. Populations of different vacuoles were apparent based on size, shape and membrane staining. Larger vacuoles had prominent tonoplasts and were often present as axially elongated, interconnecting networks with associated microfilament bundles. Endoplasmic reticulum fragmented during rest into numerous vesicular structures similar to small vacuoles, then with the transition to quiescence reformed into the smooth cisternal form.     

Isolation and ultrastructural identification of membranes from the fungusGilbertella persicaria

Summary Methods are described for isolating and identifying subcellular membranes from walled hyphae ofGilbertella persicaria. Differences in thickness and symmetry of membranes and in contents of vesicles were used to distinguish different types of membranes. Mitochondria, vacuoles, plasma membrane, and vesicles with attached ribosomes from homogenized germlings equilibrated at the 1.2/1.4 M interface in discontinuous sucrose gradients. Accelerated flotation in centrifuged Ficol-sucrose gradients resulted in the additional separation of the mixed membranes into three fractions: one contained predominantly intact mitochondria, another was composed of vacuoles and vesicles coated with ribosomes, and a third was enriched in plasma membranes. Based upon morphometric analysis, these fractions contained 92% mitochondria, 53% vacuoles, and 89% plasma membranes, respectively. The source of vesicles coated with ribosomes was investigated since rapidly growing hyphae ofG. persicaria contained little rough endoplasmic reticulum as compared with other classes of membranes. Reconstruction from electron micrographs of mitochondrial fragmentation and vesiculation suggested that most of the ribosome-coated vesicles originated from disrupted mitochondria rather than from rough endoplasmic reticulum. The study demonstrates the utility of ultrastructural markers to identify membranesin vitro independent of, or as an adjunct to, cytochemical and biochemical markers.     

ELECTRON MICROSCOPY OF A CONTRACTILE-VACUOLE MUTANT OF CHLAMYDOMONAS MOEWUSII (CHLOROPHYTA) DEFECTIVE IN THE LATE STAGES OF DIASTOLE1

Electron microscopy of a “vacuole-less” mutant of Chlamydomonas moewusii Gerloff revealed the presence of small anterior vacuoles. These vacuoles behaved like contractile vacuoles in wild-type cells, but they were apparently unable to complete diastole and discharge their contents. When wild-type and mutant cells were incubated in hypertonic medium, small coated vacuoles persisted in the region where contractile vacuoles form. When these cells were transferred to hypotonic medium, the vacuoles appeared to fill and fuse to form larger vacuoles Shortly after the appearance of full expanded contractile vacuoles, collapsed vacuoles were observed in wild-type cells suggesting the completion of diastole and the onset of systole. In mutant cells, the initial steps of filling and fusion to form larger vacuoles apparent interactions of vacuoles with the plasma membrane were not observed. New contractile vacuoles accumulated around the nucleus. When fusion of the contractile vacuole with the plasma membrane was blocked by EGTA, a similar accumulation of large vacuoles occurred. Our observations suggest that the contractile-vacuole mutant of C. Moewusii produces vacuoles which can accumulate excess water as part of the mechanism of osmoregulation but which cannot complete diastole.     

Electron microscopy of the skin of the teleost,Hippoglossoides elassodon

Summary The normal skin of the pleuronectid fish, Hippoglossoides elassodon, is described by light and electron microscopy. The epidermis consists of 5 to 9 layers of cells, the majority of which are squamous cells and the minority mucous cells. The squamous cells are characterized by numerous desmosomes and associated cytoplasmic filaments. The mucous cells accumulate mucous droplets in vacuoles of Golgi origin and are observed apparently in the process of releasing their content at the free surface. The dermis consists of alternating lamellae composed of typical collagen fibers. Pigment cells are of three types: melanophores, iridophores (guanophores), and lipophores.This work was supported by Public Health Service Research Grant CA-08158 from the National Cancer Institute.     

Kristalloide Einschlüsse im Zytoplasma pflanzlicher Zellen

Summary In healthy as well as dahlia mosaic sick plants ofVerbesina encelioides, Sanvitalia procumbens, Zinnia elegans, Calendula spec. andDahlia hybrids, leaf cell vacuoles are found in the marginal cytoplasm which contain protein crystals. They are single membrane-limited products of the endoplasmatic reticulum. They may be found mainly in the older leaves and especially in those of virus infected plants. The crystalline structures consist of tetragonally arranged tubules of 105 Å in diameter, separated by an interspace about 35 Å wide. There are similar structures in virus infected plants ofFragaria vesca, but not inChenopodium quinoa, where the vacuoles contain no bodies. This cell organelle is compared with crystalline inclusions already described. Its significance and relations to the virus disease are discussed.     

Reduction in vacuolar volume in the tapetal cells coincides with conclusion of the tetrad stage in Arabidopsis thaliana

Although the tapetum is known for its role in the removal of the tetrad wall, a morphological change in the tapetum correlating with such a role has not been described. Here we report that in two ecotypes of Arabidopsis thaliana, Landsberg erecta and Columbia, the vacuoles in tapetal cells underwent progressive enlargement prior to the separation of tetrads but became drastically reduced when tetrads just separated from one another. Such a drastic change in vacuolar volume was not observed in later anther development. We also observed that the walls of associated tetrads were much less stained with Toluidine Blue O than the walls of separate tetrads, indicating that the tetrad walls underwent an alteration during the tetrad stage. Furthermore, we identified the N-terminal propeptide signals for sorting vacuolar proteins in 15 β-1,3-glucanases, five polygalacturonases, and two endocellulases that are expressed in Arabidopsis young floral buds; all three types of the enzymes are known to participate in degradation of the tetrad wall. These results suggest that the tapetal vacuoles might be a storage site for these enzymes prior to their secretion to the anther locule.