Novel changes in the plasma membrane and cortical cytoplasm during wound-induced contraction in a giant-celled green alga

Summary Changes in the plasma membrane surface and in the cortical cytoplasm during wound healing in giant green algal cells ofErnodesmis verticillata (Kützing) Brgesen were followed using scanning and transmission electron microscopy. Microvillus-like structures that contain cytoplasmic and cytoskeletal constituents were observed emanating from the surface of the plasma membrane at the retracting/cut end of wounded cells. These delicate structures seem to be remnants of cell wall-plasmalemma connections that draw out the plasma membrane and cortical components from the contracting cytoplasm as it pulls away from the cell wall. Most of these connections break during wound healing and, when contraction stops, the microvillus-like protrusions become progressively shorter. In cells treated with a calmodulin antagonist (W-7), a number of distinctive bodies accumulate that are of unknown composition, are oblong in shape, and have a diameter slightly smaller than the protoplasmic protrusions. Ultrastructural and other data indicate that these bodies result from retrieved constituents of the plasma-membrane protrusions, as they do not accumulate in unwounded drugtreated cells or in cells treated in W-5. These findings suggest that the protoplasmic protrusions accumulate membrane and cytoplasmic components that are retrieved and recycled during wound healing inErnodesmis by a novel mechanism. The combined plasma membrane surfaces of the microvillus-like protrusions may help to account for the drastic decrease in surface area that occurs during wound healing.Abbreviations SEM scanning electron microscopy - TEM transmission electron microscopy - W-7 N-[6-aminohexyl]-5-chloro-1-naph-thalenesulfonamide - W-5 N-[6-aminohexyl]-1-naphthalenesulfonamide     

Scanning Electron Microscopy of the Extracellular Matrix of Amphibian Gastrulae by Freeze-drying

In order to demonstrate the extracellular matrix (ECM) which tends to be missing from specimens prepared by ordinary procedures, a freeze-drying method was applied to gastrulae of the newt ( Cynops pyrrhogaster ) without prefixation and the embryos were examined with a scanning electron microscope (SEM). It was revealed that a prominent amount of fibrillar ECM occupied the blastocoel and amorphous ECM covered the inner surface of the blastocoelic wall (BW) as well as the surface of migrating cells. These results were compared with those obtained from observations of specimens fixed chemically.     

Electron microscopy of thin sections of Candida utilis: The structure of the cell wall

Summary Details of the structure of the Candida utilis cell wall were described. Using intact cells, cell walls, about 0.02 m thick, have been resolved into three electron dense layers, each about 700 Å thick, made up of materials of very similar electron opacity. The central and the inner layers of the wall seem to be closely packed forming a slightly more compact structure of somewhat greater electron density. Laminations were observed in some of the inner layers of the sections, particularly in some areas where the cell wall had separated: The possibility of this lamination being associated with the presence of chitin in the framework of the cell wall is discussed. Interesting observations have also been made in the sections of the heat-killed cells confirming the existence of a central electron dense cell wall layer, differing from the inner and the outer ones. Underlying the cell wall is the cytoplasmic membrane, a not too well defined sinous structure with some invaginations.     

Structural features of mycorrhizal associations in two members of the Monotropoideae, Monotropa uniflora and Pterospora andromedea

Species in the subfamily Monotropoideae (family Ericaceae) are achlorophyllous and myco-heterotrophic. They have become highly specialized in that each plant species is associated with a limited number of fungal species which in turn are linked to autotrophic plants. This study provides an updated and comprehensive examination of the anatomical features of two species that have recently received attention with respect to their host-fungal specificity. Root systems of Monotropa uniflora and Pterospora andromedea collected from the field were characterized by light microscopy and scanning electron microscopy. All roots of both species were associated with fungi, each root having a well-developed mantle, paraepidermal Hartig net, and intracellular fungal pegs within epidermal cells. The mantle of M. uniflora was multi-layered and numerous outer mantle hyphae developed into cystidia of two distinct morphologies. Large calcium oxalate crystals were present, primarily on the mantle surface. The outer mantle of P. andromedea was more loosely organized, lacked cystidia, and had smaller plate-like as well as cylindrical crystals on the surface and between outer mantle hyphae. Fungal pegs in M. uniflora originated from inner mantle hyphae that penetrated the outer tangential wall of epidermal cells; in P. andromedea, these structures were initiated either from inner mantle hyphae or Hartig net hyphae and penetrated radial walls of epidermal cells. With respect to function, fungal pegs occurred frequently in both host species and, although presumed to be the sites of active nutrient exchange, no direct evidence exists to support this. Differences between these two monotropoid hosts, resulting from the mycorrhizal fungi with which each associates, are discussed.     

The remarkable genus<Emphasis Type="Italic"> Coptosapelta</Emphasis> (Rubiaceae): pollen and orbicule morphology and systematic implications

The pollen morphology and distribution of orbicules were investigated in ten species of the genus Coptosapelta (Rubiaceae) using light, scanning electron and transmission electron microscopy. In general, Coptosapelta pollen is three- to five-pororate and suboblate to oblate-spheroidal. The sexine ornamentation varies from psilate to reticulate, and the pollen-wall stratification lacks columellae. Droplets are observed on the inner nexine surface. Distinct orbicules are found in five species. Orbicules are often lobed and appear to be aggregated and embedded in the tapetal remnants of the locule wall. Ultrastructurally, a single, flattened core is frequently observed. Coptosapelta has a unique pollen type within Rubiaceae and does not resemble possibly related genera. Pollen characteristics provide additional evidence supporting earlier findings that Coptosapelta represents an isolated lineage in Rubiaceae.     

SEM analysis of amphibian mesodermal migration

Summary At the end of gastrulation, the lateral mesoderm of amphibian embryos migrates ventrally between the ectoderm and the endoderm. The present study is an examination of the morphology of the leading cells of the mesodermal sheet and of the substratum over which they move (the inner surface of the ectoderm). The cells of the leading edge of the mesoderm are generally round, with very short and narrow flattened projections in the forward direction. These projections do not have a ruffled morphology, regardless of whether fixation is carried out before or after the ectoderm and mesoderm are dissected away from the endoderm. The inner surface of the ectoderm is covered with fine (450–500A) filamentous extracellular material and the ectoderm cells sometimes extend cytoplasmic processes (approx. 0.1 wide) onto the leading surface of the mesoderm or onto adjacent ectoderm cells. These studies indicate that the morphology of cell migration in amphibians is closer to that seen inFundulus than to that characteristic of chick or mammalian cells.This paper is dedicated to the memory of Mac V. Edds, Jr., who warmly encouraged the developmental biologists of the Pioneer Valley     

Specialized calciferous cells in the marine algaRhodogorgon carriebowensis and their implications for models of red algal calcification

Summary Calcification inRhodogorgon carriebowensis J. Norris et Bucher was associated with a particular cell type in the cortex. Calciferous cells were 4–6 times the length of cortical assimilatory cells. The distal two-thirds of the calcifying cell was invested with a thick wall that stained with periodic acid Schiff. Thick fibrils formed a reticulum and surrounded grains of calcium carbonate that ranged in shape from rhombohedral to subspherical and were up to 200 nm in greatest dimension. The proximal third of the cell was a tapering uncalcified stalk. The narrow base of the cell was attached to the subtending cell of the fascicle by a normal septum with a pit plug. The cell within the calcified wall matrix was usually flattened and had a very small volume. Cellular contents were dense; even when organelles could be discerned, they could not be identified. X-ray microanalysis revealed that other elements commonly found mixed with calcium carbonate are virtually absent from mineral deposits inR. carriebowensis, but electron diffraction study showed d-spacings that varied from those of pure calcite. Current models of red algal calcification are discussed in light of the findings on this alga.Abbreviations CaCO³ calcium carbonate - DIG differential interference contrast - PAS periodic acid Schiff - SEM scanning electron microscopy - TEM transmission electron microscopy     

Membrane–wall attachments in plasmolysed plant cells

Summary. Field emission scanning electron microscopy of plasmolysed Tradescantia virginiana leaf epidermal cells gave novel insights into the three-dimensional architecture of Hechtian strands, Hechtian reticulum, and the inner surface of the cell wall without the need for extraction. At high magnification, we observed fibres that pin the plasma membrane to the cell wall after plasmolysis. Treatment with cellulase caused these connecting fibres to be lost and the pinned out plasma membrane of the Hechtian reticulum to disintegrate into vesicles with diameters of 100–250nm. This suggests that the fibres may be cellulose. After 4h of plasmolysis, a fibrous meshwork that labelled with anti-callose antibodies was observed within the space between the plasmolysed protoplast and the cell wall by field emission scanning electron microscopy. Interestingly, macerase-pectinase treatment resulted in the loss of this meshwork, suggesting that it was stabilised by pectins. We suggest that cellulose microfibrils extending from strands of the Hechtian reticulum and entwining into the cell wall matrix act as anchors for the plasma membrane as it moves away from the wall during plasmolysis.Correspondence and reprints: Institute of Ecology and Conservation Biology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria.     

The endobiotic thallus ofPhysoderma maydis,the causal agent ofPhysoderma disease of maize

Summary The endobiotic thallus ofPhysoderma maydis is characterized by the presence of an extremely fine rhizomycelium which passes through the host cell wall, allowing the spread of the disease, and irregularly shaped turbinate cells, which may be septate or nonseptate and which are in close association with developing resting sporangia. The formation of the resting sporangium wall is first seen as localized depositions on the rounded surface of the sporangium and only later on the flattened surface of the sporangium which will form the operculum. The substructure of the resting sporangium wall is typical for members of theBlastocladiales. The resting sporangium is contiguous with the rhizomycelium during development and is finally sealed-off from the rhizomycelium by a further deposition of wall material. After the sealing-off of the resting sporangium from the rhizomycelium the content of the sporangium is compartmentalized and the two inner wall layers are deposited. The centre of the sporangium is filled with an electron dense accretion. At the periphery of the sporangium is a layer of lipid bodies. Between the lipid bodies and the central electron dense accretion is a thin layer of cytoplasm which contains the nuclei. The outer surface of the resting sporangium is smooth.     

The fine structure of Micrococcus cyaneus

Summary Micrococcus cyaneus (strain CCM 856) was studied by electron microscopy of thin sections. The cells exhibit different forms (spherical, flattened and pear-shaped) varying in size from 0.6 to 1.1 m. The cell wall consists of one layer 40 to 60 nm thick, the surface of which is covered with, or expands as, a fuzzy material. The cytoplasmic membrane has an asymmetric triple-layered structure with a thickness varying from 8 to 10 nm, and infolds into the cytoplasm as intracytoplasmic membrane systems with configuration, size and number dependent on the fixation conditions. The shape and arrangement of the cells of M. cyaneus differs from that of other micrococci and therefore its taxonomic status should be re-evaluated.     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

Structure and function of the neck cell during fertilization in Ginkgo biloba L.

Key message

Neck cells in Ginkgo biloba contribute to archegonial opening through morphological changes and might be involved in the production of fertilization liquid to attract spermatozoids toward archegonia.

Abstract

Neck cells are an essential part of the archegonium in archegoniate gymnosperms, but their function in the sexual reproductive process remains unclear, particularly in zoidogamous gymnosperms. To clarify the structural characteristics of neck cells and their role in fertilization, we examined the neck cells of Ginkgo biloba L. by means of scanning electron microscopy and transmission electron microscopy. The two curved inner neck cells, which are covered imbricately by the two turgid outer neck cells, were pushed to two sides during fertilization, which indicated that morphological changes in these cells contribute to archegonial opening. The neck cells contained many secretory organelles with some material accumulated outside the cell wall, thus the neck cells might be involved in the production of fertilization liquid to attract spermatozoids toward the archegonium. In addition, the surrounding surface cells of the female gametophyte also cooperate to produce the liquid. Taken together, these results indicate that the neck cells provide an effective mechanism by which zoidogamous gymnosperms achieve reproductive success through altering the morphology and cellular physiology of the neck cells.     

Probable involvement of cytoskeleton in stomatal-pore formation inAsplenium nidus L.

Summary Stomatal-pore formation in the fernAsplenium nidus L. commences in postcytokinetic guard cells at the mid-region of the ventral wall, before the deposition of any cellulosic wall material on it, by the local movement of the adjacent plasmalemmata apart from each other. In this way a rudimentary internal stomatal pore is formed. At this stage the ventral wall exhibits an undulated appearance and gives a positive reaction to aniline blue. Detailed study of postcytokinetic guard cells by electron microscopy, as well as after tubulin immunolabeling and actin staining, shows that stomatal pore initiation coincides with the initiation of the organization of the anticlinal microtubule bundles along the middle of the ventral wall and the colocalization of actin filaments at the same sites. Afterwards, the stomatal pore broadens towards the periclinal walls, a phenomenon keeping pace with the further bundling of the cytoskeletal elements beneath the plasmalemmata lining the middle of the ventral wall. At this stage the anticlinal microtubule bundles lining the stomatal pore are very prominent. The above findings, as well as the fact that treatments with antimicrotubule drugs inhibit the internal stomatal-pore formation, denote that the cortical cytoskeleton lining the ventral wall and particularly the microtubules are involved in this process. Afterwards, distinct local wall thickenings are deposited at the sites of junction of the mid-region of the ventral wall with the periclinal walls as well as at the junctions of the polar ventral-wall ends with the external periclinal wall. Along the middle-lamella region of the former wall thickenings the fore- and rear-chambers of the stomatal pore are formed. The final stomatal-pore opening is achieved by disruption of the expanded thin median periclinal wall region inherited from the guard cell mother cell and of the overlying cuticle, which covers the stomatal pore externally and internally. At the same time the fore-chamber of the stomatal pore broadens by a schizogenous opening towards the polar ventral-wall ends. The observations show that the stomatal-pore formation inA. nidus is a unique process, which is probably restricted to ferns.Abbreviations Af actin filament - GC guard cell - Mt microtubule - MSB microtubule-stabilizing buffer - PBS phosphate-buffered saline - VW ventral wall     

Freezing behavior of xylem ray parenchyma cells in softwood species with differences in the organization of cell walls

Summary By cryo-scanning electron microscopy we examined the effects of the organization of the cell walls of xylem ray parenchyma cells on freezing behavior, namely, the capacity for supercooling and extracellular freezing, in various softwood species. Distinct differences in organization of the cell wall were associated with differences in freezing behavior. Xylem ray parenchyma cells with thin, unlignified primary walls in the entire region (all cells inSciadopitys verticillata and immature cells inPinus densiflora) or in most of the region (mature cells inP. densiflora and all cells inP. pariflora var.pentaphylla) responded to freezing conditions by extracellular freezing, whereas xylem ray parenchyma cells with thick, lignified primary walls (all cells inCrytomeria japonica) or secondary walls (all cells inLarix leptolepis) in most regions responded to freezing by supercooling. The freezing behavior of xylem ray parenchyma cells inL. leptolepis changed seasonally from supercooling in summer to extracellular freezing in winter, even though no detectable changes in the organization of cell walls were apparent. These results in the examined softwood species indicate that freezing behavior of xylem ray parenchyma cells changes in parallel not only with clear differences in the organization of cell walls but also with subtle sub-electron-microscopic differences, probably, in the structure of the cell wall.     

Effects of injected inhibitors of microfilament and microtubule function on the gastrulation movement in Xenopus laevis

It was suggested recently that gastrulation movements in amphibian embryos are caused by the active cell locomotion of individual cells. In order to elucidate the role of microfilaments and microtubules in the cell locomotion occurring during gastrulation, cytochalasin B, colchicine, and other microtubule-disrupting drugs were injected into the blastocoel of early gastrulae of Xenopus laevis. Hypertonic solutions of sorbitol were also injected to elucidate the influence of the internal hydrostatic pressure on the migrating cells. The effects were examined in 1-μm Epon sections of serially fixed embryos and by transmission electron microscopy. Cytochalasin B strongly inhibits cell migration even under conditions that do not cause dissociation into single cells. The cells become round, and have only a few thin cell processes. Electron microscopy shows an alteration in the cortical microfilament network. Colchicine and other microtubule-disrupting drugs have little effect on the rate of cell migration before they cause the accumulation of many mitotic cells and the dissociation of the embryo. The interphase cells are angular and have thin processes like those in the control embryos. The microtubules disappear, and bundles of 10-nm filaments are observed in the cytoplasm of colchicine-injected embryos. Hypertonic sorbitol solutions strongly inhibit cell migration.     

Some observations on the skin ofOxychilus spp. (Fitzinger), with particular reference toO. helveticus (Blum)(Mollusca,Pulmonata, Zonitidae)

Summary A sub-epithelial collagenous layer and pits in the skin have been described inOxychilus spp. The collagen is especially developed beneath the skin on the right side of the body inO. helveticus. It seems to arise from pore cells which previous authors have described in other molluscs. The pits inO. helveticus are lined by cells which together exhibit a gradation of electron densities. This probably represents an accumulation of secretory material and a macroapocrine secretory process has been observed.

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Zusammenfassung Bei denOxychilus-Arten wurden eine sub-epitheliale Kollagenschicht und Vertiefungen in der Haut beschrieben. Das Kollagen ist besonders ausgebildet unter der HautoberflÄche der rechten Körperseite inO. helveticus. Dies scheint aus den pore cells hervorzugehen, was auch von früheren Autoren in anderen Mollusken beobachtet wurde. Die Vertiefungen inO. helveticus sind umgeben von Zellen, die zusammen eine Abstufung von Elektronendichte zeigen. Wahrscheinlich representiert dies eine Ansammlung von Sekret-Material; ein macroapokriner Sekretionsvorgang wurde beobachtet.

     

Development of the resting sporangia ofSynchytrium endobioticum,the causal agent of potato wart disease

Summary An ultrastructural study of the development of the resting sporangium ofSynchytrium endobioticum (Schilb.) Perc. infecting potato cells is presented. The resting sporangium is found to have a single large, centrally placed nucleus with a prominent nucleolus through its entirein situ development. The cytoplasmic organization of the resting sporangium is further characterized by numerous membrane-bound lipid bodies and osmiophilic bodies. The latter have a characteristic sieve-like appearance, probably because certain storage components have been extracted during preparation for electron microscopy. Because of the similar location and appearance of these osmiophilic bodies it is suggested that they are identical to what has earlier (based on light microscopy) been described as chromatin granules; and the ultrastructural studies presented here show that nucleolar discharge which was described from light microscopic observations as leading to chromatin granules in the cytoplasm, and finally forming the nuclei of the zoospores (bally 1912,curtis 1921,percival 1910) simply does not occur.The appearance of dense fibrillar-like structures on the sporangial surface at an early stage of resting sporangium development ultrastructurally distinguishes the resting sporangium from the zoosporangium. The development of the layered portion of the thick sporangial wall is shown to be due to the fusion of vacuoles containing pre-made wall fibrils with the cell membrane. It is suggested that the inner compact wall layer which is essentially substructureless is formed by the membrane itself.The characteristic wings of the matureS. endobioticum resting sporangium originate from the potato host cell wall. Remnants of host cell organelles in the outermost layer of the resting sporangium wall show that degradation of the host cell cytoplasm contributes to wall formation of the parasite.     

Ultrastructural studies on migrating epidermal cells during the wound healing stage of regeneration in the adult newt, Notophthalmus viridescens

The ultrastructure of the epidermal cells which migrate over the wound surface of the amputated limb of the adult newt, Notophthalmus viridescens, was observed with transmission (TEM) and scanning (SEM) electron microscopy. In order to aid in the visualization of polyanionic surface materials on the wound epithelium and wound surface with TEM, the basic dye, ruthenium red, was introduced into the fixatives and buffer. Control limbs were processed without ruthenium red. Shortly after amputation, basal cells at the wound margin possessed elongated, flattened profiles with long pseudopodial projections (lamellipodia and filopodia) that appeared to make contact with the fibrin exudate covering the stump tissues. Epidermal cells proximal to the site of amputation were also in a state of mobilization. Large intercellular spaces and a reduction in the number of desmosomes were observed in the migrating cells. Epidermal cell nuclei became characteristically euchromatic with well-developed nucleoli. Microfilaments were seen within the cytoplasm, extending toward the plasma membrane of cellular processes. Phagocytosed material was also present in the migrating cells. By approximately 9 hours post-amputation, wound closure was complete, and the wound epithelium consisted of three to four cell layers of a non-cornified epidermis. Generally, the amount of extracellular material present on the surface and in the enlarged intercellular spaces of migrating epidermal cells remained the same throughout the period of wound closure. A layer of polyanionic material was observed consistently over the fibrin meshwork covering the wound surface with TEM.     

Absence of “void area” in freeze-etched vesicles of the Alnus crispa var. mollis Fern. root nodule endophyte

Nitrogen-fixing root nodules of Alnus crispa var. mollis Fern. were studied by transmission electron microscopy and by freeze-etching technique. Ultrathin sectioning of septate vesicles of the actinomycetal endophyte showed an electron transparent zone, the so-called void area, between the vesicle cell wall and its encapsulation material. This void area was not observed in the freeze-etching replicas of cryoprotected nodular tissue. It is suggested that the void area is the result of the coming-off of the vesicle cell wall from the capsule and that its formation reflects difficulty in fixing the voluminous mature vesicle of the root nodule endophyte.     

Patterns of cells and extracellular material of the sea urchin Lytechinus variegatus (Echinodermata; Echinoidea) embryo,from hatched blastula to late gastrula

Scanning electron microscopy of six stages of Lytechinus variegatus embryos from hatching through gastrulation reveals changes in the shapes of the ectodermal cells and morphological changes in the extracellular material (ECM) in relation to the locations and migratory activities of mesenchyme cells. The classical optical patterns in the blastular wall (Okazaki patterns) are due to differential orientations of the cells, which bend and extend sheet-like lamellipodia over adjoining cells toward the eventual location of the primary mesenchymal ring. The blastocoelic surfaces of the blastomeres become covered with a thin basal lamina (BL) composed of fibers and nonfibrous material. During primary mesenchyme cell (PMC) ingression, a web-like ECM is located in the blastocoel overlying the amassed PMCs. This ECM becomes sparse in migratory mesenchyme blastulae, and is confined to the animal hemisphere. Localized regions of intertwining basal cell processes in the blastular wall are also present during PMC migration. While a distinct BL is present during early and midgastrulation, blastocoelic ECM is absent. Late gastrulae, on the other hand, have an abundance of blastocoelic ECM concentrated near secondary mesenchyme cell protrusive activity. ECM appearing at both the early mesenchyme and late gastrula stages are probably remnants of degraded BL and intercellular matrix preserved by fixation for SEM. Thus, early mesenchyme ECM is formed of BL material whose degradation is necessary for entry of PMCs into the blastocoel. Late gastrula ECM is apparently a degradation product of BL and intercellular material whose destruction is required for fusion of the gut with oral ectoderm in formation of the mouth.