Professor Dr: Dr h.c. Ernst G. Pringsheim: 1881–1970

A study of the fine structure of Oocystis apiculata W. West has been made. The internal cytology has been described and, where appropriate, comparisons have been made with the externally similar alga, Glaucocystis. Particular attention has been paid to the structure and composition of the wall; the microfibrils immediately external to the plasmalemma are shown to be highly oriented; they are also shown by freeze-etching to close in to a ‘pole’. A brief discussion on the size difference in microfibrils as determined by shadowing and staining is also made.     

Mise en évidence par le cryodécapage de lomasomes dans la basidiospore de Hypholoma fasciculare (Huds. ex Fr.) Kummer

Summary Lomasomes were shown to be present in mature basidiospores of Hypholoma fasciculare using the freeze-etching technique. Grooves in the plasmalemma were also generally observed. It is suggested that lomasomes are related to the elaboration of the chitinous spore wall and, in accordance with the views of Campbell, plasmalemma grooves are connected with the melanisation of its outer parts.     

Further electron microscope studies of the conidium of Alternaria brassicicola

Summary The freeze-etching technique, aldehyde fixation and heavy metal shadowing of wall material were used in an electron microscope study of the maturing spores of Alternaria brassicicola (Schw.) Wiltshire. The walls are composed of fibres, probably of chitin. The plasmalemma has rectangular grooves in its outer surface and corresponding ridges on the inner one; both surfaces bear particles of two distinct sizes. Endoplasmic reticulum may be lamellated or vesicular and its involvement in wall formation is confirmed; vesicles produced by the endoplasmic reticulum fuse with the plasmalemma. The structure of nuclei, mitochondria and vacuoles is also demonstrated.     

A new type of 3‐D peripheral ultrastructure in Glaucocystis (Glaucocystales,Glaucophyta) as revealed by ultra‐high voltage electron microscopy

The coccoid glaucophyte genus Glaucocystis is characterized by having a thick cell wall, which has to date prohibited examination of the native ultrastructural features of the protoplast periphery. Recently, however, the three‐dimensional (3‐D) ultrastructure of the protoplast periphery was revealed in two divergent Glaucocystis species, with the world's most powerful ultra‐high voltage electron microscope (UHVEM). The two species exhibit morphological diversity in terms of their 3‐D ultrastructural features. However, these two types do not seem to encompass actual ultrastructural diversity in the genetically diverse genus Glaucocystis. Here, we report a new type of peripheral 3‐D ultrastructure resolved in “G. incrassata” SAG 229‐2 cells by 3‐D modeling based on UHVEM tomography using high‐pressure freezing and freeze‐substitution fixation. The plasma membrane and underlying flattened vesicles in “G. incrassata” SAG 229‐2 exhibited grooves at intervals of 200–600 nm, and the flattened vesicles often overlapped one another at the protoplast periphery. This 3‐D ultrastructure differs from those of the two types previously reported in other species of Glaucocystis. The possibility of classification of Glaucocystis species based on the 3‐D ultrastructure of the protoplast periphery is discussed.     

Zur Cytologie und taxonomischen Einordnung von Glaucocystis

Zusammenfassung Glaucocystis ist eine einzellige Alge mit endosymbiontischen Cyanophyceen, die gewöhnlich für eine apoplastidische Oocystis gehalten wird. Gegen diese taxonomische Einordnung sprechen verschiedene Befunde unserer licht- und elektronenmikroskopischen Untersuchungen, unter anderem der Besitz zweier rudimentärer Geißeln, pulsierender Vacuolen (während der Zellteilung) und die Zellsymmetrie. Wie aus einer tabellarischen Übersicht über die Anordnung des Golgi-Apparates in den verschiedenen Algengruppen hervorgeht, ist es sehr unwahrscheinlich, daß Glaucocystis überhaupt eine Chlorophycee ist, denn ihre Dictyosomen liegen im Gegensatz zu denen der Grünalgen ringförmig um die Geißelbasis, sind also parabasal angeordnet. Weil weitere spezifische Merkmale sowie eigene Pigmente fehlen, scheint uns eine taxonomische Einordnung vorerst nicht möglich zu sein.

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Summary Glaucocystis is an unicellular alga with endosymbiontic blue-green algae and is usually thought to be an apoplastidic Oocystis. However, our light and electron microscopical investigations show, that there is no relationship to Oocystis, since Glaucocystis has two reduced flagella, contractile vacuoles (during the cell division), and a different symmetry of the cell. A survey on the position of the Golgi apparatus in the different groups of algae demonstrates that Glaucocystis is most probably no chlorophycean since its dictyosomes surround the flagellar base and are therefore in contrast to that of green algae in parabasal position. Due to the lack of other specific characteristics as well as own pigments it seems us very difficult to place at present Glaucocystis in the taxonomic system of the algae.

     

THE ULTRASTRUCTURE OF SCENEDESMUS (CHLOROPHYCEAE). I. SPECIES WITH THE “RETICULATE” OR “WARTY” TYPE OF ORNAMENTAL LAYER1

The ultrastructure of the wall layers and ornamentative features of Scenedesmus pannonicus and S. longus are described using carefully correlated freeze-etched replicas, thin sections and scanning electron micrographs. The two species arc enclosed by different types of ornamented layers, S. pannonicus by the tightly filling, “warty” layer and S. longus by the loosely fitting, “reticulate” layer, held off the coenobium by 2 types of tubular propping spikelets and rosettes. The reticulate layer has an intricate substructure, especially when studied with freeze-etching. Its inner and outer surfaces appear different, as is its attachment to the 2 types of spikelets. Whole cells of S. longus subjected to acetolysis lack the cellulose wall and cytoplasm, but all other surface features survive, including the Trilaminar Sheath (TLS); this ornamentation cannot be “pectic.” The cellulose wall and ornamentation is unaffected by boiling water alone. Boiling in 6n NaOH removes the surface ornamentation, but the TLS and wall remain; the possibility that these features contain silica is discussed. The terminal spines of both species consist of closely packed spikelets enclosed within a skin of hexagonally-packed subunits. Similar subunits are seen in the propping spikelets of S. longus, and in the rows or “combs” of laterally fused spikelets of S. pannonicus. The warty layer of S. pannonicus is tightly appressed to the TLS except close to where the cells are joined, where it is suspended free. It is composed of a layer of globular subunits, and small indentations form the warts. Single, evenly distributed warts characterize the freely suspended sections of the warty layer, and the layer that encloses young coenobia soon after they have been formed: in contrast, the warts are clumped over the surface of older and larger colonies. Some of the single warts form characteristic double rows, but these latter remain single even on older cells. The surface structure of the warty layer, TLS, and plasmalemma are revealed by the freeze-etching process.     

Microfibril orientation in plant cell walls

Summary The distribution of particles on the surface of the plasmalemma in the collenchyma of Apium graveolens was studied by the freeze-etching technique. The aim was to determine whether the distribution of particles was related to the known longitudinal or transverse orientation of cellulose microfibrils in different layers of the walls of these cells. Preliminary statistical studies have shown no obvious correlation between particle distribution and microfibril orientation although the distribution appeared uniform rather than random. Qualitatively, the particle distribution on the plasmalemma of differentiating xylem fibres of Eucalyptus maculata and of the cortical parenchyma of Avena sativa coleoptiles appeared to be similar to that observed on the plasmalemma of Apium. No correlation between the particle distribution and the microfibril orientation known to exist in the walls of these cells could be discerned.The orientation of microtubules in the cytoplasm of collenchyma cells of Apium graveolens was parallel to the microfibril orientation in many instances, but exceptions were noted. A possible interpretation for this variation is discussed. It is concluded that the microtubules are the structures which are most likely to be involved in determining microfibril orientation in the cell wall.     

Almost Pure Iα Cellulose in the Cell Wall of Glaucocystis

Crystalline features of cellulose microfibrils in the cell walls of Glaucocystis (Glaucophyta) were studied by combined spectroscopy and diffraction techniques, and the results were compared with those of Oocystis (Chlorophyta). Although these algae are grouped into two different classes, by the composition of their chloroplasts for instance, their cell walls are quite similar in size and morphology. The most striking features of their cellulose crystallites are that they have the highest cellulose I_α contents reported to date. In particular, the I_α fraction of cellulose from Glaucocystis was found to be as high as 90% from ¹³C NMR analysis. The mode of preferential orientation of cellulose crystallites in their cell walls is also interesting; equatorial 0.53-nm lattice planes were oriented parallel to the cell surface in the case of Glaucocystis, while the 0.62-nm planes were parallel to the Oocystis cell surface. Such a structural variation provides another link to the evolution of cellulose structure, biosynthesis, and its biocrystallization mechanism.     

Zur systematischen Einordnung von Cyanophora paradoxa,Gloeochaete wittrockiana und Glaucocystis nostochinearum1

Die Morphologie und Feinstruktur (einschließlich Pellicula, Geißelapparat, Mitose und Cytokinese) von Cyanophora paradoxa, Gloeochaete wittrockiana und Glaucocystis nostochinearum, drei apoplastidalen Algen mit blaugrünen Endosymbionten (Cyanellen), wurde vergleichend untersucht. Gloeochaete und Glaucocystis sind in allen systematisch bedeutsamen Merkmalen gleich, während Cyanophora insbesondere durch seine andersartige Struktur der Geißelwurzeln abweicht. Die bei Gloeochaete und Glaucocystis vorkommende Kombination von Merkmalen, in geringerem Maße gilt das auch für Cyanophora, ist bei keiner anderen Klasse der Algen vorhanden. Es wird vorgeschlagen, Gloeochaete und Glaucocystis (und mit gewissen Einschränkungen auch Cyanophora) als Glaucophyceen zu führen, wie das bereits Skuja (1954) vorgeschlagen hat.     

Endoplasmic reticulum as the initiator of bud formation in yeast (S. cerevisiae)

Summary An electron microscopical investigation of synchronously dividing yeast cells (S. cerevisiae) prepared by freeze-etching revealed that ER is inducing bud formation. In the first step, ER elements join and form a nearly-closed bag-like envelope which surrounds the nucleus and vacuoles. From the small opening of the ER-envelope, vesicles are produced by a splitting or proliferation of the ER-membranes. The vesicles fuse with the plasmalemma and release their content into the cell wall. In this limited area, bud formation starts explosively by a local evagination of the cell wall. The ER-derived vesicles are concluded to contain proteindisulfide-reductase. The limited introduction of the enzyme into the cell wall explains bud formation to be initiated by a local increase of wall plasticity caused by the reduction of disulfide bonds between cell wall proteins. The wall is forced to extrude by the internal pressure (turgor) of the cell.     

Plasmatubules: fact or artefact?

Plasmatubules are tubular evaginations of the plasmalemma associated with sites where high solute flux occurs between apoplast and symplast. Plasmatubules of the scutellar epithelial cells of germinating barley (Hordeum vulgare L.) have been examined following a variety of fixation methods. Of the aqueous fixations, primary aldehyde fixation with osmium post-fixation and osmium as the primary fixative gave comparable images, whilst potassium permanganate resulted in some distortion of the tissue in general including dilation of the tubular evaginations of the plasmalemma. Freeze-fixation and substitution with acetone and acetone-osmium gave images of the plasmalemma comparable to those obtained by the aqueous aldehyde and osmium methods. The similarity of structure with aldehyde or osmium and freezing as the primary fixation is taken to indicate that plasmatubules are real and not artefacts resulting from the fixation procedure.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

Freeze-etch study of the teleostean pituitary

Summary The structure of membranes in the pituitary of several teleostean species was studied with the freeze-etching technique. Nuclear pore density is higher in hormone-secreting than in stellate cells, suggesting a higher rate of nucleo-cytoplasmic interactions in the secretory cells. The perinuclear endoplasmic reticulum (ER) in the MSH-secreting cells has a large number of fenestrae, whereas in the ER whorls of the prolactin-secreting cells the fenestration occurs only rarely. Three different types of membrane specializations are described, presumably related to exo-endocytotic events at the level of the plasmalemma.The Following Abbreviations are used in the Present Study AH adenohypophysis - ER endoplasmic reticulum - ES extravascular spaces - FE freeze-etching - HRP horseradish peroxidase - NH neurohypophysis - NS neurosecretory - SG secretory granule This work was supported by grants from the Swiss National Science Foundation Nos. 3.636.75, 3.611.75 and the Dr. Eric Slack-Gyr Stiftung in Zürich     

Freeze-fracturing in normal vacuum reveals ringlike yeast plasmalemma structures

The fine structure of the regular arrays of subunits seen on both plasmalemma fracture faces in resting and starved Saccharomyces cerevisiae (baker's yeast) has been compared using different freeze-fracture replication methods. Freeze-cleaving was carried out at 173 degrees, 133 degrees, and 108 degrees K under a vacuum of 2 X 10(-7) torr (2.6 X 10(- 7)mbar) or under liquid nitrogen at atmosphereic pressure. Independent of the preparation conditions (fracturing temperature, and whether cleaved under vacuum or liquid nitrogen), resting and starved yeast show a significant difference in the morphology of the subunits forming the regular arrays. The regularly arranged particles of the P face of the plasmalemma of starved yeast have a clear craterlike structure which has previously been reported to be demonstrated only by freeze-etching at very low temperatures in ultrahigh vacuum. A complementary structure is seen on the plasmalemma E face. Prolonged exposures of fracture faces under the protection of liquid nitrogen-cooled shrouds have shown that, because of the consequent drastic reduction of condensable gases in the specimen area, no detectable condensation contamination of exposed fracture faces occurs within 15 min at a specimen temperature of 108 degrees K. This shows that a complicated ultrahigh vacuum technology is not required for high resolution freeze- etching.     

The effect of red and far-red light on proton secretion from mesophyll-cell protoplasts of Vicia faba L.

The influence of red and far-red irradiation on the transport of H⁺ and ⁸⁶Rb⁺ through the plasmalemma was studied using parenchymal protoplasts isolated from Vicia faba leaves. The results indicate that red light stimulates H⁺ secretion and the uptake of ⁸⁶Rb⁺. Moreover, it has been demonstrated that far-red irradiation acts antagonistically with respect to red light in both these processes.     

The fine structure of frozen-etched bacillus cereus spores

Summary The fine structure of Bacillus cereus spores was studied using the freeze-etching technique and compared with that already described in chemically fixed cells. Although the basic structures did not appear different, the freeze-etching technique permitted the examination of membrane surfaces and their involvement in sporulation. In this respect, some membranes were found to be covered with small 120 Å particles believed to represent multi-enzyme complexes, while other membranes appeared relatively smooth.One feature revealed by the freeze-etching technique and not demonstrated in chemically fixed cells, was the presence of relatively large (500 Å) vesicular structures. In many instances these vesicles were closely associated with different layers or membranes in the developing spore. While the role of these vesicular structures is as yet undetermined, their possible connection with the lytic process which liberates the spore from the sporangium, is discussed.     

The fine structure of Micrococcus radiophilus and Micrococcus radioproteolyticus

The radiation resistant bacteria Micrococcus radiophilus and M. radioproteolyticus were studied by thin sectioning and freeze-etching techniques and the two species were found to be similar in the fine structure. The only significant difference was in the appearance of the surfaces of the cell walls in freeze-etched preparations.Since the two species, together with M. radiodurans, possess a unique cell wall structure and a cell wall peptidoglycan, which is different from that of other micrococci and Gram-positive cocci, it is recommended that they be reclassified into a new genus.     

Lomasomes and plasmalemmasomes in fungi

Summary Electron microscope observations of fungal hyphae and yeast-like cells, using conventional fixation methods and freeze-etching, demonstrate that plasmalemmasomes are not fixation artifacts. The small invaginations of the plasmalemma observed in aldehyde-fixed preparations are also present in frozen-etched samples. The morphology of plasmalemmasomes in the species examined is variable and ranges from vesicles or tubules within the plasmalemma invagination to parallel arrays of membrane lamellae. Plasmalemmasomes thus appear to be primarily excess plasma membrane that has accumulated, perforce, endocellularly. Lomasomes, in contrast, appear to be accumulations of ejected material between the plasmalemma and cell wall that have become sequestered by the deposition of wall material.     

Light-Dependent Chloroplast Reorientation in Mougeotia and Mesotaenium: Riased by Pigment-Regulated Plasmalemma Anchorage Sites to Actin Filaments?

Conjugatophycean green algae, such as Mougeotia and Mesotaenium, are presumably the most ancient organisms to show phytochrome-mediated photomodulatory processes, i.e. chloroplast reorientational movements. Experiments have provided striking evidence for a dichroic mode of light absorption by the phytochrome molecules located at the periphery of the cylindrical cell; in addition, the transition moment of the chromophoric group of phytochrome has been shown to change by a fixed angle upon conversion of P_r to P_fr and vice versa. Consequently, a hypothesis has been put forward involving a tetrapolar phytochrome gradient at the plasmalemma. This presumed pigment pattern precisely controls chloroplast reorientation in the low-irradiance response. Intriguingly, a blue-light absorbing pigment is expressed in Mougeotia as well, which also mediates low-irradiance response via a presumed tetrapolar gradient, apparently independent of the phytochrome. Two hypotheses for the controlling mechanism of chloroplast reorientation have been put forward:

• a) Coupling of the influx of calcium through the plasmalemma to the tetrapolar gradient of the sensor pigment proper, resulting in a tetrapolar gradient of calcium in the cytoplasm. This is the “reorientation via calcium” hypothesis.
• b) Coupling of actin anchorage sites on the plasmalemma to the tetrapolar gradient of the sensor pigment proper, resulting in a tetrapolar gradient of actin anchorage sites. Cytoplasmic calcium, released from internal stores or taken up through the plasmalemma, triggers actomyosin interaction. This is the “reorientation via anchorage sites” hypothesis.

Consistent with the latter hypothesis, photoregulation by two steps seems to be indicated, (i) cytoplasmic initiation of actomyosin interaction, (ii) the graded formation of plasmalemma anchorage sites for actin filaments.     

The spermatozoa of ascidians: Acrosome and nuclear envelope

Summary The spermatozoon of Ascidia callosa has a head with a wedge-shaped tip. Between the nuclear envelope and the plasmalemma, at the tip of the head, there are one or two previously undescribed vesicles, 45 to 55 nm in diameter. These vesicles have the characteristics of an acrosome. Their role in the process of fertilization has not been determined. Ultrastructural studies of sperm activation are needed, but claims that the spermatozoa of ascidians do not have an acrosome should be reconsidered.Behind the tip of the sperm there are pores in the nuclear envelope. This part of the envelope also contains a dense band of amorphous material that may have a supportive function. A nearly identical structure, associated with pores has been found in the spermatozoon of Boltenia villosa. An analysis of the nuclear envelope of Ascidia callosa indicates that the same structure has previously been misinterpreted as an acrosome in the spermatozoon of Ascidia nigra.