THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : III. The Surface Layers of Chondrococcus columnaris

An electron microscope study of the myxobacterium Chondrococcus columnaris has revealed the following structures in the peripheral layers of the cells: (1) a plasma membrane, (2) a single dense layer (probably the mucopeptide component of the cell wall), (3) peripheral fibrils, (4) an outer membrane, and (5) a material coating the surfaces of the cells which could be stained with the dye ruthenium red.The ruthenium red-positive material is probably an acid mucopolysaccharide and may be involved in the adhesive properties of the cells. The outer membrane and plasma membrane both have the appearance of unit membranes: an electron-translucent layer sandwiched between two electron-opaque layers. The peripheral fibrils span the gap between the outer membrane and the mucopeptide layer, a distance of about 100 A, and run parallel to each other along the length of the cell. The fibrils appear to be continuous across the ends of the cells. The location of these fibrillar structures suggests that they may play a role in the gliding motility of these bacteria.     

THE CELL WALL OF PYROCYSTIS SPP. (DINOCOCCALES)1,2

The cell of Pyrocystis spp. is covered by an outer layer of material resistant to strong acids and bases. Internal to this layer much of the cell wall is composed of cellulose fibrils. The presence of cellulose fibrils was established by staining raw and ultra-violet–peroxide-cleaned cell walls and by combining X-ray diffraction spectroscopy with electron microscope observation. Carbon replicas of freeze-etched preparations and thin sections of P. lunula walls show outer layers, inside them ca. 24 layers of crossed parallel cellulose fibrils (4–5 nm thick, ca. 12 nm wide), then a region of smaller (ca. 6–12 nm diameter) fibrils in a disperse texture, and then the plasma membrane. Cellulose fibrils in the parallel texture are constructed of 3–5 elementary fibrils ca. 3 nm in diameter. Walls of P. fusiformis and P. pseudonctiluca also have cellulose fibrils in a crossed parallel texture similar to those of P. lunula. The Gymnodinium-type swarmer from lunate P. lunula appears to have a cell wall ultrastructure typical of other “naked” dinoflagellates.     

Deposition of glucuronoxylans on the secondary cell wall of Japanese beech as observed by immuno-scanning electron microscopy

Summary Glucuronoxylans (GXs), the main hemicellulosic component of hardwoods, are localized exclusively in the secondary wall of Japanese beech and gradually increase during the course of fiber differentiation. To reveal where GXs deposit within secondary wall and how they affect cell wall ultrastructure, immuno-scanning electron microscopy using anti-GXs antiserum was applied in this study. In fibers forming the outer layer of the secondary wall (S₁), cellulose fibrils were small in diameter and deposited sparsely on the inner surface of the cell wall. Fine fibrils with approximately 5 nm width aggregated and formed thick fibrils with 12 nm width. Some of these thick fibrils further aggregated to form bundles which labelled positively for GXs. In fibers forming the middle layer of the secondary wall (S₂), fibrils were thicker than those found in S₁ forming fibers and were densely deposited. The S₂ layer labelled intensely for GXs with no preferential distribution recognized. Compared with newly formed secondary walls, previously formed secondary walls were composed of thick and highly packed microfibrils. Labels against GXs were much more prevalent on mature secondary walls than on newly deposited secondary walls. This result implies that the deposition of GXs into the cell wall may occur continuously after cellulose microfibril deposition and may be responsible for the increase in diameter of the microfibrils.Abbreviations GXs glucuronoxylans - PBS phosphate-buffered saline - RFDE rapid-freeze and deep-etching technique - FE-SEM field emission scanning electron microscope - TEM transmission electron microscope     

STRUCTURE AND DEVELOPMENT OF WALLS IN FUNARIA STOMATA

The development and structure of the guard cell walls of Funaria hygrometrica Hedw. (Musci) were studied with the light and electron microscopes. The stoma consists of only one, binucleate guard cell as the pore wall does not extend to the ends of the cell. The guard cell wall is thinnest in the dorsal wall near the outer wall but during movement is most likely to flex at thin areas of the outer and ventral walls. The mature wall contains a mottled layer sandwiched between two, more fibrillar layers. The internal wall layer has sublayers with fibrils in axial and radial orientations with respect to the pore. During substomatal cavity formation, the middle lamella is stretched into an electron dense network and into strands and sheets. After stomatal pore formation, the subsidiary cell walls close to the guard cell become strikingly thickened. The functional implications of these results are discussed.     

Wall texture in the spore of a vesicular-arbuscular mycorrhizal fungus

Summary InGlomus epigaeum Daniels and Trappe, a vesicular-arbuscular mycorrhizal fungus, the mature spore has a complex multi-layered wall containing a regular pattern of wall subunits.The outer wall (2–4 m thick) consists of a simple layer of parallel microfibrils. The inner wall (5–6 m thick) is built from two layers possessing different organization. The innermost layer, near the plasmalemma has a texture of apparently dispersed fibrils, whereas the second layer is regularly organized with an arced texture. Ten to twelve bundles of fibrils connected by apparently bow-shaped fibrils are consistently observed. The appearance of this arced organization depends on the section plane and on the angle of observation in the electron microscope as confirmed by tilting experiments. Wall subunits are evident as straight electron transparent fibrils; particularly well-defined in negatively stained frozen sections: their diameter is about 3.5nm.The regular pattern of wall subunits in this fungal cell wall is compared with the textures shown by cellulose fibrils in algae or higher plants and by chitin fibrils in arthropod cuticle.Research work supported by CNR, Italy. Special grant I.P.R.A.—Sub-project 1. Paper No. 55.     

THE FINE STRUCTURE OF CHONDROCOCCUS COLUMNARIS : I. Structure and Formation of Mesosomes

Cells of Chondrococcus columnaris were sectioned and examined in the electron microscope after fixation by two different methods. After fixation with osmium tetroxide alone, the surface layers of the cells consisted of a plasma membrane, a dense layer (mucopeptide layer), and an outer unit membrane. The outer membrane appeared distorted and was widely separated from the rest of the cell. The intracytoplasmic membranes (mesosomes) appeared as convoluted tubules packaged up within the cytoplasm by a unit membrane. The unit membrane surrounding the tubules was continuous with the plasma membrane. When the cells were fixed with glutaraldehyde prior to fixation with osmium tetroxide, the outer membrane was not distorted and separated from the rest of the cell, structural elements (peripheral fibrils) were seen situated between the outer membrane and dense layer, and the mesosomes appeared as highly organized structures produced by the invagination and proliferation of the plasma membrane. The mesosomes were made up of a series of compound membranes bounded by unit membranes. The compound membranes were formed by the union of two unit membranes along their cytoplasmic surfaces.     

THE MICROFIBRILLAR STRUCTURE OF THE CELL WALLS OF THE FILAMENTOUS FUNGUS, Allomyces

Cell walls of the fungus, Allomyces, were isolated by chemical procedures, using either potassium permanganate oxidation or glacial acetic acid-hydrogen peroxide treatment followed by dilute mineral acid. The structure of the treated walls was investigated by means of electron microscopy and electron diffraction analysis which showed that rhizoidal walls were especially suitable for observation. Chitin microfibrils exist in the extreme tips of rhizoidal walls, and tend to lie in a preferred longitudinal orientation. Older rhizoidal wall segments show a crossed fibrillar structure under a thin layer of short randomly arranged microfibrils. In the possession of systems of crossed fibrils these walls are like the cell walls of certain green algae. Walls of branch rhizoidal filaments were observed in the early stages of development, in which case the observed microfibrillar orientations are such that it is possible to envisage their origin from pre-existing fibrils that have passively reoriented. With respect to the continued growth of the filaments, however, it is difficult to explain the observed microfibrillar arrangements in terms of the "multi-net" theory. Hyphal walls usually show two layers, the outer consisting of microfibrils arranged randomly, and the inner consisting of well oriented microfibrils running parallel with the longitudinal axis of the hypha. The oriented inner layer appears to be similar in structure to the secondary wall of the Phycomyces sporangiophore.     

LIGHT AND ELECTRON MICROSCOPE STUDIES OF THE CELL WALL STRUCTURE OF THE ROOT HAIRS OF RAPHANUS SATIVUS

Dawes , Clinton J., and Edwin Bowler . (U. of California, Los Angeles.) Light and electron microscope studies of the cell wall structure of the root hairs of Raphanus sativus. Amer. Jour. Bot. 46(8): 561–565. Illus. 1959.—The structure and development of the cell wall of the root hair of Raphanus sativus were studied under the light and electron microscopes. The outer layer of the root hair consists of mucilage which covers the entire hair and forms a thick cap at the tip. Beneath the mucilage a thin cuticle covers the inner layers of the cell wall. These layers consist of cellulose microfibrils, varying in pattern, in a granular matrix, presumably pectic in nature. The microfibrils of the outer layer, apparently laid down at the tip, are reticulate in arrangement. In mature regions of the root hair, the wall is thickened by an inner layer of parallel and longitudinally orientated microfibrils. Pores in the cellulose wall are evident and increase in number and size near the base of the hair.     

Zur Epidermisaußenwand der Fühlborsten von Dionaea muscipula

Summary The outer epidermal wall of the podium of the trigger hair of Dionaea muscipula reveals an unusual ultrastructure under the electron microscope. The cuticular layer is penetrated by numerous radially arranged fibrils of about 2 nm in diameter inserting in a fibrillar network beneath the cutinized part of the wall. Both the fibrils and the fibrillar network are heavily stained after treatment with lead citrate. Possibly these specific wall structures make the podium elastic and enable it to undergo repeated bendings.     

ELECTRON MICROSCOPY OF CENTRIFUGED HYPHAE OF NEUROSPORA

Normal and centrifuged hyphae of Neurospora were studied with the electron microscope. The following cell structures could be identified: nuclei with nucleoli, mitochondria, endoplasmic reticulum, ribosomes, glycogen, fat bodies, vacuoles, and vesicles with an inner canalicular system, of unknown nature. In centrifuged hyphae, the glycogen layer appeared as a light area, with a slight indication of granular structure. The ribosome layer consisted of densely packed ribosomes without any membranes. The mitochondrial layer contained spaces filled with ribosomes. The nuclei were loosely packed, with endoplasmic reticulum between them. The "enchylema" layer was composed of vesicles belonging to the endoplasmic reticulum. The vacuolar layer was poorly preserved and consisted of double-walled vesicles. Fat appeared as stellate osmiophilic droplets. These observations were compared with previous observations under the optical microscope and their meaning for cell physiology was discussed.     

DEMONSTRATION OF A FIBRILLAR COMPONENT IN THE CELL WALL OF THE YEAST SACCHAROMYCES CEREVISIAE AND ITS CHEMICAL NATURE

The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-β-(1 → 3)-glucanase and endo-β-(1 → 6)-glucanase produced by Bacillus circulans; purified exo-β-glucanase and endo-β-(1 → 3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-β-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-β-(1 → 3)- or endo-β-(1 → 6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-β-(1 → 3)-glucanase it can be concluded that this fibrillar component is composed of β-(1 → 3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.     

ON THE DEVELOPMENT OF MACROSCLEREIDS IN SEED COATS OF PISUM SATIVUM L.

Macrosclereid differentiation was investigated by light and electron microscopy in pea testae during the transformation of protodermal precursors to the mature sclereids. The protodermal cells divide anticlinally and elongate into the macrosclereid layer during seed coat development. Young sclereids have elongate nuclei, plastids become somewhat granal during cellular maturation, vacuolation appears to be an autolytic process, and the cells have dense arrays of endoplasmic reticulum and ribosomes. Considerable dictyosome activity and microtubule development is observed as the secondary wall is produced. Many coated vesicles are associated with and fuse with the plasmalemma. During development, the outer tangential wall area of the macrosclereids acquires a definite cuticle and subcuticular layer. Also, at this time the sclereid walls under the subcuticular layer display semicircular microfibril orientation. The sclereid walls adjacent to the hypodermis become multilayered. As the macrosclereids near maturity, the “light line” becomes discernable in the light microscope at the junction of the cellulosic tips of the macrosclereids and the subcuticular layer. This “light line” is prominent using interference optics and is an osmiophilic layer in the electron microscope. This layer may represent the suberin “caps” reported by earlier workers.     

INTRACELLULAR CENTRIFUGAL SEPARATION OF ORGANELLES IN PHYCOMYCES

Live sporangiophores of Phycomyces blakesleeanus were centrifuged at 35,000 rpm. The cell contents sedimented into distinct layers, and each layer was studied with an electron microscope and with cytochemical methods. The following layers were found (their volumes and their densities are shown in Fig. 3): 1. polyphosphates; 2. polyphosphates and protein crystals; 3. glycogen; 4. yellow layer with ferritin; 5. ribosomes; 6. protein crystals; 7. mitochondria; 8. mitochondria and fibrils; 9. nuclei; 10. endoplasmic reticulum; 11. vesicles, membranes, and reticulum; 12. vacuole; 13. lipoproteins, membranes; 14. fat droplet. The densities of the various layers were determined by the injection of droplets of inert oils of known density into the sporangiosphores before centrifugation. Sedimented cell organelles could be isolated. Centrifuged nuclei of a lycopene-producing mutant were injected into the intact sporangiophore of an albino host where they induced color formation. The ensuing spores, when plated, gave a mixture of white and colored colonies. It was concluded that cell organelles, sedimented by centrifugation of living sporangiophores, remain alive and can be used for biochemical studies. Microspectrophotometric examination of the layers indicated the presence of cytochromes and flavines in the mitochondria and of cytochromes in the nuclei. No pigments corresponding to the action spectrum for the light growth response were found.     

Laminin Fibrils in Newt Gastrulae Visualized by the Immunofluorescent Staining

An anastomosing network of extracellular fibrils on the inner surface of the ectoderm layer of amphibian gastrulae has been shown to provide an adequate substratum for attachment and migration by the mesodermal cells. These fibrils contain fibronectin as shown by immunostaining at the light and electron microscope levels. Now we report the presence of laminin, another cell adhesion glycoprotein, as a fibrillar network on the inner surface of the ectoderm layer in gastrulae of the Japanese newt ( Cynops pyrrhogaster ), but its absence on the blastula ectoderm layer, by the immunofluorescent staining using an antiserum specific for mouse laminin. The same antiserum was shown to stain basement membranes of adult newt organs as expected.     

Electron microscopy of germinating ascospores ofSaccharomyces cerevisiae

The wall of mature ascospores ofSaccharomyces cerevisiae showed in sections under the electron microscope a dark outer layer and a lighter inner layer. The latter was composed of a greyish inner part and a light outer part. During germination, the spore grew out at one side and the dark outer layer was broken. Of the light inner layer, the inner greyish part became the wall of the vegetative cell, but the extented part of the cell had a new wall.     

Cucullosporella mangrovei, ultrastructure of ascospores and their appendages

The ultrastructure ofCucullosporella mangrovei ascospores is described. Mature ascospores possess two wall layers, an outer electron-dense episporium and an innermost tripartite mesosporium. Episporial elaborations form electrondense spore wall ornamentations from which extend fibrils that may constitute a highly hydrated exosporium which was not visualised at either the scanning electron microscope or light microscope level. Ascospores possess a hamate appendage at each pole which unfolds in seawater to form a long thread. Ultrastructurally the polar appendage comprises folded fibro-granular electron-dense material and fine fibrils. The fibrils form a matrix around and within the fibro-granular appendage and around the entire unreleased ascospore. These fibrils have not been observed associated with the ascospore appendages in other species of the Halosphaeriales and are a discrete and new appendage component. The fibro-granular appendage and fibrils are bounded by the outer delimiting membrane which is absent around released ascospores. The nature of the spore appendage is compared with that of other marine and freshwater ascomycetes and the taxonomic assignment of the species is discussed.     

On the cross-sectional shape of cellulose crystallites in Valonia ventricosa

The cross-sectional shape of the cellulose crystallites from the Valonia ventricosa cell wall has been investigated by electron microscopy, using negative staining and Bragg contrast in the bright field mode, with emphasis on this latter technique. The appearance of the cellulose crystallites in the electron microscope depends on their orientation. The cross-section of each cellulose crystal is almost square, with an average side of 18 nm. Sub-units corresponding to elementary fibrils were not detectable within the crystals. The differences between these results and those of earlier workers are discussed.     

Dorsal-ventral differentiation in Simonsiella and other aspects of its morphology and ultrastructure

The morphology and ultrastructure of the aerobic, Gram-negative multicellular-filamentous bacteria of the genus Simonsiella were investigated by scanning and transmission electron microscopy. The flat, ribbon-shaped, multicellular filaments show dorsal-ventral differentiation with respect to their orientations to solid substrata. The dorsal surface, orientated away from the substrate, is convex and possesses an unstructured capsule. The ventral surface, on which the organisms adhere and glide, is concave and has an extracellular layer with fibrils extending at right angles from the cell wall. The cytoplasm in the ventral region contains a proliferation of intracytoplasmic membranes and few ribosomes in comparison to the cytoplasm in other parts of the cell. Centripetal cell wall formation is asymmetrical and commences preferentially in the ventral region. Quantitative differences in morphology and cytology exist among selected Simonsiella strains. Functional aspects of this dorsalventral differentiation are discussed with respect to the colonization and adherence of Simonsiella to mucosal squamous epithelial cells in its ecological habitat, the oral cavities of warm-blooded vertebrates.List of Abbreviations SEM scanning electron microscope - TEM transmission electron microscope     

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.