Ultrastructural Architecture and Organization of the Spore Envelope during Development in Thelohania bracteata (Strickland, 1913) after Freeze-Etching*

An ultrastructural study was made of the spore envelope during development in the microsporidan, Thelohania bracteata. The frozen-etched outer (convex) face of the relatively thin spore coat in the earliest immature stage of development has a granular structure in regular array. The inner (concave) face bears particles as well as depressions arranged in a net-like pattern. The mature spore coat has a substructure of numerous microfibers, ～8 nm in diameter, arranged in a matrix and forming thin layers which run parallel to the spore surface. The mature spore coat possesses both outer and inner limiting layers. The outer (convex) face of the outer limiting layer is granular. The convex face of inner limiting layer bears many particles as well as many long, narrow depressions. The concave face of the inner limiting layer carries many stud-like projections, ～40 nm long and 30 nm high, which are complementary to the depressions observed on the convex face. In addition, the concave face has subunits ～15 nm in diameter, apparently arranged in a hexagonal pattern with a center to center distance of ～18 nm. The change in size of these projections, depressions, and subunits presumably is related to spore maturation.     

Die Feinstruktur der lamellären Einschlußkörper in den Zellkernen des Nektariums von Catalpa bungei

Summary In the nectary of Catalpa bungei the gland cells contain nuclear inclusions consisting of stacks of lamellae 12.7 nm thick. Each lamella is composed of globular particles with a diameter of approximately 12.7 nm. The particles are arranged in a monolayer, revealing a regular square pattern in face view. In adjacent lamellae the globular subunits are almost exactly superimposed; each of them is probably built up of smaller subunits having a diameter of 0.4 nm.

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Herrn Prof. Dr. Horst Drawert nachträglich zum 60. Geburtstag gewidmet     

A new putative cellulose-synthesizing complex ofColeochaete scutata

Summary Cells of the charophycean alga,Coleochaete scutata active in cell wall formation were freeze fractured in the search for cellulose synthesizing complexes (TCs) since this alga is considered to be among the most advanced and a progenitor to land plant evolution. We have found a new TC which consists of two geometrically distinctive particle complexes complementary to one another in the plasma membrane and occasionally associated with microfibril impressions. In the E-fracture face is found a cluster of 8–50 closely packed particles, each with a diameter of 5–17 nm. Most of these particles are confined within an 80 nm circle. In the P-fracture face is found an 8-fold symmetrical arrangement of 10 nm particles circumferentially arranged around a 28 nm central particle. The TCs ofC. scutata are quite distinctive from the rosette/globule TCs of land plants. The 5.5×3.1 nm microfibril inC. scutata is also distinctive from the 3.5×3.5 nm microfibril typical of land plants. The phylogenetic implications of this unique TC in land plant evolution are discussed.     

Ultrastructure of Inclusions Formed by Ribgrass Mosaic Virus in Leaf Cells

Diffrent types of cytoplasmic inclusions were observed in ultrathin sections of plants systemically infected with three different strains of ribgrass mosaic virus (RMV) (tobamovirus group). Tissue from uninoculated plants did not contain such inclusions. Most common were “rounded plates” consisting of layers of aligned virus particles 300 nm long. RMV also induced angled layer aggregates in Capsicum annuum plants. A novel type of inclusion for the tobamovirus group were the abundant spiral aggregates found in Digitalis purpurea, systemically infected with strain D of RMV. In these aggregates the virions become circularly arranged around a center. The orientation of the particles changes in such a way that virions being 300 nm apartare cut in the longitudinal and in the transverse direction respectively.     

Insect UV-, and green-photoreceptor membranes studied by the freeze-fracture technique

Summary The membranes of the microvilli of UV- and green-photoreceptors of the ant Myrmecia gulosa have been studied with the freeze-fracture technique. Both inner fracture faces, the cytoplasmic P-face and the extracellular E-face, are covered by globular particles. The P-face particles appear to be randomly distributed, occasionally forming clusters. Their density is about 7,000/m², and their mean diameter is 8.5 nm. The E-face particles, however, are arranged in an ordered square pattern with a center-to-center spacing of 9 nm. The density and distribution of P- and E-face particles are the same in both the UV- and the green-photoreceptor membranes. No differences were found in the ultrastructural organization of photoreceptor membranes after dark or light adaptation. It is suggested that the P-face particles represent rhodopsin molecules.     

Die Feinstruktur der Zellwand und Cytoplasmamembran von Clostridium nigrificans,dargestellt mit Hilfe der Gefrierätz- und Ultradünnschnittechnik

Zusammenfassung Der mit Hilfe der Gefrierätztechnik dargestellte Feinbau der Zellwand und Cytoplasmamembran von Clostridium nigrificans wurde mit den Ergebnissen der chemischen Fixation verglichen. Die Zellwand von chemisch fixierten Zellen zeigt einen Aufbau aus drei stark und zwei schwach elektronenstreuenden Schichten, wobei die innerste, der Cytoplasmamembran angrenzende Schicht nicht immer darstellbar ist. Die Cytoplasmamembran hat eine asymmetrische Elementarmembran-Struktur. Mit Hilfe der Gefrierätztechnik konnte die Zellwand in drei Schichten aufgespalten werden: Einer äußeren aus globulären, rechtwinkelig geordneten, etwa 9 nm messenden Partikeln aufgebauten Schicht und zwei darunter liegenden, 15 und 5 nm dicken Schichten. Die Cytoplasmamembran wird in jungen Kulturen vollkommen, in alten teilweise mit 5–15 nm großen Teilchen bedeckt. An Stellen wo die Teilchen abgespalten wurden oder entsprechend locker angeordnet waren, konnte die Oberfläche der Cytoplasmamembran sichtbar gemacht werden. Sie ist sowohl auf der der Zellwand als auch auf der dem Cytoplasma zugekehrten Seite mit systemlos verteilten, scheinbar verschieden tief eingebetteten Teilchen bedeckt.

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The fine structure of the cell wall and cytoplasmic membrane of Clostridium nigrificans demonstrated by means of freeze-etching and chemical fixation techniques

Summary A comparison was made between the results obtained using the freeze-etching technique in demonstrating the fine structure of the cell wall and the cytoplasmic membrane of Clostridium nigrificans with those using the chemical fixation technique. In chemically fixed cells, the cell wall appears to consist of three dense layers separated by two layers of low electron scattering power, whereby it is not always possible to observe that layer immediately bordering on the cytoplasmic membrane. The cytoplasmic membrane has an asymmetrical unit membrane structure. It was possible, to separate the cell wall in three distinct layers using the freeze-etching technique. The outermost is composed of globular, rectangularly arranged particles, approximataly 9 nm in size, the two inner layers are 15 and 5 nm wide respectively. The cytoplasmic membrane is covered with particles 5 to 15 nm in size, in younger cultures completely, in older cultures partially. On those places where the particles have been split off or where they are loosly arranged, it was possible to observe the surface of the cytoplasmic membrane. It appears to be covered with unsystematically scattered particles imbedded at different depths, not only on that side turned to the cell wall but also on that facing the cytoplasm.

     

Heliobacterium chlorum: cell organization and structure

The basic cellular organization of Heliobacterium chlorum is described using the freeze-etching technique. Internal cell membranes have not been observed in most cells, leading to the conclusion that the photosynthetic apparatus of these organisms must be localized in the cell membrane of the bacterium. The two fracture faces of the cell membrane are markedly different. The cytoplasmic (PF) face is covered with densely packed particles averaging 8 nm in diameter, while the exoplasmic (EF) face contains far fewer particles, averaging approximately 10 nm in diameter. Although a few differentiated regions were noted within these fracture faces, the overall appearance of the cell membrane was remarkably uniform. The Heliobacterium chlorum cell wall is a strikingly regular structure, composed of repeating subunits arranged in a rectangular pattern at a spacing of 11 nm in either direction. We have isolated cell wall fragments by brief sonication in distilled water, and visualized the cell wall structure by negative staining as well as deep-etching.Abbreviations PF protoplasmic fracture face - EF exoplasmic fracture face     

Ultrastructural Observations of Experimental Naegleria Meningoencephalitis in Mice: Intranuclear Inclusions in Amebae and Host Cells*

SYNOPSIS. Primary amebic meningoencephalitis was experimentally produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality, with average time of onset of symptoms or death occurring on the 7–8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.     

Freeze fracture studies of reaction centers from Rhodospirillum rubrum in chromatophores and liposomes

In freeze-fractures of chromatophores of Rhodospirillum rubrum the reaction centers are seen as hexagonal arranged particles of 13 nm diameter with a density of around 5,500 particles per m². Similar regions on the cytoplasmic membrane suggest that these parts are the prospective invagination sites.Isolated reaction centers are easily incorporated into liposomes. In freeze fractures of liposomes particles similar in shape and size, although less dense as in chromatophores are observed. In negative staining much smaller units of only 5 nm in diameter are found indicating that reaction centers occur in the membrane as tri- or tetramers. There is a strong correlation between particle density in chromatophores and titratable reaction centers remaining in these membranes after extraction of reaction centers by detergents; both values are in good agreement with the yield of reaction centers at a given detergent concentration.Abbreviations LDAO Lauryldimethylamine oxide - PF protoplasmic fracture face - EF exoplasmic fracture face     

Cytoskeleton-membrane interactions in the cnidocil complex of hydrozoan nematocytes

Summary Each cnidocil complex of the hydrozoans Tubularia larynx and Hydra vulgaris consists of 9 or 7–10 large stereovilli (=stereocilia), respectively, and a modified cilium, the cnidocil. The cnidocils comprise the regular 9 microtubule doublets, up to 30 additional microtubules, as well as a central filament body. Adjacent stereovilli are linked together by intermembrane connectors forming the stereovillar cone. The distal tips of the stereovilli surround the cnidocil in a closed tubular arrangement measuring up to 0.7 m in length. Within this contact region the cnidocil is linked to the stereovillar tube by another set of intermembrane connectors, which seem to hold the cnidocil in a central position within the stereovillar cone. Stereovillar membrane and actin core are linked by 16-nm long cross bridges, which display a periodicity of 16 nm and emerge from the actin core. Within the cnidocils periodically arranged membrane-cytoskeleton bridges are uniformly restricted to the contact region. Here, 24-nm long cross bridges, which are spaced by a regular distance of 20 nm, interconnect the A-tubules of the microtubule doublets and the membrane. The cnidociliary membrane is differentiated into distinct domains as revealed by freeze-fracturing. Within the contact region of the nematocytes of Tubularia larynx, intramembrane particles are arranged in 9 rows of 700 nm length and 50 nm width, separated by particlefree areas. Intramembrane particles are irregularly distributed distal to the contact region. Considering recent physiological results we presume that the latter represent chemoreceptor units, while mechanical stimuli are transmitted via the intermembrane connectors and the microtubule-membrane bridges to mechanosensitive channels within the domain of the cnidociliary membrane in the contact region.     

Striped projections of the outer membrane of the generative cell in Convallaria majalis pollen

In pollen grains of Convallaria majalis the outer membrane of the generative cell (GC) is the inner membrane of the vegetative cell (VC). Striped projections (SP) at the cytoplasmic face of the outer membrane of the GC were revealed by chemical fixation and also by a rapid freeze-fixation and freeze-substitution. The projections, located in groups on the protruding lobes of the GC, were arranged parallel to each other and were equally spaced (40 nm apart). The length of the SP, estimated from grazing sections of GC, was 400 nm. Each projection was composed of T-shaped elements, about 35 nm high, spaced at an average distance of 25 nm. SP were observed in mature, hydrated, activated and germinated pollen grains and seemed to be associated with microtubules and microfilaments of the VC. No evidence exists yet of SP on the sperm cell membrane. Immunogold labelling with anti-myosin antibodies showed many gold particles attached preferentially to the surface of the protruding lobes of the GC in the area of the projections. These results may suggest that the SP of Convallaria GC contain myosin-like protein and play an important role in the motility of the GC during pollen tube growth.     

Ultrastructure of a cave-wall cyanophyte-Gloeocapsa NS4

Gloeocapsa strain NS4, a cyanophyte (cyanobacterium) which grows in low light levels inside cave entrances, was studied in the electron microscope by thin sectioning and freeze-etching. The cells are surrounded by a microfibrillar sheath divided by dense lamellae, which are probably an acidic mucopolysaccharide. Inside this is a typical Gramnegative cell wall. Double-replica freeze-fracture showed that the outer envelope of the wall fractures to give two faces each consisting of densely-packed particles; the particles of the outer leaflet seem to consist of subunits arranged in a hollow cylinder. A structural model of the outer envelope is proposed. The plasma membrane fractures to give a PF face with 3000 9 nm particles m^-1 and an EF face with 150–700 11–12 nm particles m^-1. The thylakoids are arranged in a pattern not previously found in a unicellular cyanophyte, parallel arrays which intersect, and may fuse with, the plasma membrane. The thylakoid membranes have 2,850 particles m^-1, mean size 10.9 nm, on the PF face and 560 particles m^-1, mean size 12.3 nm, on the EF face. Phycobilisomes are difficult to see, but may be unusually large. These ultrastructural features may be adaptations to a very low light habitat.     

Virus-like particles in Urastoma cyprinae, a turbellarian parasite of Mytilus galloprovincialis

Urastoma cyprinae is a turbellarian parasite infecting the gills of several marine bivalves. We observed the presence of virus-like particles (24 to 30 nm in diameter; icosahedral symmetry) arranged in paracrystalline arrays in the cytoplasm of subepidermal dorsal cells. These particles appear to be RNA viruses, possibly related to the Picornaviridae. This is the first report of viral particles in the parasite U. cyprinae.     

Ultrastructure of a Hexagonal Array in Exosporium of a Highly Sporogenic Mutant of Clostridium botulinum Type A Revealed by Electron Microscopy Using Optical Diffraction and Filtration

The ultrastructure of a hexagonal array in the exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A strain 190L was studied by electron microscopy of negatively stained exosporium fragments using optical diffraction and filtration. The exosporium was composed of three or more lamellae showing an equilateral, hexagonal periodicity. Images of the single exosporium layer from which the noise had been filtered optically revealed that the hexagonally arranged, morphological unit of the exosporium was composed of three globular subunits about 2.1 nm in diameter which were arranged at the vertices of an equilateral triangle with sides of about 2.4 nm. The morphological units were arranged with a spacing of about 4.5 nm. The adjacent globular subunits appeared to be interconnected by delicate linkers.     

Plasma-membrane rosettes in root hairs of Equisetum hyemale

Particle arrangement in the plasma membrane during cell wall formation was investigated by means of the double-replica technique in root hairs of Equisetum hyemale. Particle density in the protoplasmic fracture face of the plasma membrane was higher than in the extraplasmic fracture face. Apart from randomly distributed particles, particle rosettes were visible in the PF face of the plasma membrane. The rosettes consisted of six particles arranged in a circle and had an outer diameter of approx. 26 nm. No gradient in the number of rosettes was found, which agrees with micrifibril deposition taking place over the whole hair. The particle rosettes were found individually, which might indicate that they spin out thin microfibrils as found in higher-plant cell walls. Indeed microfibril width in these walls, measured in shadowed preparations, is 8.5±1.5 nm. It is suggested that the rosettes are involved in microfibril synthesis. Non-turgid cells lacked microfibril imprints in the plasma membrane and no particle rosettes were present on their PF face. Fixation with glutaraldehyde caused, probably as a result of plasmolysis, the microfibril imprints to disappear together with the particle rosettes. The PF face of the plasma membrane of non-turgid hairs sometimes showed domains in which the intramembrane particles were aggregated in a hexagonal pattern. Microfibril orientation during deposition will be discussed.Abbreviations EF extraplasmic fracture face - PF protoplasmic fracture face     

Fine structure of the dioptric apparatus in the stalk eye of Onchidium verruculatum (Gastropoda, Stylommatophora): a distinct lamellar substructure of the lens

 The dioptric apparatus of the stalk eye in Onchidium verruculatum, including a tentacular epidermis, a cornea, and a lens, was examined using transmission electron microscopy. The tentacular epidermis was formed by columnar epidermal cells, sensory dendrites, and glandular cells. The cornea was an anterior part of the eye vesicle and consisted of corneal cells which contained abundant glycogen particles but no dark pigment granules in their cytoplasm. An acellular, transparent, ellipsoidal lens was located in the center of the eye vesicle. The lens showed a marginal zone, an outer zone, a transitional zone, an inner zone, and a central region arranged concentrically. The outer zone was the most intense electron-dense region and was finely granular in structure. The marginal zone was also finely granular and surrounded the outer zone with many hair-like slender strands on the retinal side. Toward the center of the lens this homogeneous fine granularity gradually changed into globular or rod-like substructures, about 30 nm in diameter, and then abruptly transformed into a lamellar substructure of about 30 nm in thickness. The inner zone contained a mosaic of lamellar substructures which were arranged in a fingerprint pattern that was particularly enhanced with periodic acid methenamine silver proteinate staining. The center itself consisted of deformed lamellar substructures. The concentric arrangement of substructures inside the lens of the O. ver-ruculatum stalk eye is probably responsible for the concentration and/or refraction of light. Accepted: 5 December 1997     

VIRUS-LIKE PARTICLES AND NUCLEAR INCLUSIONS IN THE RED ALGA PORPHYRIDIUM PURPUREUM (BORY) DREW ET ROSS1

Ultrastructural examination of the unicellular red alga Porphyridium purpureum has revealed cytoplasmic and nuclear inclusions termed concentrosomes. These bodies are morphologically distinct from the irregular membranous inclusions previously reported by others as concentric bodies. In thin section, the morphology of concentrosomes varies from a simple set of 3 or 4 (or rarely 5) concentrically arranged, electron-opaque, circular profiles to elongate, sinuous forms and particulate aggregations, the majority clearly within the nucleus but separated front the nucleoplasm by what appears to be the nuclear envelope. Although simple concentrosomes may be observed in either the cytoplasm or the nuclei, the more elaborate forms occur only in nuclei. In addition to the concentrosomes, subspherical to polygonal virus-like particles of approximately 40 nm diameter have been observed in P. purpureum. These particles are characterized by an electron-opaque perimeter that, in approximately equal numbers, surrounds an “empty” or an opaque core. Dense arrays of the virus-like particles appear in the cytoplasm but not in the nuclei. Similarities between certain forms of the concentrosomes and the virus-like particles are suggestive of an ontogenetic relationship. The infrequency with which either the concentrosomes or the virus-like particles are observed has hampered attempts to verify a developmental sequence or to establish unequivocally the infectious nature of the virus-like particles.     

A Structural Analysis of the Regularly Arranged Porin on the Outer Membrane of Campylobacter jejuni Based on Correlation Averaging

A negatively stained electron micrograph of regularly arranged porin proteins of Campylobacter jejuni on the isolated outer membrane of bacteria was analyzed in detail by the correlation averaging method using a computer-assisted program. The results showed that the porin of C. jejuni had a trimeric structure separated by about 10.4±0.15 nm. In addition, the pores in the trimers were also separated by about 4.3 ±0.1 nm.     

High resolution analysis of the formation of cellulose synthesizing complexes inVaucheria hamata

Summary Ultrastructure and assembly of cellulose terminal synthesizing complexes (terminal complexes, TCs) in the algaVaucheria hamata (Waltz) were investigated by high resolution analytical techniques for freeze-fracture replication.Vaucheria TCs consist of many diagonal rows of subunits located on the inner leaflet of the plasma membrane. Each row contains about 10–18 subunits. The subunits themselves are rectangular, approx. 7×3.5 nm, and each has a single elliptical hole which may be the site of a single glucan chain polymerization. The subunits are connected with extremely small filaments (0.3–0.5 nm). Connections are more extensive in a direction parallel to the subunit rows and less extensive perpendicular to them. Nascent TC subunits are found to be packed within globules (15–20 nm in diameter) which are larger than typical intramembranous particles (IMPS are 10–11 nm in diameter) distributed in the plasma membrane. The subunits in the globule, which may be a zymogenic precursor of the TC, are generally exhibited in the form of doublets. Approximately 6 doublets are connected to a center core with small filaments. The globules are inserted into the plasma membrane together with IMPS by the fusion of cytoplasmic (Golgi derived) vesicles. Two or three globules attach to each other, unfold, and expand to form the first subunit rows of the TC on the inner leaflet of the plasma membrane. More globules attach to the structure and unfold until the nascent TC consists of a few rows of subunits. These rows are arranged almost parallel to each other. Two formation centers of subunits appear at both ends of an elongating TC. New subunits carried by the globules are added at each of these centers to create new rows until the elongating TC structure is completed. On the basis of this study, a model of TC assembly and early initiation of microfibril formation inVaucheria is proposed.Abbreviations IMPS intramembranous particles - MF microfibril - TC terminal complex