Combined Fluorescent-Antibody and Electron Microscopy Study of Marek''s Disease Virus-Infected Cell Culture

Duck embryo fibroblast (DEF) and chicken embryo fibroblast (CEF) cultures infected with Marek's disease virus were studied by combined fluorescent antibody and electron microscopy techniques. In both DEF and CEF cultures, cells containing immunofluorescent (IF) antigen also contained herpesvirus particles; conversely, cells lacking this antigen lacked herpesvirus particles. Two morphologically distinct IF antigens were detected in the cytoplasm. (i) A granular antigen in the perinuclear region was brightly stained with the conjugated antibody. This antigen was composed of a granular mass of osmiophilic material and did not contain virions. (ii) A diffuse antigen, present throughout the cytoplasm of infected cells, was less brightly stained. The area of the cell with the highest concentration of this antigen contained small vesicles, folded membranes, and fine electron-dense granules. Naked virions were occasionally seen in these areas. A diffuse nuclear IF antigen was occasionally seen in infected cells. This antigen was often separated from the nuclear membrane and the nucleolus by a clear margin. The intranuclear IF antigen was composed of a fine granular aggregate and naked herpesvirus particles which were randomly distributed throughout the nucleus. Viral capsids in antibody-treated cells were coated with fine filamentous material.     

鸟类原生殖细胞的研究 Ⅰ.鸡胚生殖新月区原生殖细胞超微结构的观察

作者观察了鸡胚生殖新月区的原生殖细胞(PGC)的超微结构。PGC为圆形或椭圆形,13—16μm,有丰富的伪足和微绒毛,尚可见到相邻PGC存在桥粒样结构。细胞核为圆形、椭圆形及分叶状,并呈多处凹陷。与同期胚的其它细胞相比,胞质内细胞器相当丰富且较成熟。观察到有大量微丝。上述PGC的形态,除了细胞桥粒样结构及微丝很少见到报道外,其它特征与鸟类PGC的超微记载相一致。 作者首次观察到PGC中有一种特殊颗粒(即电子致密小体),它自核内产生,进入核周池,并借核膜破裂的方式进入胞质。这种颗粒可能就是生殖颗粒,而由该颗粒在胞质中聚集所构成的特殊高电子致密区可能就是生殖质。从而从形态学上提供了鸟类具有生殖质的证据。     

Cortical differentiation of the animal pole during maturation division in fertilized eggs of Tubifex (Annelida,Oligochaeta): I. Meiotic apparatus formation

The fine structure of the animal pole cortex is examined in the fertilized Tubifex egg undergoing the formation of the second meiotic apparatus (MA). The fully formed MA which orients its axis at right angles to the surface is found at the animal pole about 40 min after formation of the first polar body. It is composed of a spindle and asters at its poles; a centriole is found in the inner aster, but not in the peripheral aster adjacent to the surface. During the formation of the MA, the animal pole surface is lined with a 0.15-μm-thick, electron-dense cortical layer, which is rich in microfilaments. The arrangement of the filaments in the layer changes from a parallel array to a meshwork with progressive formation of the MA. Microtubules of the peripheral aster terminate in the cortical layer. When a jet stream of glycerol/dimethyl sulfoxide solution is applied to an egg fragment glued on a polylysine-coated coverslip, an egg cortex-MA complex is isolated on the coverslip; the MA appears to be tethered to the egg surface by the structural connection between the filamentous cortical layer and microtubules of the peripheral aster. Cytochalasin B (50 μg/ml), when administrated at early phase of the MA formation, does not show any effect on the structure of the cortical layer and the MA; however, if eggs shortly before the termination of the first polar body formation are immersed in the same test solution, the cortical layer of the animal pole becomes thinner, and the filamentous material is not observed in it. Furthermore, in these eggs, the peripheral aster and the spindle are not structurally discernible because of the suppression of microtubule assembly, whereas microtubules on kinetochores and in the inner aster are normally developed. These results are discussed in relation to the role of the animal pole cortex in fixing of the MA to the egg surface and in forming of the MA.     

Ultrastructure of the haemocytes of Tetrodontophora bielanensis Waga (Collembola)

Five types of haemocytes: prohaemocytes, plasmatocytes, granular haemocytes, spherule cells and phagocytes, have been distinguished on the basis of ultrastructural studies. Prohaemocytes are ovoid cells with a simple structural organization. Plasmatocytes are larger; their cytoplasm contains well-developed rough endoplasmic reticulum, numerous mitochondria and free ribosomes. Granular haemocytes are the most numerous of the blood cells, characterized by the presence of electron-dense granules. The cytoplasm of spherule cells contains many spherules made up of filamentous material of medium electron density. Rough endoplasmic reticulum, free ribosomes and mitochondria are also found in the cytoplasm. Phagocytes are the largest haemocytes. Their cytoplasm contains an abundance of lysosomes and myelin structures. In addition to haemocytes, cells intermediate between plasmatocytes and granular haemocytes have been observed, which indicates that the granular haemocytes are derived from plasmatocytes.     

Embryonic development of the heart. I. A light and electron microscopic study of myocardial development in the early chick embryo

Embryonic chick myocardium (stages 8+ to 12?) was studied by light and electron microscopy. The myocardium, which is initially comprised of radially oriented cells with large intercellular spaces gradually becomes more tightly packed. Intercellular spaces decrease and the cells assume a circumferential orientation. Myocardial cells remain epithelial throughout formation of the functional tubular heart and specialized epithelial junctions (apical junctional complex or terminal bars) undergo modification to form intercalated discs. Embryonic myocardial cells contain large amounts of free ribosomes and particulate glycogen, the latter often associated with portions of granular reticulum. Unlike developing skeletal muscle. The amount of granular reticulum contained in the myocardial cell cytoplasm is large and, along with a hypertrophied Golgi apparatus, suggests that these cells may have a secretory function. These organelles persist during the initial period of fibril formation. Myofibrils apparently form from non filamentous precursor material and not by alignment of sequentially synthesized components.     

Formation of the karyosome in developing oocytes of weevils (Coleoptera, Curculionidae)

A complex structure termed the karyosome forms in the nuclei of the developing oocytes of Anthonomus pomorum, Hylobius abietis and Phyllobius sp. (Coleoptera: Curculionidae). It is composed of highly condensed chromosomes, fused with an electron-dense granular material. There are two types of nuclear body associated with the karyosome. The smaller bodies are found in the immediate vicinity of the karyosome. The larger, and more electron-dense, bodies originate next to the condensing chromosomes. During vitellogenesis, the latter bodies disperse in the karyoplasm, and at least some of them locate in the characteristic irregular projections of the germinal vesicle. Morphologically, these projections resemble the accessory nuclei described in other insects. In the studied species, a proteinaceous sheath, the so-called karyosome capsule, surrounds the karyosome. The formation of the karyosome and its capsule occurs during previtellogenesis, so that these structures are fully formed at the onset of vitellogenesis. An extraction of the oocyte cytoplasm with Triton X-100 showed that the material constituting the karyosome capsule is filamentous. Staining with rhodamine-conjugated phalloidin reveals large amount of F-actin in the karyosome capsule.     

New Model for the Genesis and Maturation of Viroplasms Induced by Fijiviruses in Insect Vector Cells

Plant reoviruses are thought to replicate and assemble within cytoplasmic, nonmembranous structures called viroplasms. Here, we established continuous cell cultures of the white-backed planthopper (Sogatella furcifera Horváth) to investigate the mechanisms for the genesis and maturation of the viroplasm induced by Southern rice black-streaked dwarf virus (SRBSDV), a fijivirus in the family Reoviridae, during infection of its insect vector. Electron and confocal microscopy revealed that the viroplasm consisted of a granular region, where viral RNAs and nonstructural proteins P6 and P9-1 accumulated, and a filamentous region, where viral RNAs, progeny cores, viral particles, as well as nonstructural proteins P5 and P6 accumulated. Our results suggested that the filamentous viroplasm matrix was the site for the assembly of progeny virions. Because viral RNAs were produced by assembled core particles within the filamentous viroplasm matrix, we propose that these viral RNAs might be transported to the granular viroplasm matrix. P5 formed filamentous inclusions and P9-1 formed granular inclusions in the absence of viral infection, suggesting that the filamentous and granular viroplasm matrices were formed primarily by P5 and P9-1, respectively. P6 was apparently recruited in the whole viroplasm matrix by direct interaction with P9-1 and P5. Thus, the present results suggested that P5, P6, and P9-1 are collectively required for the genesis and maturation of the filamentous and granular viroplasm matrix induced by SRBSDV infection. Based on these results, we propose a new model to explain the genesis and maturation of the viroplasms induced by fijiviruses in insect vector cells.     

Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes

The Pseudomonas aeruginosa genes pilB-D and pilQ are necessary for the assembly of type 4 fimbriae. Homologues of these genes and of the subunit (pilin) gene have been described in various different bacterial species, but not always in association with type 4 fimbrial biosynthesis and function. Pil-like proteins are also involved in protein secretion, DNA transfer by conjugation and transformation, and morphogenesis of filamentous bacteriophages. It seems likely that the Pil homologues function in the processing and export of proteins resembling type 4 fimbrial sub-units, and in their organization into fimbrial-like structures. These may either be true type 4 fimbriae, or components of protein complexes which act in the transport of macromolecules (DNA or protein) into or out of the cell. Some PilB-like and PilQ-like proteins are apparently also involved in the assembly of non-type 4 polymeric structures (filamentous phage virions and conjugative pili). The diverse studies summarized in this review are providing insight into an extensive infrastructural system which appears to be utilized in the formation of a variety of cell surface-associated complexes.     

The fine structure of the axial organ of the feather star Nemaster rubiginosa (echinodermata: crinoidea)

The fine structure of all the constituent cell type of the crinoid axial organ is described (coelomic epithelial cells, muscles, free cells, gland cells and neurons) ; also described is the fine structure of the extracellular haemal fluid. The gland cells of the glandular tubules of the axial organ have the characteristic fine structure of protein exporting cells and may produce granular and filamentous components of the haemal fluid. The neurons (perikarya and axons) of the axial organ may possibly be neurosecretory, since they are filled with electron-dense granules. Homologies between the axial organs of different echinoderm classes are discussed.     

Salivary gland ultrastructure of the unfed adult and feeding female of Haemaphysalis (Rhipistoma) leachi (Ixodoidea: Ixodidae)

The paired salivary glands of unfed adult Haemaphysalis (Rhipistoma) leachi contain one type of agranular and three types of granular alveoli connected to a salivary duct system. Type I agranular alveoli consist of one large, central cell surrounded by peripheral cells with numerous basal membrane infoldings indicative of epithelia involved in fluid transport. Glycogen particles, lipid-like droplets, and the parallel pattern of infolded membranes disappeared from the peripheral cells during feeding. Types II, III, and IV granular alveoli contain some agranular interstitial epithelial cells, cap cells, and fundus cells, but are predominantly composed of structurally different granular cell types a, b, c, d, e, and f. Agranular cells develop during the early stages of feeding. Granular a, c, e, and f cells release their granules directly after attachment to the host and possibly are involved in cement secretion required for firm attachment to it. The b cell granules are replaced by b₁ filamentous granules during feeding. Golgi bodies and rough endoplasmic reticulum (RER) participate in the formation of most types of granules. The d cells contain lamella-like structures and condensing vacuoles, probably responsible for lysosome formation. The main salivary duct and all types of alveoli are innervated by neurosecretory axons.     

Assembly site of bacteriophage f1 corresponds to adhesion zones between the inner and outer membranes of the host cell.

Morphogenesis of the filamentous bacteriophage f1 occurred at adhesion zones between the inner and outer membranes of the host cell. Quantitation of adhesion zones in cells infected with mutant phage strains suggested that the phage gene I protein may be involved in the formation of adhesion zones for phage assembly.     

Ultrastructure of proteinaceous bladder plugs in male rats

Proteinaceous plugs in the bladder (bladder plugs) were found in male rats with an incidence of 14.1 to 17.8% in ages ranging from 10 weeks to 2 years. No evidence of urinary obstruction was found due to the plugs, but they appeared to irritate the bladder epithelium mechanically causing denudation. Consequently, exfoliated epithelial cells were incorporated into the plugs. Early in development, the plugs consisted of loosely organized eosinophilic masses with fine eosinophilic granules and fenestrated filaments in which eosinophilic globules were suspended. The components of plugs were similar to that contained in seminal vesicles. Subsequently, the plugs became more compact in structure with formation of densely interwoven amphophilic trabeculae containing exfoliated cells and spermatozoa. The periphery of the plugs was surrounded by exfoliated cells, cellular debris, eosinophilic granular materials and spermatozoa. Under electron microscopy, the eosinophilic granules surrounding the plugs were dense aggregates of electron-dense globules and vesicles derived from disintegrated bladder epithelium. The amphophilic trabeculae had a dense compact granular structure consisting of densely aggregated protein globules with a filamentous network. The intertrabecular proteinaceous material had a spongy like structure consisting of sparsely scattered protein globules with fine fenestrated filaments. Proteinaceous plugs having exfoliated cells and spermatozoa were found also in the male accessory sex glands. The plugs in the urinary bladder or male sex accessory glands appeared to be developed from back-flow of semen following ejaculatory disturbance.     

Release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells

Summary The release of chromaffin granular content from staphylococcal enterotoxin B (SEB)-treated and-untreated PC12 cells was studied by electron microscopy. The treatment of the cells with SEB at the concentration of 20 μg/ml caused marked increase of the chromaffin granules that either bound to the plasma membrane by the characteristic rods, measuring 15 to 20 nm in length and showing a tubular structure, or budded off at the free cell surface, surrounded by a layer of rod-containing cytoplasm and enclosed by the plasma membrane. The binding between the granular and plasma membranes by the rods did not lead to membrane fusion and exocytosis of the granular content. Many of the bound granules showed vesiculation with loss of the electron-dense core material; at the same time, some of the binding rods contained intraluminal electron-dense material similar to the granular core material. These findings suggested that the electron-dense material (i.e., norepinephrine) of the bound granules was released extracellularly through channels within the rods. Although the granules were bound to the plasma membrane with equal frequency at the free and contiguous cell surfaces, the granular budding occurred only at the free cell surface, indicating that it occurred incidentally to some granules bound at the free cell surfaces. On the basis of the morphological observations, it is postulated that the electron-dense material of the bound granule is selectively released extracellularly through the rods, leaving the vesiculated granules behind in the cytoplasm. The same mode of release of the granular content was observed, though less frequently, in the untreated control cells. No morphological evidence that indicated that the granular content was released extracellularly by exocytosis was found in the treated and control cells. The present observations indicated that the SEB treatment of PC12 cells stimulated the binding of chromaffin granules to the plasma membrane by the rods and the budding of the bound granules at the free cell surface.     

Two types of virus-like particles in the bean leaf beetle,Cerotoma trifurcata

Two types of virus-like particles were observed in the cytoplasm of hemocytes of the bean leaf beetle, Cerotoma trifurcata. The polyhedral particles were 37–40 nm in diameter and were usually in a crystalline array. They were often associated with granular and laminated structures. The enveloped, spherical particles were 70–75 nm in diameter and were composed of three parts: an outer envelope, a central electron-dense core, and an electron-lucent space between the envelope and the central core. The envelope was similar in structure to the membranes of the cell organelles. These particles were also associated with granular and filamentous structures which were distinct from those associated with the nonenveloped, smaller, polyhedral particles. The nonenveloped particles were recovered in large amounts from partially purified preparations from beetles that contained the particles in thin sections and from soybean loopers, Pseudoplusia includens, which were injected with partially purified preparations from beetles.     

Single charge change on the helical surface of the paramyosin rod dramatically disrupts thick filament assembly in Caenorhabditis elegans

Charge interactions between alpha-helical coiled-coil proteins have been postulated to determine the alignment of many filamentous proteins, such as myosin heavy-chain rod, paramyosin and alpha-keratin. Here we determined the sequence changes in nine mutations in the unc-15 paramyosin gene of Caenorhabditis elegans, including one nonsense, four missense, one deletion and three suppressor mutations. These mutation sites were located on a molecular model, constructed by optimizing charge interactions between paramyosin rods. Remarkably, single charge reversals (e.g., glutamic acid to lysine) were found that either disrupted or restored filament assembly in vivo. The positions of the mutations within the paramyosin molecule support the models of paramyosin assembly and further suggest that the C-terminal region containing a cluster of five mutations, and a site interacting with it, play a key role in assembly. One amino acid substitution in this C-terminal region, in which there is a "weak spot", led to a loss of reactivity with one monoclonal anti-paramyosin antibody. The results demonstrate how a single amino acid substitution can alter the assembly properties of alpha-helical molecules.     

Some ultrastructural observations on the mode of association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

Summary— The ultrastructural aspects of the association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Polamon dehaani, were studied by thin-section electron microscopy combined with detergent treatment. In the tendon cell, MTs were linked laterally by anchoring filaments to the plasmalemma via a submembranous electron-dense layer called the plasmalemmal undercoat. To further clarify how such anchoring filaments are spatially related to the plasmalemma through the undercoat, we carefully examined and compared thin-section images obtained from various specimen preparations using saponin and Triton X-100. When the tissues were treated with saponin or Triton, electron-dense materials in the undercoat were extracted in varying degrees to expose internal substructures. The undercoat appeared to show a two-layer organization, the inner and outer layers. In more extracted samples, filamentous networks became prominent in the outer layer. Anchoring filaments were seen to attach to such filamentous networks, which in turn were linked to the plasmalemma proper. Thus, it may be reasonable to consider that the filamentous network constitutes the core structure of the plasmalemmal undercoat which is structurally reinforced by extractable electron-dense materials.     

The ultrastructure of the byssal apparatus of Mytilus galloprovincialis

The ultrastructure of the byssus of Mytilus galloprovincialis was analysed by transmission electron microscopy in thin sections of either embedded or frozen samples. All parts of the byssus (stem core laminae, stem outer laminae, threads proximal and distal parts) appear to be formed by the same basic filamentous components organized in different ways at the submicroscopic level and embedded in a variable quantity of matrix. The filaments appear to consist of a central electron-lucent zone (3 nm in diameter), surrounded by an electron-dense rim (total diameter 7 nm). The matrix has a granular or microfilamentous structure. The stem and the threads differ greatly in their submicroscopic organization, but their basic constituents (filaments and matrix) are similar. Peculiar filamentous banded elements (FBE) were found mainly in the stem outer laminae. A relation between the ultrastructure and mechanical properties of the different parts of the byssus was established. The presence of collagen is discussed; since no morphological evidence of any of the known forms of collagen organization was revealed by electron microscopy, it is suggested that byssus collagen may be localized in the matrix and in the FBE.     

The effect of the Bacillus thuringiensis exotoxin on the fine nucleolar morphology and ultrastructure

Depending on the dose administred to the experimental mice, the Bacillus thuringiensis exotoxin produces striking changes in the nucleolar morphology of hepatocytes such as the formation of ring-shaped nucleoli, micronucleoli and the segregation of nucleolar components. Such changes are apparently related to the decrease and inhibition of nucleolar biosynthetic activities in the production of the nucleolar RNA. In addition, the Bacillus thuringiensis exotoxin causes the formation of nucleolar peripheral dense plaques and, at higher concentrations, the segregation of two distinctly separated granular areas in the nucleolus. Both these light and dense granular areas showed positive staining with Bernhard's EDTA procedure for the preferential demonstration of RNA-containing structures. In some segregated nucleoli the granular components of light granular areas seemed to leave the nucleolus. The presence of discontinuous filamentous shell around micronucleoli produced by the high dose of exotoxin suggests the nucleolar origin of nuclear granular bodies which are surrounded by similar but continuous filamentous shell characteristic of these structures.     

Dependence of alpha-synuclein aggregate morphology on solution conditions

Alpha-synuclein is the major component of Lewy bodies and Lewy neurites, which are granular and filamentous protein inclusions that are the defining pathological features of several neurodegenerative conditions such as Parkinson's disease. Fibrillar aggregates formed from alpha-synuclein in vitro resemble brain-derived material, but the role of such aggregates in the etiology of Parkinson's disease and their relation to the toxic molecular species remain unclear. In this study, we investigated the effects of pH and salt concentration on the in vitro assembly of human wild-type alpha-synuclein, particularly with regard to aggregation rate and aggregate morphology. Aggregates formed at pH 7.0 and pH 6.0 in the absence of NaCl and MgCl(2) were fibrillar; the pH 6.0 fibrils displayed a helical twist, as clearly evident by scanning force and electron microscopy. Incubations at pH 7.0 remained transparent during the process of aggregation and exhibited strong thioflavin-T and weak 8-anilino-1-naphthalenesulfonate (ANS) binding; furthermore, they were efficient in seeding fibrillization of fresh solutions. In contrast, incubating alpha-synuclein at low pH (pH 4.0 or pH 5.0) resulted in the rapid formation of turbid suspensions characterized by strong ANS binding, reduced thioflavin-T binding and reduced seeding efficiency. At pH 4.0, fibril formation was abrogated; instead, very large aggregates (dimensions approximately 100 microm) of amorphous appearance were visible by light microscopy. As with acidic conditions, addition of 0.2M NaCl or 10mM MgCl(2) to pH 7.0 incubations led to a shorter aggregation lag time and formation of large, amorphous aggregates. These results demonstrate that the morphology of alpha-synuclein aggregates is highly sensitive to solution conditions, implying that the fibrillar state does not necessarily represent the predominant or most functionally significant aggregated state under physiological conditions.     

Development and fine structure of the yolk nucleus of previtellogenic oocytes in the medaka Oryzias latipes

The development and fine structure of yolk nuclei in the cytoplasm of previtellogenic oocytes were examined by electron microscopy during several stages of oogenesis in the medaka, Oryzias latipes. Shortly after oogenesis starts, oocytes 20-30 microm in diameter have much electron-dense (basophilic) cytoplasm, within which a continuous or discontinuous, irregular ring-shaped lower electron-dense area of flocculent appearance (LF) begins to emerge around the nucleus. The yolk nucleus is first recognized within an LF area as a few fragments of dense granular thread measuring 20-25 nm in width. The threads consist of two rows of very dense granules resembling ribosomes or ribonucleoprotein (RNP)-like particles in size and electron density. These thread-like fragments gradually increase in number and length until they assemble into a compact, spherical mass of complicated networks. Analysis of serial sections suggests that the yolk nucleus is a complicated mass of numerous, small deformed vacuoles composed of a single lamella with double layers of ribosomes or RNP-like granules, rather than a mass of granular threads. When oocytes develop to greater than 100 microm in diameter, the yolk nucleus begins to fragment before dispersing throughout the surrounding cytoplasm, concomitantly with the disappearance of LF areas. At this stage of oogenesis, a restricted region of the granulosa cell layer adjacent to the yolk nucleus becomes somewhat columnar in morphology, fixing the vegetal pole region of the oocyte.