Ultrastructure of Escherichia coli cells infected with bacteriophage R17

Franklin, Richard M. (Institut de Recherches sur le Cancer, Villejuif, Seine, France), and Nicole Granboulan. Ultrastructure of Escherichia coli cells infected with bacteriophage R17. J. Bacteriol. 91:834-848. 1966-Ultrastructural changes in Escherichia coli cells infected with ribonucleic acid (RNA) bacteriophage R17 were studied under conditions of one-step growth. No morphological alterations were seen during the latent period. During the period of rapid viral synthesis, a fibrillar lesion surrounded by ribonucleoprotein particles was observed in a polar region. Late in infection, paracrystalline arrays of virions were found in over 90% of the cells. When protein synthesis was blocked by in over 90% of the cells. When protein synthesis was blocked by chloramphenicol at 20 min postinfection, allowing continued viral RNA synthesis without production of coat protein, a dense fibrillar area appeared in a paranuclear region. Cytochemical studies were done on cells embedded in hydroxypropyl methacrylate, a water-miscible embedding agent. The paracrystalline arrays of virions were digested after extensive treatment with either pepsin or ribonuclease. Shorter digestion with the pepsin resulted in better definition of the crystal regions. The fibrillar area found in chloramphenicol-treated cells was digested by ribonuclease but not by pepsin, and was also resistant to lead extraction. This region probably represents a pool of virus-specific RNA.     

The karyosphere during late oogenesis in Rana ridibunda.

The organization of the nucleus in the oocytes of Rana ridibunda was examined during late diplotene at the light and electron microscopic level. At this stage the chromosomes are relatively condensed and assembled in the centre of the nucleus, constituting a karyosphere. The chromosomes here are associated with the central "protein sphere" (15--20 microns in diameter), obviously at their telomeres. Numerous nucleoli are accumulated around the chromosomes, forming a karyosphere capsule and contain segregated fibrillar and granular components; structures resembling perinucleolar chromatin and fibrillar bodies (spherules) are associated with the nucleoli. Granules 30 to 40 nm in diameter are seen to surround the fibrillar spherules. "Nucleolus-like bodies" consisting of granules 10 to 15 nm in diameter which are embedded in finely fibrillar material are often associated in contact with the chromosomes. The central sphere is an accumulation of annular structures similar to those of the pore complexes of the nuclear envelope. These structures are bound to the chromosome material, the "nucleolus-like bodies" and the fibrillar bodies. A participation of "nucleolus-like bodies" in the formation of the central sphere is suggested. A possible role of the nuclear protein matrix in the construction of the karyosphere elements is discussed.     

Ultrastructural Changes of the Nucleolus During Cell Cycle in Triticum aestivum

The ultrastructural changes of the nticleolus during cell cycle in common wheat (Triticum aestivum L. ) were studied by an "en bloc" silver-staining method. It was observed that in interphase, the nucleolus was heavily stained, within which fibrillar centres, dense fibrillar component, granular component and nucleolar vacuoles could be identified. A large quantity of argentine fine granules were distributed in the condensed chromatin. Dur-ing prophase, along with the disintegration of the nucleolus and condensation of the chromatin, the larger heavily-stained granules gradually appeared at the periphery of the chromatin. At late prophase, the materials derived from the nucleolus were spread and deposited on the surface of the chromosomes. The silver-stained, larger granules, deriving from the disintegrated nucleolus, accumulated at the periphery of the metaphase chromosomes and formed an uneven and discontinuous "sheath"-like structure. This "sheath"-like structure was also observed at anaphase. In telophase, the silver-stained nucleolar materials were progressively separated from the "sheath' and fused with each other to form prenucleolar bodies, and at last, participating in the formation of new nucleoli. The results showed that the nucleolar materials were transferred directly to the surface of the chromosomes and formed a discontinuous coat, but not incorporated into the interior of the chromosomes. The silverstained granules inside the chromosomes were neither related to the nucleolus nor to the materials from the disintegrated nucleolus.     

Nucleolar fibrillar centers in mouse spermatid nucleoli

The nucleoli of young spermatids of mice are described. They exhibit a very special shape resembling a "padlock" in which three different areas can be distinguished: (a) a compact zone corresponding to the fibrillar component, (b) the granular component and (c) a fibrillar center of low density. Fibrillar and granular components usually appear segregated. This nucleolus has been reconstructed based on serial sectioning. When the silver impregnation technique is employed, both fibrillar and granular components show a positive reaction, although the fibrillar center is free of granules. The morphology of the fibrillar center seems to be similar to that reported in other cells. The possibility that these fibrillar centers correspond to the nucleolar organizer is discussed.     

Whole mount electron microscopy of the nucleolus in salivary gland cells of Drosophila melanogaster.

The nucleolus of Drosophila melanogaster salivary gland cells, examined by whole mount electron microscopy, consists of a fibrillar core region and a peripheral region containing both fibres and granules. These regions appear to correspond to the fibrillar and granular components, respectively, seen in thin sections. Most of the nucleoli were attached to the chromocenter region of the polytene chromosomes, containing the nucleolar organizer. Bundles of relatively straight chromatin fibres, 13 nm in diameter, extended from the chromocenter into the core region of the nucleolus, however it was not possible to trace the path of these chromatin fibres through the nucleolus since they were obscured within the mass of nucleolar fibres. The nucleolar fibres in both the core and peripheral regions were irregular and knobby, with a diameter of about 15 nm. In the core region, the fibres appeared to be of considerable length and were characteristically clustered together to form small interconnected masses. The fibres in the peripheral region were relatively short and some appeared to blend with amorphous, poorly-defined pools of material. Electron dense granules 15-20 nm in diameter were also associated with this amorphous substance. It is hypothesized that the formation and subsequent packaging of the 28s rRNA may be represented by a morphological transition of the peripheral fibres, via an amorphous pool-like intermediate stage, into the nucleolar granules. The results of this study indicate that whole mount electron microscopy may be a useful alternative to thin sectioning in high resolution studies of the nucleolus.     

Atomic force microscopy of the morphology of the matrix and mineral components of the otolith of Hyperoglyphe antarctica

The sagittal otolith of Hyperoglyphe antarctica (Centrolophidae: Teleostei) has a prismatic structure in which the anti-sulcal growth axes of each prism consist of a series of nested cones each composed of a mineral layer followed by an organic matrix layer. Broken sections show the mineral layers to be composed of stacks of crystals. Otolith matrix that has been decalcified and air-dried, or critical-pont-dried, retains a periodic structure of repeating high and low matrix density. At high magnifications, both broken whole crystal surfaces and decalcified matrix surfaces have a granular structure. Chloroxbleached whole otoliths also show a granular crystalline structure. At higher magnifications, the air-dried matrix showed a parallel fiber structure with similar dimensions to keratin fibers. © 1995 Wiley-Liss, Inc.     

X-ray and dissolution studies of paramylon storage granules fromEuglena

Summary Paramylon is the -1,3 glucan storage carbohydrate in the euglenoid algae. Mature paramylon granules are highly crystalline, fibrillar, and have a complex substructure. X-ray diffraction was used to demonstrate that mature paramylon granules are much more crystalline than immature granules. Freeze-etch electron microscopy showed that in mature granules, the microfibrils are organized in highly ordered arrays while the microfibrils of immature granules are less organized. The data suggest that the high crystallinity of paramylon is due to higher-order aggregates of microfibrils and the interaction of water with the microfibrils. The dissolution of paramylon was recorded by darkfield videomicroscopy. In a 0.5 N NaOH solution, paramylon dissociates in a regular manner into its constituent 4 nm microfibrils, and the central region of the granule is the last remaining refractile area during the dissolution process.Abbreviation LN liquid nitrogen     

Further cytochemical studies on the perichromatin granules

Summary The perichromatin granules were studied in hepatocytes of experimental rats injected with cycloheximide because the increased number of these nuclear components after such treatment facilitated their cytochemical investigation. Most perichromatin granules were sensitive to the digestion with pepsin and ribonuclease. In contrast, small population of perichromatin granules was resistent to such digestion under conditions which remove known RNA containing components such as ribosomes, nucleolar RNP components and interchromatin granules. The size of these resistent perichromatin granules was reduced and they consisted of filaments the width of which was similar to that of filaments in the chromatin. Moreover, a small population of perichromatin granules was sensitive to the digestion with pepsin and deoxyribonuclease. The size of these granules was only slightly reduced. All these observations indicate that most perichromatin granules contain the RNA and some the DNA. A possibility also exists that the perichromatin granules might contain both RNA and DNA but in various proportions. In addition, partial digestion with pepsin followed by a complete digestion with ribonuclease and deoxyribonuclease removed perichromatin granules as well as other nucleoprotein structures. On the other hand, such digestion facilitated the visualization of the nuclear and cytoplasmic skeleton (matrix) in situ.Dedicated to the memmory of Dr. W. Bernhard     

Ultrastructural analyses of acellular glomerular basement membranes and mesangial matrix in a spontaneously diabetic rhesus monkey

Renal changes similar to those considered diagnostic for diabetes in humans are infrequently observed in spontaneous noninsulin-dependent (NID) diabetic monkeys. In the current study, renal cortical tissue blocks are rendered acellular to demonstrate glomerular basement membrane (GBM) and mesangial matrix (MM) changes in a naturally occurring NID diabetic rhesus monkey (Macaca mulatta). Transmission electron micrographs of these specimens show axial MM accumulations with numerous striated collagen fibrils that frequently extend onto internal (endothelial) surfaces of peripheral GBM. The outer (epithelial) component, although compact, appears bilaminar due to folded external surfaces not coinciding with similar irregularities on internal surfaces. By scanning electron microscopy, external surfaces of sclerotic GBMs are extensively wrinkled and, following cryofracture, show congestion and expanded MM. A fenestrated meshwork of MM, which appears less dense and compact than epithelial BM, extends from axial regions onto GBM internal surfaces. The true thickness of randomly sampled peripheral GBM thickness (approximately 400 nm) is approximately double that of normal rhesus GBM. Diabetic GBMs exhibit ruthenium red positivity for surface polyanions with linear site densities not significantly different from normal. These observations indicate that sclerotic GBMs in diabetic rhesus monkeys closely resemble those seen in human end-stage diabetic glomerulopathy and suggest that this nonhuman primate may offer an excellent model for studies of chronic diabetic BM disease.     

Cell differentiation during early development of Nassarius reticulatus L. (Gastropoda,prosobranchia)

Summary In Nassarius reticulatus the nuclei and nucleoli undergo important morphological changes from the zygote to the 16-cell stage. In the zygote and in the trefoil (2-cell) and 4-cell stage, several agranular, fibrillar nucleolus-like bodies 1 m diameter are present in the interphase nucleus. Granular nucleoli first appear at the 8-cell stage. These nucleoli have fibrillar and granular regions. The granular regions are made up predominantly of ribosome-like osmiophilic granules. From the 16 and 32-cell stage onwards, the one or two spherical nucleoli of each nucleus measure 2.5 m in diameter and show a concentric organization with a very dense central region surrounded by a broad peripheral zone containing numerous granules, possibly of ribonucleoprotein. At the same time the number of ribosome-like particles increase in the karyoplasm and that of ribosomes in the cytoplasm. These findings are surprising because in eggs with radial cleavage, which have been subjected to more detailed analysis, the first granular nucleoli appear after the end of the cleavage, at the blastula or gastrula stage. The early appearance of granular nucleoli which seem to be characteristic of several eggs with spiral cleavage is discussed in connection with biochemical data on RNA-synthesis.This investigation was supported by the Deutsche Forschungsgemeinschaft and the Stiftung VolkswagenwerkWe wish to thank Mrs. C. Mehlis for valuable technical assistance and Prof. J. Bergerard for the excellent working conditions at the Station biologique at Roscoff (France)     

Nuclear ultrastructure of the oocytes from human antral follicles. Formation of the karyosphere

The organization of the nucleus in the oocytes from human antral follicles was examined at the electron microscopic level. At this time all the chromosomes are aggregated around an inactivated nucleolus forming a karyosphere 5-7 micron in diameter. The nucleolus bears no granular component and consists of densely packed delicate fibrillar material. The peripheral zone resembling a ring 0.5 micron thick is separated in the nucleolus. Nucleolus-like bodies (NLB), consisting of granules 20 nm in diameter embedded in finely fibrillar material, are constantly observed in contact with the chromatin. The eventually formed karyosphere is a complex of intimately interconnecting structures--the nucleolus, chromosomes and NLB. However, the chromatin surrounding the nucleolus does not form a continuous (compact) mass as it is observed at the light microscopic level. It is determined that the human karyosphere is formed during the preovulatory period when the connection between oocyte and follicular cells of cumulus oophorus is lost. The duration of karyosphere existence in the human oocytes, and relation of the karyosphere to the processes of antral follicle atresia are discussed.     

Effects of bromodeoxyuridine substitution on metaphase chromosome structures examined by scanning electron microscopy

Chinese hamster chromosomes were differentially substituted with 50 M 5-bromodeoxyuridine (BrdU) to obtain chromosomes with bifilarly and unifilarly substituted (BB-TB) and unifilarly and non-substituted (TB-TT) chromatid constitutions. To avoid the effect of Giemsa staining on the ultrastructure of chromosomes, unstained preparations were exclusively used. When TB-TT chromosomes were prepared with the conventional air-drying method followed by the osmium tetroxide-thiocarbohydrazide (OsO₄-TCH) technique and examined by scanning electron microscopy (SEM), the TB-chromatid appeared somewhat more slender and showed more conspicuous spiral structures, thereby appearing more loosened compared to the TT-chromatid. At higher magnifications, however, 30 nm chromatin fibres which were seen to constitute both chromatids showed no discernible differences in dimension between the TT- and TB-chromatids. On the other hand, TB-TT chromosomes specially prepared for SEM without the process of air-drying appeared in their entirety less extended and no spiral configuration was observed even in the TB-chromatid. The TB-chromatid instead appeared rather less loosened than the TT-chromatid whereas thick fibre-like structures which in turn seemed to consist of 30 nm fibres were more easily discernible in the TT-chromatid compared to the TB. Such seemingly contradictory results obtained from the two different preparatory procedures were tentatively explained on the basis of our multiple coiling model (Taniguchi and Takayama 1986).     

小麦核仁在细胞周期中的变化

运用Bernhard染色方法研究了小麦根端分生组织细胞核仁在细胞周期中的变化。结果显示,间期核仁染色很深,能够区分出纤维中心（FC）、致密纤维组分（DFC）和颗粒组分（G）,而染色质被漂白,在染色质间可以观察到细小的RNP颗粒。进入前期,在染色质的边缘有小的RNP颗粒分布。中期,染色体周边分布着类似于间期核仁的深染的大RNP颗粒,形成一个不完全连续的“鞘”状结构;在染色体内部看不到类似核仁的深染颗粒。到了后期时,仍可见RNP“鞘”状结构的存在。进入末期,这些RNP植物逐渐由“鞘”脱离,最后参与新核仁的形成。这些结果表明,核仁解体后的物质直接转移到了中期染色的表面,并形成不连续的表层,没有进入染色体的内部。     

Tridimensional architecture of the Golgi apparatus and its components in mucous cells of Brunner's glands of the mouse

The three-dimensional structure of the Golgi apparatus and its components has been analyzed in thin and thick sections of mucous cells of mouse Brunner's glands by using low- and high-voltage electron microscopes and a stereoscopic approach. In thick sections of glands impregnated with osmium or treated to detect nicotinamide adenine dinucleotide phosphatase (NADPase) or thiamine pyrophosphatase (TPPase) activity, the Golgi apparatus appeared, at low magnification, as a continuous network located in the supranuclear region. At higher magnifications and in thin sections of tissue postfixed with reduced osmium and stained with lead citrate or treated to demonstrate phosphatase activity, the following components were observed: on the cis-face of the Golgi stacks, an osmiophilic tubular network referred to as the cis-element; a cis-saccular-compartment composed of a distended porous saccule slightly reactive for NADPase and three or four underlying NADPase-positive, flattened, poorly fenestrated saccules; a trans-saccular-compartment consisting of four to six TPPase-positive saccules or sacculo-tubular elements, prosecretory granules, and "peeling off" trans-tubular networks. The saccules of the cis-compartment were often perforated by large pores in register. The cavities thus formed in the stacks were called wells and were pan-shaped with a mouth directed toward the cis-face of the stacks and a bottom closed by TPPase-positive saccules. The wells always contained 80-nm vesicles. The saccules of the trans-compartment were involved in the formation of secretory granules according to the following proposed sequence of transformation. The secretion product appeared initially as a granular material evenly distributed throughout a slightly distended, poorly fenestrated saccule. These saccules appeared to transform into fenestrated elements with irregular pores and with parts of them taking on the appearance of a tubular network; they were thus referred to as sacculotubular elements. The secretory material initially distributed throughout these elements accumulated in nodular dilatations randomly distributed along the tubular portions of the elements. The dilatations, considered as prosecretory granules, increased in size as they drained the secretory material from the rest of the sacculotubular elements. Such prosecretory granules, large and irregular in shape, "peeled off" from the stacks of saccules with residual saccular or tubular structures still attached to them, some of the latter forming trans-tubular networks. The prosecretory granules detached from such membranous residues, condensed, and finally transformed into spherical secretion granules.     

Immunoelectronmicroscopic localization of extracellular matrix components produced by bovine corneal endothelial cells in vitro

Bovine corneal endothelial cells deposit an extracellular matrix in short-term cultures, which contains various morphologically distinct structures when analysed by electron microscopy after negative staining. Amongst these were long-spacing fibers with a 150 nm periodicity, which appeared also to be assembled into more complex hexagonal lattices. Another structure was fine filaments, 10-40 nm in diameter, which occasionally exhibited 67 nm periodic cross-striation. Non-striated 10-20 nm filaments sometimes formed radially oriented bundles arranged in networks and fuzzy granular material was associated with the filaments in the bundles. Often, these bundles extended into solitary filaments, 10-20 nm in diameter, with a smooth surface. In addition, amorphous patches were seen, which contained dense aggregates of fibrillar and granular material. In longer-term cultures, some of the structures coalesced to form large fibrillar bundles. By using specific antibodies to various extracellular matrix components and immunolabeling with gold some of these structures could be identified as to their protein composition. Whereas fibronectin antibodies labeled a variety of structures--fine filaments with granular materials, radially oriented bundles, patchy amorphous aggregates and small granular material scattered throughout the background--type III collagen antibody predominantly labeled filaments with periodic banding (10-40 nm in diameter). A small amount of type III specific labeling was also observed over the networks of radially oriented fibrils and fine filaments associated with granular material. Type IV collagen and laminin antibodies localized in areas of the patchy amorphous aggregates. Type VI collagen antibodies, on the other hand, labeled fine filaments and the gold particles showed a pattern of 100 nm periodicity. Many of the fine 10-20 nm filaments exhibited a tubular appearance on cross-section, but they were not reactive with any of the antibodies used. Also negative were the long-spacing fibers and assemblies--including hexagonal lattices--containing this structural element.     

Imaging of the surface of living cells by low-force contact-mode atomic force microscopy.

The membrane surface of living CV-1 kidney cells in culture was imaged by contact-mode atomic force microscopy using scanning forces in the piconewton range. A simple procedure was developed for imaging of the cell surface with forces as low as 20-50 pN, i.e., two orders of magnitude below those commonly used for cell imaging. Under these conditions, the indentation of the cells by the tip could be reduced to less than l0 nm, even at the cell center, which gave access to the topographic image of the cell surface. This surface appeared heterogeneous with very few villosities and revealed, only in distinct areas, the submembrane cytoskeleton. At intermediate magnifications, corresponding to 20-5 microm scan sizes, the surface topography likely reflected the organization of submembrane and intracellular structures on which the plasma membrane lay. By decreasing the scan size, a lateral resolution better than 20 nm was routinely obtained for the cell surface, and a lateral resolution better than 10 nm was obtained occasionally. The cell surface appeared granular, with packed particles, likely corresponding to proteins or protein-lipid complexes, between approximately 5 and 30 nm xy size.     

Intercellular fibrillar skeleton in the basal interdigitations of kidney tubular cells

Summary The tubular cells from the thick ascending limb of the loop of Henle in rabbit kidney medulla contain in their basallateral surfaces a complex system of interdigitations. Within these interdigitations, the plasma membranes are separated by extracellular spaces of relatively constant width that contain a previously undescribed fibrillar system. The structural organization and distribution of this intercellular fibrillar skeleton was studied using freeze-fracture etch and then section electron microscopy. The skeleton is comprised of discrete strands with a density of 300 to 400 per m² evenly distributed along the entire basal-lateral region. Each strand has the shape of a brace and it is constructed from up to eight finer filaments each having a width of about 2 nm. The filaments are tightly joined together along their shafts for about 30 nm but they separate at both ends for about 10 nm before contacting the external surface of the plasma membrane. We propose that this intercellular fibrillar skeleton is responsible for maintaining the wide (about 50 nm) and uniform plasma membrane separation along the entire length of the basallateral region of the tubular cells of the thick ascending limb.