The nucleolonema changes its three-dimensional conformation during interphase in root-tip meristems of onion

Summary The three-dimensional architecture of a filamentous nucleolar structure, called the “nucleolonema”, was investigated in onion root-tip cells by applying a silver impregnation technique to air-dried cells and serial ultrathin sections. The entire configuration of the nucleolonema was revealed when silver staining was applied to air-dried cells. The nucleolonema was knobbly or segmented along its entire length and showed great variation in thickness. Three categories of nucleolonema were discriminated depending on thickness; each had an average value of 0.5, 1.0, and 1.3 μm, respectively. Some root tips were embedded in Lowicryl K4M resin and cut into serial ultrathin sections about 100 nm thick. When these sections were subjected to silver impregnation, segments of nucleolonema were visualized. Most of them were found to contain achromatic holes. These holes apparently corresponded to the fibrillar centres seen with the electron microscope. According to the profiles of the holes, nucleolonema structures were classified into three types: (1) nucleolonema with no distinct holes, (2) those with beaded holes, and (3) those with cylindrical holes. The thicknesses were 0.7–0.8, 0.9–1.2, or 1.2–1.4 μm for nucleolonemata with no holes, beaded holes, or cylindrical holes, respectively. The argyrophilic wall of nucleolonemata with holes was about 0.4 μm thick, roughly compatible with the thinnest nucleolonema seen in air-dried specimens. The crescent-shaped segments were sometimes observed when the nucleolonema was sectioned transversely, suggesting that the achromatic holes are exposed to the nucleoplasm, in other words, the nucleolonema is partially degraded. Thus, the nucleolonema was not always structurally stable during interphase. The results suggest that the nucleolonemata gradually become knobbly and increase their thickness, with concomitant expansion of the fibrillar centres sometimes degrading into approximately 0.5 μm thick strands.     

Identification of actin-, alpha-actinin-, and vinculin-containing plaques at the lateral membrane of epithelial cells

In this paper, a new type of spot desmosome-like junction (type II plaque) is described that is scattered along the entire lateral plasma membrane of rat and human intestinal epithelium. Ultrastructurally type II plaques differed from the classical type of epithelial spot desmosome ("macula adherens", further denoted as type I desmosome) by weak electron density of the membrane-associated plaque material, association of the plaques with microfilaments rather than intermediate filaments, and poorly visible material across the intercellular space. Thus, type II plaques resemble cross-sections of the zonula adherens. Immunofluorescence-microscopic studies were done using antibodies to a main protein associated with the plaques of type I desmosomes (desmoplakin I) and to the three major proteins located at the plaques of the zonula adherens (actin, alpha-actinin, and vinculin). Two types of plaques were visualized along the lateral surface of intestinal and prostatic epithelium: (a) the type I desmosomes, which were labeled with anti-desmoplakin but did not bind antibodies to actin, alpha-actinin, and vinculin, and (b) a further set of similarly sized plaques, which bound antibodies to actin, alpha-actinin, and vinculin but were not stained with anti-desmoplakin. Three-dimensional computer reconstruction of serial sections double-labeled with anti-desmoplakin and anti-alpha-actinin further confirmed that both types of plaques are spatially completely separated from each other along the lateral plasma membrane. The computer graphs further revealed that the actin-, alpha-actinin-, and vinculin-containing plaques have the tendency to form clusters, a feature also typical of type II plaques. It is suggested that the type II plaques represent spot desmosome-like intercellular junctions, which, like the zonula adherens, appear to be linked to the actin filament system. As the type II plaques cover a considerable part of the lateral cell surface, they might play a particular role in controlling cellular shape and intercellular adhesion.     

PLOIDAL CHANGES IN CLONAL CULTURES OF SPIROGYRA COMMUNIS AND IMPLICATIONS FOR SPECIES DEFINITION

A clonal culture of Spirogyra filaments of initially uniform width produced filaments of three additional significantly different widths. Group I filaments of the original clone were 30.9 ± 0.7 μm wide (mean ± SD, N = 50). Group I filaments produced Group II filaments (22.0 ± 1.1 μm) through vegetative growth and sexual reproduction. Zygospores from homothallic Group I filaments produced germlings representative of Groups I and II; zygospores from homothallic Group II filaments produced germlings representative of Group II only. Germlings of Groups III (27.7 ± 1.0 μm) and IV (44.9 ± 0.8 μm) were produced in the cross of I × II. Viable zygospores from homothallic Group III filaments were obtained. Cells of Group IV filaments were initially binucleate and did not conjugate. Of the six intergroup crosses possible, four resulted in conjugation-tube formation only; two crosses yielded zygospores (I × II and III × IV). Germlings from the successful cross of Groups III and IV produced filaments of all four groups. Chromosome counts were: Group I (24), Group II (12), Group III (18), and Group IV (24, one nucleus). Relative nuclear fluorescence values of mithramycin-stained DNA were (mean ± SD, N ≥ 30): Group I (11.1 ± 1.4), Group II (5.7 ± 0.7), Group III (8.8 ± 1.3), and Group IV (10.0 ± 0.9, one nucleus). Cytologically, Group II appears to be a diploid (2x), Group I a tetraploid (4x), and Group III a triploid (3x). Systematically, Groups I, II, and III key out to Spirogyra singularis, S. communis, and S. fragilis, respectively, using Transeau's mongraph of the family Zygnemataceae. These species are interpreted to represent a species complex of S. communis (whose name has priority) with the ancestral haploid (x = 6) missing.     

THE MYOFILAMENT ARRANGEMENT IN THE FEMORAL MUSCLE OF THE COCKROACH, LEUCOPHAEA MADERAE FABRICIUS

The structure of the femoral muscle of the cockroach, Leucophaea maderae, was investigated by light and electron microscopy. The several hundred fibers of either the extensor or flexor muscle are 20 to 40 µ in diameter in transverse sections and are subdivided into closely packed myofibrils. In glutaraldehyde-fixed and epoxy resin-embedded material of stretched fibers, the A band is about 4.5 µ long, the thin filaments are about 2.3 µ in length, the H zone and I band vary with the amount of stretch, and the M band is absent. The transverse sections of the filaments reveal in the area of a single overlap of thick and thin filaments an array of 10 to 12 thin filaments encircling each thick filament; whereas, in the area of double overlap in which the thin filaments interdigitate from opposite ends of the A band, the thin filaments show a twofold increase in number. The thick filament is approximately 205 to 185 A in diameter along most of its length, but at about 0.2 µ from the end it tapers to a point. Furthermore, some well oriented, very thin transverse sections show these filaments to have electron-transparent cores. The diameter of the thin filament is about 70 A. Transverse sections exhibit the sarcolemma invaginating clearly at regular intervals into the lateral regions of the A band. Three distinct types of mitochondria are associated with the muscle: an oval, an elongate, and a type with three processes. It is evident, in this muscle, that the sliding filament hypothesis is valid, and that perhaps the function of the extra thin filaments is to increase the tensile strength of the fiber and to create additional reactive sites between the thick and thin filaments. These sites are probably required for the functioning of the long sarcomeres.     

A study on the structure of paracrystals of F-actin.

The structure of three types of paracrystals formed by a muscle protein, actin, was studied by electron microscopy using the technique of optical diffraction and filtering methods.The type I paracrystal of F-actin⁴ had a flat net structure and each thread of the net appeared to be made of a single double-stranded filament of F-actin. Its unit cell was rhombic with sides of about 340 Å in length. The narrower angle of the rhomb was about 30 °. A side of the rhomb corresponded to one repeating unit of F-actin. The cross-connecting point of the net appeared to occur at a cross-over point of the double helical F-actin filament when the paracrystal plane was observed perpendicularly. A set of parallel filaments running in one direction seem to simply overlie another set of parallel filaments running in another direction.The type II paracrystal also had a flat net structure with a unit cell of the same size and shape as type I, but had twice the amount of material in the unit cell in comparison with that of type I; a thread of type II was made of a pair of F-actin filaments. The type II paracrystal seemed to be made by attaching the F-actin filaments side-by-side to filaments of the type I paracrystal. These newly associated filaments cross-connected with each other in the same manner as those of the type I paracrystal.The type III paracrystal was a side-by-side aggregate of F-actin filaments. There was no lateral order between the neighbouring filaments.     

Isolation of intermediate filament assemblies from human hair follicles

We used developing human hair follicle cells for the isolation of hard alpha-keratin structural components. Intracellular dispersions examined by electron microscopy contained both individual alpha-keratin filaments and the tactoid-like filament assemblies observed in situ organized along subfibrillar arms of macrofibrils. The assemblies of average width 47 nm were composed of closely packed alpha-keratin filaments and originated from the initial filament arrays observed in sections of developing mammalian hair follicles. We have distinguished two types of assemblies: the para-like or hexagonally packed and the ortho-like spiral or whorl type. Axial banding extended across the width of filament assemblies, which suggested that hard alpha-keratin filaments pack in lateral register and form a lattice that contains interfilamentous bridges. We observed axial banding patterns with periods ranging from 20 to 22 nm, consistent with the 22-nm periodic structure deduced from x-ray diffraction studies and present in models proposed for hard alpha-keratin and other intermediate filaments. Preliminary biochemical studies of filaments and filament assemblies indicate that they consist of the closely related group of proteins (low-sulfur proteins) ubiquitous among extracts of hard mammalian keratins. Isolated hard alpha-keratin filament assemblies provide a new and valuable structural entity for investigating the assembly mechanisms involved in the formation of the filament-matrix framework found in hard mammalian keratin appendages.     

Subfilament organization in myosin filaments of the fast abdominal muscles of the lobster, Homarus americanus

The myosin filaments of the fast abdominal muscle of the lobster are about 2.7 microns long with a diameter of about 20 nm. They have a low density core in transverse sections except for a short portion in the middle of the filaments about 140 nm in length which is solid. In the solid region the diameter of the filaments is 25 nm. The wall of the filaments is made up of 12 subfilaments arranged in six pairs in a single layer around the wall. The spacing between the subfilaments of a pair is 3.4 nm and the spacing between successive pairs is 8.4 nm. An extra density is present on the inner surface of the wall of the filament along the entire length of the tubular portion of the filament. This density is always adherent to the wall and in serial transverse sections of the same filament its position changes from section to section without any apparent pattern to the change. No structural organization could be detected in this extra density.     

The myosin filament: V. Intermediate voltage electron microscopy and optical diffraction studies of the substructure

Using a 200 kV electron microscope (JEM 200 A), thick (up to 0.4 μm) crosssections of the myosin filaments of vertebrate striated muscle were studied. It was found that: (a) with increasing section thickness the cross-sectional profiles of the shaft of the filament were increasingly more triangular and in sections 0.4 μm thick each apex of the triangle was clearly blunted. This unique cross-sectional profile is predicted by the model proposed by Pepe (1966,1967) in which 12 parallel structural units are packed to form a triangular profile with a structural unit missing at each apex of the triangle. (b) With increasing section thickness the substructure of the myosin filament was enhanced, with the best substructure visible in sections 0.2 μm to 0.3 μm thick. This strongly supports parallel alignment of structural units in the shaft of the filament as proposed by Pepe (1966,1967). (c) The substructure spacing, determined by optical diffraction from electron micrographs of cross-sections of individual myosin filaments or groups of filaments is about 4 nm. (d) The different optical diffraction patterns observed from individual myosin filaments can be explained if the projection of each structural unit in the plane of the section has an elongated profile. With a substructure spacing of 4 nm an elongated cross-sectional profile could be produced by having two myosin molecules per structural unit. Models drawn with two myosin molecules per structural unit in the model proposed by Pepe (1966,1967) gave optical diffraction patterns similar to those observed from individual filaments. (e) The different optical diffraction patterns observed from individual myosin filaments can be explained if the elongated profiles in each structural unit are similarly oriented but with the orientation changing along the length of the filament. The change in orientation per unit length of the filament must be small enough to maintain an elongated profile for the projection of the structural unit in the plane of the sections 0.3 μm thick. All of these observations and conclusions strongly support the model for the myosin filament proposed by Pepe (1966,1967).     

Forming and removing stain precipitates on ultrathin sections

Stain precipitates resulting from the use of lead or uranyl salts, or both, on ultrathin sections can be classified as belonging to one of three morphological types: I) extremely electron-dense particles caused by prolonged use of lead salts only, II) amorphous networks formed following double staining with either aqueous or alcoholic uranyl and lead salts, and III) crystalline needles sometimes resulting from double staining with alcoholic uranyl and lead salts. It has been found, however, that either acetic acid or aqueous uranyl acetate can be used to remove type I and type II precipitates from sections, and that oxalic acid and alcoholic uranyl solution will remove type II precipitates. Unfortunately, type III precipitates are unaffected by any agents tested so far.     

Forming and Removing Stain Precipitates on Ultrathin Sections

Stain precipitates resulting from the use of lead or uranyl salts, or both, on ultrathin sections can be classified as belonging to one of three morphological types: I) extremely electron-dense particles caused by prolonged use of lead salts only, II) amorphous networks formed following double staining with either aqueous or alcoholic uranyl and lead salts, and III) crystalline needles sometimes resulting from double staining with alcoholic uranyl and lead salts. It has been found, however, that either acetic acid or aqueous uranyl acetate can be used to remove type I and type II precipitates from sections, and that oxalic acid and alcoholic uranyl solution will remove type II precipitates. Unfortunately, type III precipitates are unaffected by any agents tested so far.     

Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.     

Morphology of the excretory system of Hysterothylacium haze (Nematoda: Anisakidae: Raphidascaridinae)

The morphology of the excretory system of Hysterothylacium haze was examined by serial histological sections. The excretory system was H-shaped and glandular, consisting of lateral filaments and a commissure, with the exretory pore opening posterior to the nerve ring. A large excretory nucleus was present in the left filament. The cuticularized excretory duct was confined to the left side of the commissure. The glandular excretory system is rare among the Raphidascaridinae.     

The fine structure of myocytes in the sponges microciona prolifera (Ellis and Solander) and tedania ignis (Duchassaing and Michelotti)

Myocytes are long, fusiform cells found in the osculum and other contractile areas of many sponges. Myocytes in the oscular sphincter of Tedania ignis and the osculum and dermal membrane of Microciona prolifera were studied with light- and electron-microscopes to compare their structure to that of muscles. Salient points of similarity between myocytes and smooth muscles were their long, fusiform shape, their red color after staining with Mallory's triple stain, and the presence of filaments running longitudinally in the cytoplasm. Microciona myocytes have thick filaments of 150–250 Å diameter and thin filament of 50–70 Å diameter, and in transverse sections the thin filaments occasionally appear as a ring of dots around a thick filament. Longitudinal sections of Tedania myocytes show only one type of filament, which varies from 100 Å to 200–300 Å diameter in thick regions of the filament. Although transverse sections show light material around the dense filaments, a distinct pattern of thick and thin filaments is not seen in Tedania. Due to infrequent contacts between cells, the large extra-cellular space observed with the electron microscope (49% in Tedania, 57% in Microciona), and the absence of nerves, each myocyte probably acts as an independent contractile unit.     

The myosin filament: VII. Changes in internal structure along the length of the filament

Improved fixation procedures have enabled substructure to be observed by electron microscopy in transverse sections of vertebrate skeletal muscle thick filaments as thin as 140 nm. Optical diffraction combined with digital autocorrelation analysis, focal series and tilting experiments have confirmed the presence of a regular substructure having a repeat near 4 nm and shown that it is highly unlikely to be an artifact associated with the electron microscope imaging system. The results obtained strongly suggest that the thick filament is constructed from a bundle of rod-like subfilaments arranged parallel to the thick filament axis to within less than a degree. This cannot easily be reconciled with the general theory of thick filament structure proposed by Squire (1973), but it is consistent with the model proposed by Pepe, 1966, Pepe, 1967. Optical diffraction of 140 nm thick serial transverse sections has also suggested a structural change along the length of the filament that is manifest by a variation in the proportion of filaments showing strong diffraction maxima in one, two or three directions.     

Growth of flagellar filaments of Escherichia coli is independent of filament length

Bacterial flagellar filaments grow at their distal ends, from flagellin that travels through a central channel ～2 nm in diameter. The flagellin is extruded from the cytoplasm by a pump powered by a proton motive force (PMF). We measured filament growth in cells near the mid-exponential-phase with flagellin bearing a specific cysteine-for-serine substitution, allowing filaments to be labeled with sulfhydryl-specific fluorescent dyes. We labeled filaments first with a green maleimide dye and then, following an additional period of growth, with a red maleimide dye. The contour lengths of the green and red segments were measured. The average lengths of red segments (～2.3 μm) were the same regardless of the lengths of the green segments from which they grew (ranging from less than 1 to more than 9 μm in length). Thus, flagellar filaments do not grow at a rate that decreases exponentially with length, as formerly supposed. If flagellar filaments were broken by viscous shear, the broken filaments continued to grow. Identical results were obtained whether flagellin was expressed from fliC on the chromosome under the control of its native promoter or on a plasmid under the control of the arabinose promoter.     

Modulation of the Shape of Epithelial Lens Cells in vitro Directed by a Retinal Extract Factor

We have shown previously [1] that bovine epithelial lens cells can be stimulated to divide and elongate by a retinal extract (RE). In this report we show that the morphological response to the stimulatory factor is directly related to the target-cell shape, and we describe how the cell shape can be modulated into morphologically different types. If the cells are grown continuously from the explant in the presence of the RE factor, they keep a typical regular pavement-like epithelial shape (type I), even after serial passages. If the same cells are cultured in the absence of the factor, they become extremely irregular in shape and enlarge enormously (type II), and during serial passage elongate spontaneously to a fibroblast-like pattern. However, when type II cells are stimulated by RE, they elongate dramatically into type III cells as described in [1], provided they are stimulated at the optimal cell density. We show that the transformation of one type to another is directly under the control of RE, and we demonstrate that the changes in cell morphology are accompanied by alterations in cytoplasmic actin filaments. Type I cells contain few microfilaments, while type II cells display actin-tropomyosin polygonal fibre networks that reform during conversion to type III cells and then to elongated stress fibres. The change from type I to type II cells is also accompanied by massive accumulation of surface-associated fibronectin. We conclude that factors obtained directly from the eye have a direct ability to control morphology and proliferation of ocular cells like lens cells perhaps by modulation of cellular adhesiveness mediated by surface fibronectin and reorganization of cytoplasmic actin-based filaments.     

Ultrastructural studies of Austramphilina elongata (Cestoda,Amphilinidea)

Summary The fine structure of larval Austramphilina elongata is described using serial semithin and ultrathin sections. Densely packed germ cells with many ribosomes and mitochondria and with large Golgi complexes fill the middle third of the body. Some necrotic nuclei were observed near the anterior end. The neodermis consists of a subepidermal syncytium connected to pericarya in the parenchyma by means of cytoplasmic processes containing peripheral microtubules; electron-dense ovoid bodies condense in these processes. Myoblasts are connected to muscle fibres by means of cytoplasmic connections rich in mitochondria. Twelve (exceptionally eleven) type I gland cells containing large secretory granules and extensive granular endoplasmic reticulum are located in the dorso-posterior part of the body; they open through 12 (or 11) discrete ducts into an anterior invagination of the tegument which is covered by epidermis and not connected to the outside. Ten type II gland cells containing elongate secretory granules with regularly arranged longitudinal microtubules are located ventral to the type I cell bodies; they open on a ventral papilla a short distance behind the anterior end. Ten type III gland cells containing irregularly round-oval secretory granules with coiled microtubules are located anterior and ventral to the type I gland cells; they open through five discrete ventro-anterior openings on each side of the body. Ducts of all gland cells have mitochondria and microtubules. The spermatozoon has a basic pattern of two axonemes, each with a single central filament, a mitochondrion (mitochondria), and a row of surface microtubules interrupted by the axonemes. In the tips of epidermal cilia, doublet 1 and doublets adjacent to it lose their microtubules B first and close in on the central pair of filaments in a spiral fashion, enclosing an electron-dense rod. Presence of a neodermis and ultrastructure of the spermatozoon support the validity of the taxa Neodermata Ehlers and Trepaxonemata Ehlers and are strong evidence against a phylogenetic relationship of the cestodarians — cestodes with the Acoelomorpha; this is also indicated by the ultrastructure of sense receptors and epidermal ciliary rootlets.     

Organization of Actin Filaments in the Axial Rod of Abalone Sperm Revealed by Quick Freeze Technique

An axial rod in abalone ( Haliotis discus ) sperm is a structure composed of a bundle of actin filaments, which elongates anteriorly to form the acrosomal process during the acrosome reaction. The ultrastructure of the actin filament bundle constituting the axial rod was examined using quick freeze technique followed by either freeze-substitution or deep-etch electron microscopy. Thin sections of quick freeze and freeze-substituted sperm revealed that the actin filaments in the axial rod are hexagonally packed in a paracrystalline array through its almost entire length with an average center-to-center spacing of 12 nm. Periodic transverse bands were also observed across the actin filament bundle, which may reflect the cross-bridges interconnecting the adjacent filaments. Quick-freeze deep-etch analysis provided the three-dimensional view of the axial rod. Actin filaments exhibiting 5.5–6 nm spaced striations were observed to run in parallel with each other inside the axial rod. The existence of cross-bridging structures was also displayed between adjacent filaments. These results suggest that the actin filaments in the axial rod are probably held together by regularly spaced cross-bridges to form a well ordered hexagonally packed bundle, and also cross-linked by fibrous structure to the lateral inner acrosomal membrane which closely surrounds the anterior half of the actin filament bundle.