Electron microscopic analysis of protoplast fusion in Streptomyces hygroscopicus and consideration on structural alterations in fusing membranes

Protoplasts of Streptomyces hygroscopicus were treated with polyethylene glycol and prepared for electron microscopic investigation as ultrathin sections. About 5% binary fusion products and 0.9% multicellular fusion products have been obtained in the sections. Three main types may be differentiated among binary fusion products, characterized by a successive loss of the bispherical shape and of continuous membrane structures in fusion zones.Analysing the membrane alterations a contact zone characterized by intact cytoplasmic membranes in both protoplasts, a fusion zone with a trilaminar fusion membrane of about 13–17 nm in thickness, and a fusion zone without continuous membrane structure can be distinguished. The different fusion areas are considered as stages in the fusion process. The data will be discussed in conjunction with a model for membrane alterations during fusion at the molecular level.     

Fine structure ofMicrococcus denitrificans andM. halodenitrificans in relation to their taxonomy

A study of ultrathin sections ofMicrococcus denitrificans andM.halodenitrificans has shown similar cell structures. The cell wall consists of several layers corresponding to those of the cell wall of gram-negative bacteria. The thickness of the cell wall is 250 – 350 Å; that of the cytoplasmic membrane 70 Å. The cytoplasm in both species contains ribosomes and inclusions of polymetaphosphate. Comparison with ultrathin sections ofThiobacillus novellus shows too much difference to consider the former two species to be identical with the latter one. The taxonomic position ofM.denitrificans andM.halodenitrificans is discussed.Deceased 3 July 1967.     

Polyhedral inclusion bodies in cells of nitrosomonas spec

Polyhedral inclusion bodies were observed in cells of a Nitrosomonas species. They were present in growing cells as well as in resting cells. In thin sections their size was about 130 nm in growing cells and about 185 nm in diameter in resting cells. The bodies were commonly located in the nucleoplasm. They appeared to be bounded by a nonunit membrane and had a granular substructure.In thin sections about 70% of the exponentially grown cells and about 20% of the resting cells of the investigated strain showed 1–7 respectively 1–3 inclusion bodies.     

Filipin-sterol complexes in bean rust- and oat crown rust-fungal/plant interactions: Freeze-etch electron microscopy

Summary Treatment with the polyene antibiotic filipin resulted in the formation of granular protuberances of the plasmamembranes of the mesophyll cells of bean (Phaseolus vulgaris) and oat (Avena sativa) plants, and of intercellular hyphal cells of the rust fungiUromyces appendiculatus var.appendiculatus andPuccinia coronata var.avenae, as seen by freeze-etch electron microscopy. The granules were also occasionally seen in intracellular vesicles ofU. appendiculatus. None were found in any intracellular organelles of plant or fungal tissue. The granules ranged in size from about 20–25 nm in the plant tissue and 21–27 nm in the fungal tissue. They were concluded to be filipin-sterol (FS) complexes. The extrahaustorial membranes of either bean or oat rustinfected tissue were generally devoid of FS complexes. The extrahaustorial membrane is continuous with the host plasmamembrane but appeared to have a lower sterol content as compared to the non-invaginated plasmamembrane. The results are discussed in relation to membrane associations at the host-parasite interface.Contribution No. 1011. Winnipeg Research Stn.     

A freeze-fracture study of the tegumental membrane of Schistosoma mansoni (Platyhelminthes:Trematoda).

Two fracture faces in each half of the freeze-fractured tegumental membrane of adult Schistosoma mansoni indicate the presence of two trilaminate membranes. This result is compatible with the heptalaminate appearance of the tegumental membrane in ultrathin sections. Intramembranous particles are located mainly in the outermost leaflet of the outer membrane and in the cytoplasmic leaflet of the inner membrane. The tegumental membrane of the cercaria (infective larva) has a single fracture plane, which conforms with its trilaminate appearance in sections. Intramembranous particles are extremely numerous and are almost all located in the cytoplasmic leaflet.     

Distribution, histochemistry and ultrastructure of somatostatin-like immunoreactive cells in the gastroenteric tract of the cartilaginous fish Scyliorhinus stellaris (L.)

Summary Somatostatin-like immunoreactive cells of an open type have been identified in the digestive tract of the cartilaginous fishScyliorhinus stellaris (L.) by the use of immunocytochemical techniques. In the stomach these cells are numerous both in the corpus (neck zone and tubular glands) and in the pyloric portion (crypts). In the spiral valve, somatostatin-like cells are rare, situated in the intestinal epithelium and without any particular localization. Using semithin serial sections,somatostatin-like cells are found to be Davenport-negative and weakly positive towards the Grimelius silver reaction, and using the semithin and ultrathin technique have been identified at the ultrastructural level; their secretory granules appear electron dense, round or slightly polygonal, and with a limiting membrane tightly adherent to the core. The mean diameter varies from 250–300 nm.     

The structure of Gloeobacter violaceus and its phycobilisomes

The fine structure of the atypical cyanobacterium Gloeobacter violaceus has been studied on frozen-etched replicas and compared to that of a typical unicellular strain: Synechocystis 6701. The complementary fracture faces of G. violaceus cytoplasmic membrane contain particles less numerous and more heterogenous in size than either the cytoplasmic membrane or the thylakoid membranes of Synechocystis. The most frequently observed particles of the exoplasmic fracture (EF) face of the G. violaceus cytoplasmic membrane are 11 nm in diameter and occasionally form short alignments. This particle class is similar in appearance to the numerous, aligned EF particles of Synechocystis thylakoid membranes. In replicas of cross-fractured G. violaceus, a layer 50–70 nm thick, composed of rod-like elements, underlies the inner surface of the cytoplasmic membrane. The rods, 12–14 nm in diameter, are oriented perpendicularly to the cytoplasmic membrane and show a 6 nm repeat along their length.Isolated phycobilisomes of G. violaceus appear, after fixation and negative staining, as bundles of 6 parallel rodshaped elements connected to an ill-defined basal structure. The bundles are 40–45 nm wide and 75–90 nm long. The rods are 10–12 nm in width; their length varies between 50 and 70 nm. These rods are morphologically similar to those observed at the periphery of hemidiscoidal phycobilisomes of other cyanobacteria, with a strong repeat at 6 nm intervals and a weaker one at 3 nm intervals along their length.The calculated molar ratio of phycobiliproteins in isolated G. violaceus phycobilisomes corresponds to 1:3.9:2.9 for allophycocyanin, phycocyanin and phycoerythrin respectively. When excited at 500 nm, isolated phycobilisomes exhibit a major fluorescence emission band centered at 663 nm.Abbreviations PBS phycobilisome(s) - PBP phycobiliprotein(s) - AP allophycocyanin - PC phycocyanin - PE phycoerythrin - K–PO₄ buffer KH₂PO₄ titrated with KOH to a given pH     

Die Verschiedenartigkeit von Haftorganen einiger Flechten

Summary The rhizines of the lichens, Parmelia caperata, Parmelia trichotera and Lobaria pulmonaria were studied with the Stereoscan scanning electronmicroscope and in ultrathin sections with the transmission electronmicroscope. The rhizines are composed of fungal hyphae.The fungal hyphae in the rhizines of the thallus of Parmelia caperata and in the cilia at the thallus border of Parmelia trichotera are connected by a glue-like substance. The ends of the Parmelia caperata rhizines are flattened and enlarged. With these footlike rhizines the thalli are in good connection with the substratum. The cilia at the thallus border of Parmelia trichotera have a tip by which the thallus is fixed on bark or rocks. The cell walls of the fungi hyphae in the Parmelia caperata rhizines and in the Parmelia trichotera cilia are 150–400 nm thick.The rhizines of Lobaria pulmonaria consist of fungi hyphae which are interlaced without a gluey substance. The thallus of Lobaria pulmonaria is connected to the substratum through the tips of single hyphae. The hyphae walls of Lobaria pulmonaria are 800–1800 nm thick.     

Characean charasome-complex and plasmalemma vesicle development

Summary We report on an unusual phenomenon which occurs in some characean algae as a normal plasma membrane activity and also in association with charasome formation. The phenomenon of formation of coated invaginations of the plasma membrane was observed in twoChara and 6Nitella species. These invaginations are coated on their cytoplasmic surface, are 50–60 nm in diameter and rarely exceed 60 nm in length. They are abundant in the young cells ofChara andNitella and also occur in mature cells, but at a lower frequency.N. translucent is an exception in that coated invaginations were few in the young cells and absent in mature cells. Coated vesicles (50–60 nm diameter) were closely associated with these invaginations. Our observations suggest the vesicles may be derived from the invaginations by endocytosis.A close relationship was noted between the development of charasomes (plasmalemma modifications) and coated invaginations. Numerous coated invaginations are seen along the membranes of young charasomes; these invaginations appear to be associated with growth of the charasomes. Coated vesicles were not associated with the coated invaginations of the charasome membrane. The tubular network of cytoplasm and wall space seen in the mature charasome may be formed by fusion of coated invaginations of the developing charasomes, leaving cytoplasmic strands between the fused portions. Coated invaginations were not present along charasomes of the mature cells.     

Ultrastructure of the synaptic ribbons in photoreceptor cells of Rana catesbeiana revealed by freeze-etching and freeze-substitution

Summary The three-dimensional structure of synaptic ribbons in photoreceptor cells of the frog retina was studied with freeze-etching and freeze-substitution methods, combined with a rapid-freezing technique. Although the synaptic ribbon consisted of two electron-dense plaques bisected by an electron-lucent layer in conventional thin sections, such lamellar nature was not so evident in freeze-etched replicas. The cytoplasmic surfaces of the synaptic ribbon presented an extremely regular arrangements of small particles 4–6 nm in diameter. Fine filaments 8–10 nm in diameter and 30–50 nm in length connected synaptic vesicles and the ribbon surface. These connections were mediated by large particles on both ends of the filaments. Approximately 3–5 filaments attached to one synaptic vesicle. Synaptic ribbons were anchored to a characteristic meshwork underlying the presynaptic membrane via another group of similar fine filaments. The meshwork seemed to be an etched replicated image of the presynaptic archiform density observed in thin sections.     

Extracellular protein fibrils in Chrysochromulina breviturrita (Prymnesiophyceae) and their serological relationship to fungal fimbriae

An extensive network of extracellular fibrils was revealed by negative staining in the greenish gold algal flagellate, Chrysochromulina breviturrita. These fibrils were of uniform diameter (4–5 nm), sometimes exceeding 5 m in length. In addition there were short, narrower fibrils (2–3 nm) on the surface of the flagella. Six protein bands were isolated from spent culture medium by SDS-PAGE and one of 80,000 Da was found to polymerize after dialysis into 4–5 nm fibrils identical to those found on the cell surface. Two other proteins of 58,000 Da and 65,000 Da also formed 4–5 nm fibrils but these were either rare or of a shorter length and different appearance. An antiserum directed against the surface 7 nm fibrils (fimbriae) of fungi agglutinated cells of C. breviturrita and some other Prymnesiophyceae and Chrysophyceae, but did not agglutinate cells of algal species in other groups. Immunofluorescence and protein A gold labelling confirmed that antigens related to fungal fimbriae were present on the surface of cells of C. breviturrita. Only the 80,000 and 58,000 Da proteins labelled heavily following protein A gold labelling. Some individual 4–5 nm fibrils labelled with gold were observed in the material prepared from the 80,000 Da band. These results therefore establish that C. breviturrita produces a surface network of fibrils that are serologically related to the fimbriae of fungi, and suggest a previously unrecognized relationship between members of the Prymnesiophyceae, Chrysophyceae and fungal groups.     

The fine structure of Chromatium buderi

Summary The fine structure of the phototrophic sulfur bacterium Chromatium buderi was studied in ultrathin sections and freeze-etch preparations.In addition to an intracytoplasmic membrane system common to all species of the Chromatiaceae, C. buderi contained extended lamellar membrane structures possibly due to too high light intensities during growth. The cell wall of C. buderi was found to be covered by a honeycomb-like outer layer consisting of macromolecular wine-glass shaped subunits 60–80 nm by 60 nm in size. This outer cell wall layer appears to be a typical property of the large cell Chromatium species.Contribution No. 3008 of the Woods Hole Oceanographic Institution.     

Identification of the sulfur inclusion body inBeggiatoa alba B18LD by energy-dispersive X-ray microanalysis

Dense inclusion bodies were observed under the STEM mode of a scanning electron microscope to occupy peripheral locations in air-dried filaments ofBeggiatoa alba B18LD, and they were determined by energy-dispersive x-ray microanalysis to consist almost entirely of sulfur. These inclusions conform in position and size (220–275 nm in diameter) to bodies seen in thin sections to be both membrane-bounded and enclosed within pockets penetrating the individual cell from the cytoplasmic membrane.     

Immuno-electron-microscopic study of the prolactin cells in the pituitary gland of male Wistar rats during aging

Summary Prolactin cells were identified by means of immunocytochemistry with protein-A gold as a marker on ultrathin sections of the pituitary gland of young (3–4 months), middle-aged (16–19 months), and aged (26–30 months) male Wistar rats. Point-counting volumetry revealed that the prolactin (PRL) cell-volume density in middle-aged rats was significantly increased in comparison to the volume densities in young and aged rats. Within the PRL-cell population, four types of PRL cells were distinguished on the basis of the shape and size of their secretory granules. During aging, dramatic changes occurred in the relative volumes of the four cell types. The volume percentage of cells with round granules (type I, granule diameter 150–250 nm, and type IIA, granule diameter 250–350 nm) increased from ±30% in young rats to ±90% in old rats. The volume percentage of cells with round and polymorphic granules (type IIB; granule diameter 350–400 nm and type III; granule diameter 500–600 nm) decreased from ±70% in young rats to ±7% in old rats. Age-related changes in serum PRL levels were not found. It is concluded that although during the life span of the male Wistar rat considerable changes in PRL-cell volume densities and in the ratios of PRL-cell types occur serum, PRL levels remain more or less constant.     

Correlation between ultrastructural and functional changes in sarcoplasmic reticulum during chronic stimulation of fast muscle

Summary Chronic indirect stimulation of fast twitch rabbit muscle (tibialis anterior and extensor digitorum longus) with a frequency of 10 Hz induced a progressive transformation of the sarcoplasmic reticulum (SR). Ultrastructural changes as studied by electron microscopy of freeze-fractured vesicles consisted in a decrease of intramembranous particles of the concave (A) face and an increase of particles in the convex (B) face. The asymmetry of the membrane proved to be lowered. Changes in the particle density of theA face were mainly confined to the 7–9 nm particles. Electrophoretic analyses revealed a decrease in the 115,000-M _r Ca²⁺ transport ATPase. The reduced density of the 7–9 nm particles correlated well with decreased activities in Ca²⁺-dependent ATPase as well as with decreases in initial and maximum Ca²⁺ uptake.     

Three-dimensional ultrastructure of anionic sites of the glomerular basement membrane by a quick-freezing and deep-etching method using a cationic tracer

Summary The ultrastructure of anionic sites in the lamina rara externa (LRE) of rat glomerular basement membrane (GBM) was studied in three dimensions by a quick-freezing and deep-etching method using polyethyleneimine (PEI) as a cationic tracer. Results were compared with those obtained with conventional ultrathin sections examined by transmission electron microscopy. Examination with the quick-freezing and deep-etching method was done without (group 1) or with (group 2) contrasting/fixation with a phosphotungstic acid and glutaraldehyde mixture and post-fixation with osmium tetroxide, which were necessary for visualization of PEI particles by conventional ultrathin sections. Using the quick-freezing and deep-etching method without following contrasting/fixation and post-fixation (group 1), many PEI particles were observed to decorate around fibrils, which radiated perpendicularly from the lamina densa to connect with the podocyte cell membrane. The arrangement of PEI particles was not as regular as that previously reported using conventional ultrathin sections. In contrast, the tissue that was studied with quick-freezing and deep-etching followed by contrasting/fixation and post-fixation (group 2) showed a shrunken appearance. The arrangement of PEI particles was regular (about 20 particles/1000 nm of LRE) as that previously observed using conventional ultrathin sections. However, the number of PEI particles on the LRE was markedly decreased and interruption of decorated fibrils was prominent, as compared with group 1. Ultrastructural examination using conventional ultrathin sections with contrasting/fixation and post-fixation (group 3) demonstrated PEI particles on the LRE in reasonable amounts (18–21 particles/1000 nm of LRE) with fairly regular interspacing (45–65 nm) as reported previously.This is the first report to identify the three-dimensional ultrastructure of anionic sites of GBM, and provides new information on the location and distribution of anionic sites in the glomerular capillary wall. In addition, these studies suggest that several chemical procedures used in conventional transmission electron microscopy to visualize PEI tracers, may produce structural changes and disarrangement of PEI particles that can be avoided with the quick-freezing and deep-etching method.     

Ultrastructure of cell wall and thylakoid membranes of the thermophilic cyanobacterium Synechococcus lividus under the influence of temperature shifts

The ultrastructure of the cell wall and the thylakoid membranes of the thermophilic cyanobacterium Synechococcus lividus was studied by freezefracture electron microscopy after temperature shifts. Different fracture faces of the outer, the cytoplasmic and the thylakoid membranes were demonstrated when the preparation-temperature was in the range of the optimal growth temperature at 52°C or after fixation at 52°C. In the outer membrane of the cell wall two fracture faces with holes and 7.5 nm intramembrane particles were detected. On both the outer (EF) and inner (PF) leaflet of the cytoplasmic membrane randomly distributed particles were demonstrated. The particle density on the PF-face was approx. three times that of the EF-face. The EF-face of the thylakoid membrane exposed rows of particles with an average diameter of 10 nm. The spacing between the particle rows was 35–50 nm. This regular particle arrangement on the EF-face was demonstrated only in a few cases. Mostly the intramembrane particles were distributed randomly on the thylakoid fracture faces. The particle density of thylakoids with a random distribution was approx. in the same range both on the EF-and PF-face. The EF-particles fall into four groups of 9,10,11, and 12.5 nm. The main particle class was the 10 nm class. The PF-face exposed smaller particles with two maxima at 8.5–9 nm and 10 nm. When Synechococcus lividus OH-53s was chilled to temperatures below 30–35°C before the freeze-etch preparation a phase transition took place after the temperature shift. On the fracture faces of the thylakoid and cytoplasmic membranes particle depleted areas occurred. The size of the areas were different in both membranes and dependent on the velocity of cooling. Contrary to Synechococcus lividus OH-53s in the mesophilic Synechococcus strain 6910 the phase transition point was 15°C. The lower phase transition point may be due to a higher content of unsaturated fatty acids.Dedicated to Prof. D. Peters (Hamburg) on the occasion of the 65th anniversary of his birthday     

Chromosome organization revealed upon the decondensation of telophase chromosomes inAllium

Chromosomes of root tip cells ofAllium cepa andAllium sativum were studied in early, middle and late telophase to examine the organization of mitotic chromosomes, taking advantage of the naturally occurring chromosome dispersion during the process of decondensation in telophase. Longitudinal and transverse sections of telophase chromosomes viewed under the transmission electron microscope showed that mitotic chromosomes inAllium were composed of helically coiled 400–550 nm chromatin fibres. In some regions of the longitudinal sections, these chromatin fibres were seen to be orientated parallel to one another but formed roughly a right angle to the long axis of the chromosome. In transverse sections, the telophase chromosome appeared to have a hollow centre encircled by the 400–550 nm chromatin fibre which in turn was a hollow tube structure formed by the coiling of a thinner fibre of 170–200 nm. In addition, cross views of chromatin fibres of 170–200 nm and 50–70 nm were also identified in telophase chromosome preparations. These two organizational levels of chromatin fibres also showed a hollow centre. The process of decondensation of telophase chromosomes is described, and some morphological characteristics associated with the activities of chromosome decondensation are analysed. Based on the observations made onAllium chromosomes in this study, various models of chromosome organization are discussed.     

Formation of the plasma membrane during regeneration ofParamecium caudatum

Summary Fragments ofParamecium caudatum cells obtained by merotomy were fixed in 1% OsO₄ within 5 seconds after cutting. The ultrastructure of the damaged area of the fragment was studied in oriented ultrathin sections and by scanning electron microscopy. The cytoplasm exposed by merotomy was covered during a few seconds with a new membrane. This was a typical trilaminar membrane continuous with the plasma membrane covering the undamaged surface of the cell. The surface over the wound was wrinkled into irregular grooves and ridges. The cytoplasm, mitochondria and trichocysts in the injured region were electron translucent. The cytoplasm under the new membrane contained an unusually high amount of small membrane vesicles, 20–90 nm in diameter. These were probably the remnants of subpellicular alveoli and the plasma membrane destroyed by microsectioning. The possibility that the exposed cytoplasm would be covered by mere shifting of the existing plasma membrane can be excluded. The complex structure of the cortex with its subpellicular alveoli and regularly distributed cilia provide a strong argument against this notion. It seems probable that the new membrane was built up from the available molecular material,e.g., phospholipids and proteins present in the cytoplasm. Fragments of the membrane and alveolar membranes in the form of small vesicles may have also been included into the new membrane.     

Localization of gastrin-releasing peptide (GRP)-like immunoreactivity in the lysosomal compartment of the trigeminal ganglion cells of the rat

Summary Cellular and subcellular localizations of gastrin-releasing peptide-like immunoreactivity (GRP-LI) were examined in the perikarya of trigeminal ganglion cells. By immunolight microscopy using semi-thin sections, GRP-LI was observed in almost all the neuronal somata with various intensity as granular profiles distributing widely in the cytoplasm. By immunoelectron microscopy using ultrathin frozen sections and protein A-gold, GRP-LI was found predominantly in rounded or oval membrane-bound structures which were 300–800 nm in diameter and displayed various electron-density and heterogenous contents. Double-labeling immunoelectron microscopy using antibodies for GRP and cathepsin L, a well-characterized lyosomal proteinase, clearly demonstrated that these GRP-immunoreactive intracytoplasmic structures were lysosomes. In contrast, GRP-LI was detected only occasionally in the large granular vesicles (100–200 nm in diameter). These findings strongly suggest that considerable amount of GRP or GRP-like peptide is subject to intracellular degradation in the lysosome rather than to the regulatory secretion pathway, and this is the reason why no fibers immunoreactive for GRP have been detected in the peripheral sensory field.