A COMPARATIVE STUDY ON THE FINE STRUCTURE OF THE BASALAR MUSCLE OF THE WING AND THE TIBIAL EXTENSOR MUSCLE OF THE LEG OF THE LEPIDOPTERAN ACHALARUS LYCIADES

Basalar and tibial extensor muscle fibers of Achalarus lyciades were examined with light and electron microscopes. Basalar muscle fibers are 100–150 µ in diameter. T-system membranes and sarcoplasmic reticulum make triadic contacts midway between Z lines and the middle of each sarcomere. The sarcoplasmic reticulum is characterized by a transverse element situated among myofilaments halfway between Z lines in every sarcomere. The morphology of Z lines, hexagonal packing of thin and thick myofilaments, and thin/thick myofilament ratios are similar to those of fast-acting insect muscles. Tibial extensor muscle fibers are 50–100 µ in diameter. Except for a lack of the transverse element, the T system and sarcoplasmic reticulum are similar to those of basalar muscle. Wavy Z lines, lack of a hexagonal packing of myofilaments, and larger thin/thick myofilament ratios are similar to those of other postural muscles of insects. The morphology of basalar and tibial extensor muscle is compared to that of other insect muscle with known functions, and reference is made to the possible contribution of the transverse element of sarcoplasmic reticulum in basalar flight muscle to speed and synchrony in this muscle.     

Fine structure of smooth muscle and neuromuscular junctions in the optic tentacles of Helix aspersa and Limax flavus

Summary The smooth muscle cells studied contain a central core of thick and thin myofilaments surrounded by a peripheral layer of myofilament-free cytoplasm. Numerous vesicles, tubules, microfilaments, mitochondria and fine granules are present in the peripheral cytoplasm. Glycogen particles are distributed in large or small groups in both the peripheral cytoplasm and among the myofilaments. In contracted muscle cells the peripheral cytoplasm bulges out at regular intervals into the intercellular connective tissue. Numerous close contacts between single, usually naked, axons and these cytoplasmic protrusions occur. The axons at these contacts contain numerous small (500 Å in diameter) and large vesicles (800–1000 Å in diameter). Sometimes a number of axons simultaneously form close contacts with a muscle cell. These close contacts are considered to be the sites at which transmitter is released and acts on the muscle cell membrane.I wish to thank Professor G. Burnstock for making laboratory facilities available. This work has been supported by the Australian Research Grants Committee.     

A comparative electron microscope study of visceral muscle fibers in Cambarus,Drosophila and Lumbricus

Summary The arrangement of myofilaments in the striated visceral muscle fibers of two arthropods (crayfish and fruitfly) and in the unstriated visceral fibers of one annelid (earthworm) was studied comparatively. Transverse sections through the A bands of arthropod visceral fibers indicate that each thick myofilament is surrounded by approximately 12 thin filaments. The myofilaments are less organized in the visceral fibers of the earthworm than in muscle fibers of the crayfish and fruitfly. The thick myofilaments of the earthworm are composed of subunits, 20–30 Å in diameter. The presence of two distinct sets of myofilaments in these slowly contracting striated and unstriated visceral muscle fibers suggests that contraction is accomplished via a sliding filament mechanism.In crayfish visceral fibers the sarcolemma invaginates at irregular intervals to form a long and unbranched tubular system at any level in the sarcomere. Dyads formed by the apposition of T and SR membranes are observed frequently. The distribution of the T and SR systems in the visceral fibers of the fruitfly and the earthworm is markedly reduced and dyads are infrequently observed. The reduced T and SR systems may be related to the slow contraction of these fibers. Transport of specific substances across the sarcolemma could initiate contraction or relaxation in these fibers.This study was supported by a training grant GM-00582-06 from the U.S. Public Health Service.     

Fine structure of the autonomic nerves of the rabbit myometrium

Summary The fine structure of the preterminal nerve fibers of the rabbit myometrial smooth muscle was studied using potassium permanganate fixation or glutaraldehyde fixation with postosmification. The preterminal fibers were mostly formed by 2–10 axons enveloped by Schwann cells. Two kinds of axons and axon terminals were found. (1) Adrenergic axons, which contained many small, granular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å) which represented ca. 2% of the total count of the vesicles. (2) Nonadrenergic axons, which contained small agranular vesicles (diameter 300–600 Å) and large granular vesicles (diameter 700–1200 Å). Both types of axons formed preterminal varicosities along their course. The real terminal varicosities, representing the anatomical end of the axons, were usually larger than the preterminal ones and showed close contact to the plasma membranes of the smooth muscle cells. Both adrenergic and nonadrenergic terminals were found close to the smooth muscle cells, but a gap of at least 2000 Å was always present between the two cell membranes. The axons and preterminal varicosities of both types of nerves were in intimate contact with each other within the preterminal nerve fiber. Axo-axonal interactions between the two types of axons are possible in the rabbit myometrium. The relative proportion of the nonadrenergic axons from the total was about one fourth.     

A neurofibrillar body in the perikaryon of nervus terminalis ganglion cells in teleosts

Summary In three species of Teleosts (Tinea tinea L., Leuciscus cephalus cabeda Risso, Epinephelus guaza L.) a round strongly argentophilic body of considerable size occurs in the cytoplasm of the nervus terminalis ganglion cells. In Tinea, surgical interruption of functional connections of the ganglion cells does not produce any apparent change either in the number or in the size of these cytoplasmic bodies.Electron microscopical observations show that the neurofibrillar body is made up of densely packed and irregularly arranged bundles. In cross section, each of these bundles appears to be composed of neurofilaments (100 Å in thickness) and neurotubules (diameter: 300 Å). Each tubule is surrounded by 9–10 filaments equi-distant from one another, and at a distance of 30–40 Å from the central tubule.The authors are indebted to Prof. G. Palladini for helpful histochemical advice, to Prof. B. Bertolini for electron micrographs and to Mr. D. Scorsini for skilful technical assistance.     

The fine structure of the kinetochore of a mammalian cell in vitro

The chromosomes of Chinese hamster cells were examined with the electron microscope and the following observations were made concerning the structure and organization of the kinetochore. — The kinetochore consists of a dense core 200–300 Å in diameter surrounded hy a less dense zone 200–600 Å wide. The dense core consists of a pair of axial fibrils 50–80 Å in diameter which may be coiled together in a cohelical manner. The less dense zone about the axial elements is composed of numerous microfibrils which loop out at right angles to the axial fibrils. Together the structures comprise a lampbrush-like filament which extends along the surface of each chromatid. Some sections suggested that two such filaments may be present on each chromatid. The fine structure of kinetochores associated with spindle filaments was essentially the same as those free of filaments. The structure and organization of the kinetochore of these mammalian cells was compared to that of lampbrush chromosomes of certain amphibian oöcytes, dipteran polytene chromosome puffs, and the synaptinemal complex seen during meiotic prophase.The authors also wish to thank Dr. Arthur Cole of the Department of Physics for the use of his electron microscope facilities and for his helpful criticism.     

The fine structure of muscle in a marine turbellarian

Summary The fine structure of the myofibers of Notoplana acticola as studied by electron microscopy indicates that they are composed of thick myofilaments about 200 Å wide with tapering ends and thin myofilaments about 50 Å wide, arranged alongside each other parallel to the long axis of the cell. There is no orderly transverse arrangement of filaments; instead they appear staggered in the fiber. In cross sections 6 to 10 thin filaments form an orbit around one thick filament with possible cross-linkage between the two types of filaments.Dense bodies are associated with the sarcolemma and with the sarcoplasmic reticulum, and appear to serve as attachments for the thin filaments. Dense bodies are compared to elements forming a fragmented Z-disc.Mitochondria, situated in the periphery or the center of fibers, are associated with granules interpreted as glycogen.The sarcoplasmic reticulum consists of: sacs or cisternae in close proximity to the sarcolemma, longitudinal tubular elements between and parallel to the myofilaments, and a tubular network around the filaments. There is no well-defined sarcolemmal-derived transverse tubule system as described in striated muscles. It is hypothesized that in these muscles, the functional equivalent of the T system may be the area of sarcolemma in contact with the cisternae of the sarcoplasmic reticulum.This work was supported by Grant No. GM 10292 from the U. S. Public Health Service to Professor Richard M. Eakin, Department of Zoology at the University of California, Berkeley, USA, where this investigation was conducted during the author's sabbatical leave of absence from the University of Illinois.I wish to thank Professor Eakin for valuable discussions and for his kind hospitality in extending the facilities of his laboratory and the use of the electron microscope to me, and the John Simon Guggenheim Memorial Foundation for the Fellowship which I held during 1964–65.     

Fine structure of smooth muscle and neuromuscular junctions in the foot of Helix aspersa

Summary The smooth muscle cells in the foot of Helix aspersa are arranged in bundles which interweave to form a complex mesh. In the peripheral cytoplasm of the muscle cells there is a system of interconnected obliquely and longitudinally orientated tubules. The full extent of this system has not been determined; its possible function in relation to Ca⁺⁺ storage and excitation-contraction coupling is discussed. Longitudinal tubules are present among the myofilaments and in association with mitochondria. Distributed throughout the myofilaments are elliptically shaped dense bodies, the fine structure of which resembles an accumulation of thin filaments. Located on the plasma membrane of the muscle cells are dense areas; the fine structure and relationships of these cellular elements resemble desmosomes. They may serve as attachment points for thin, cytoplasmic filaments (not necessarily myofilaments). The muscle cells are innervated by axons which diverge from a coarse, neural plexus (the sole plexus). The axons initially come into close contact with the muscle cells and then pass over their surfaces for up to 35 before being gradually enveloped by flange-like protrusions of the muscle cells. These axons contain either, (i) agranular vesicles (600 Å in diameter), (ii) agranular and very dense granular vesicles (1000 Å in diameter) or (iii) agranular and less dense, granular vesicles (1000 Å in diameter). The possible role of these inclusions as sites of excitatory and inhibitory transmitters is discussed.I wish to thank Professor G. Burnstock for making laboratory facilities available. This work has been supported by the Australian Research Grants Committee.     

The ultrastructure of organelles appearing in spermatids and nutritive cells of Cipangopaludina malleata

Summary The ultrastructure of organelles appearing in the early typical and atypical spermatids, and the nutritive cells of Cipangopaludina malleata has been examined by a Siemens' electron microscope Elmiskop I.Mitochondria appearing in the early typical spermatid have doughnut-like profiles in which the internal ridges appear as triple-layered membranes arranged radially and extending into the interior of the organelle without reaching the other side. Each membrane 40–60 Å in width, separated by a clear interspace 60–90 Å wide, is characterized by a porous structure 20–30 Å in diameter which suggests a filtration apparatus for enzymes.Walls of the flattened saccules consisting the Golgi apparatus are calculated 35–60 Å thick, in which an electron-lucent, porous structure about 30 Å wide has been revealed.The smooth-surfaced endoplasmic reticulum is bordered by a triple-layered membrane consisting of two opaque layers with a less opaque interspace 20–30 Å wide. The outer membrane ca. 15 Å wide presents a more linear appearance than the dotted arrangement of the inner membrane 20–25 Å thick.The plasma membrane is composed of a triple-layered structure where two dense lines 15 Å wide are separated by a layer 20–30 Å thick of less density.The electron micrographs for the present studies were taken with the Siemens electron microscope, model Elmiskop I, at the Anatomical Institute of Kiel University, Germany. The one of the authors, G. Yasuzumi is deeply grateful to Prof. Dr. W. Bargmann and Dr. A. Knoop for the privilege of using this instrument and other equipments in the Laboratory.     

The ultrastructural effects of triparanol on rat atrial muscle

Summary The atrial musculature of rats given the cholesterol inhibitor triparanol (MER/29) (250 mg/kg daily) for 8 days was examined under the electron microscope and compared with that from untreated animals. The sarcoplasmic core of muscle fibers from animals given triparanol exhibited a new formation of sarcoplasmic granules which displayed a crystalline latticework with opaque lines approximately 40–60 Å separated by clear spaces 50–70 Å. They were partially or completely surrounded by a membrane. The crystalline bodies in cardiac muscle fibers were not as numerous as those observed in adrenocortical, testicular interstitial, or luteal cells as reported earlier by the investigators.This research was supported by USPHS Grants HE 12751, NS 05665, and 00690.Recipient of Career Research Development Award 1 K 3 GM 28064.     

Ultrastructure of the neurohaemal region of the toad median eminence

Summary According to the internal structure and size of the granules, six types of nerve endings can be distinguished in the toad median eminence: 1. Endings containing mostly dense granules of 600 Å in diameter; 2. Endings containing dense granules of about 800 Å in diameter; 3. Endings which contain dense granules 1,000–2,000 Å in diameter, with the peak at 1,200–1,400 Å; 4. Endings containing granules with a characteristic structure, which differentiate them from the other three types; 5. Scarce endings containing granules 2,000 to 3,800 Å in diameter; and 6. Endings containing only vesicles 400–500 Å in diameter. Types 3 and 4 endings are mainly found in the outer pericapillary zone, and are probably responsible for the strong Gomori-positive reaction observed in this zone. The other four types of endings occur mainly in the inner pericapillary zone, and appear to be Gomori-negative.The probable origins of the different types of endings, and their possible relations with the different releasing factors is discussed.The subendothelial basement membrane has numerous long processes which form a complicated network in contact with all the nerve endings, some nerve fibres and glial cells.Two types of glial cells are described. Pinocytotic vesicles are frequently seen at the points where these cells contact the basement membrane. All the ultrastructural features suggest that these cells are carrying out transport functions.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina.The author is very grateful to Professor H. Heller for his continued encouragement and criticism and to Mr. J. Lane and Mr. P. Heap for their valuable help.     

The fine structure of neuromuscular junctions and contact zones between body wall muscle cells of Ascaris lumbricoides (var. suum)

Summary Neuromuscular junctions and close membrane apposition between body wall muscle cells of Ascaris lumbricoides (var. suum) have been examined with the light and electron microscopes. It was found that the body wall muscle cells send out elongate processes from their basal, myofibril containing portion to terminate on dorsal and ventral nerves. When observed with the aid of the electron microscope the neuromuscular junctions were seen to consist of several muscle cell processes in apposition to a single axon. The intersynaptic cleft was approximately 350–500 Å wide. Both the axolemma and sarcolemma were triple layered membranes which were 75–80 Å thick. Electron dense patches were observed at intervals on the apposed membranes which were due to increased thickness of the inner membrane leaflets of axolemma and sarcolemma. Muscle cell membranes, at the level of the neuromuscular junction, were in close apposition resulting in an apparently five-layered membrane complex which was 170–210 Å thick. The sarcolemmata in these regions were separated by 10–50 Å. Presynaptic axons contained mitochondria, microtubules which were 180–270 Å in diameter, and two, morphologically distinct types and sizes of synaptic vesicles. One was 200–600 Å in diameter, with a single, triple-layered membrane bounding a center of low electron density. The other was 600–1200 Å in diameter, with a single, triple-layered membrane bounding a central, electron dense granule of 500–800 Å size.The functional significances of the close membrane appositions between body wall muscle cells and of the two types of synaptic vesicles found at the neuromuscular junctions of Ascaris lumbricoides were discussed with respect to their possible role in neuromuscular physiology.Supported by U.S.P.H.S. Grant No. NB-01528 and Research Career Development Award No. 9-K3-NB-15255. — The author wishes to express his grateful appreciation for the excellent technical assistance given by Miss Gabrielle Rouiller during the course of this investigation.     

Chromosome fibers studied by a spreading technique

Chromosomes and interphase nuclei can be spread on the surface of water in a simplified Langmuir trough. Interphase nuclei of Triturus erythrocytes display fibers with a diameter of about 250–300 Å. Very similar fibers are seen in metaphase chromosomes of cultured human cells. Fibers from grasshopper spermatocyte chromosomes (prophase) are more variable in diameter, and many fibers thinner than 200 Å extend laterally from the chromosome. In the grasshopper spermatocyte, fibers align in parallel to form plates. It is suggested that the 250–300 Å fibers may represent an inactive state of the chromosome material, and that only the thinner fibers are involved in RNA synthesis. The 250–300 Å fibers may result from the folding or coiling of a thinner fiber having the approximate dimensions of the nucleohistone molecule.     

Ultrastructure of the human submaxillary gland

Summary Myoepithelial cells in the human submaxillary gland are stellate in form, with long, tapering processes. They are interposed between the base of the secretory cells and the basement membrane, and are bound to the secretory cells by desmosomes. Their cytoplasm contains numerous myofilaments measuring approximately 40 Å in diameter, which frequently aggregate to form structures similar to the dark bodies seen in smooth muscle cells. The myofilaments are anchored to the plasma membrane by attachment devices. The myoepithelial cells are often accompanied by cells which have a similar shape, but possess an extremely electron-lucent cytoplasm that contains almost no organelles or inclusions. Electron microscopical observations indicate that these cells, termed clear cells, are transformed directly into myoepithelium, since all morphological intergrades between the two cell types have been recognized.This work was supported in part by a grant from the Henry Spenadel Trust. The technical assistance of Mr. Roy R. Keppie and Mrs. Mona Brandreth is gratefully acknowledged.     

Morphology of egg phosphatidylcholine-cholesterol single-bilayer vesicles,studied by freeze-etch electron microscopy

Summary Homogeneous, small, single-bilayer vesicles were prepared from egg phosphatidylcholine with various concentrations of cholesterol by ultrasonic dispersion in 0.1m KCl, 0.01m Tris, pH 8.0, buffer, followed by gel chromatography. The shape and size distributions of the fractionated vesicles were investigated for preparations with cholesterol compositions from 0 to 50 moles/100 moles, using freeze-etch electron microscopy. The size distribution was estimated from the shadow width of vesicles which were exposed by etching and the vesicle shape was checked by comparing the images obtained by tilting the replicas. The widths of the vesicle diameter distributions were relatively broad, corresponding to standard deviations in the range 60–90 Å, but showing no systematic variation with cholesterol composition. In all cases it was found that 70% of the vesicle diameters lay within 150 Å of the modal value. The apparent vesicle diameters remained constant for cholesterol compositions up to 20 moles/100 moles (modal diameter=330 ± 20 Å, mean diameter = 350 ± 3 Å), but there was a sharp net increase in diameter at 30 moles cholesterol/100 moles reaching a model diameter of 430 ± 20 Å (mean diameter = 430 ± 3 Å) at 50 moles cholesterol/100 moles. Using the tilted microscope stage it was found that all vesicles were spherical at all cholesterol compositions studied, including those above 30 moles cholesterol/100 moles. The molecular mechanism by which cholesterol controls the vesicle size is discussed in terms of the asymmetric distribution of cholesterol across the vesicle bilayer.     

Electron microscopic identification of sex chromatin bodies of tissue culture cells

Summary A method is described for the unequivocal identification of sex chromatin bodies in electron micrographs of thin sections of tissue culture cells derived from human skin. Fibers, rodlets, and circular profiles having a similar diameter of about 200 Å appear to be the only components of the sex chromatin bodies. The fibers and rodlets are sometimes resolvable into two similar subunits and the circular profiles often have a less electron dense center. The overall density of sex chromatin bodies is found to be considerably less than that of metaphase chromosomes. The fibers of the sex chromatin bodies found lying away from the nuclear periphery appear to be organized more compactly than the fibers of the sex chromatin bodies found lying at the nuclear periphery.This paper is based on investigations supported by a research grant No. GM-04738 from the National Institutes of Health, Public Health Service, to Dr. H. Ris, Department of Zoology, University of Wisconsin.     

The structure of a mesokaryote chromosome

Condensed and dispersed forms of the chromosomes of the dinoflagellate, Prorocentrum micans, deposited on grids by the microcentrifugation technique were studied by electron microscopy. In the normally condensed form, the chromosomes appear as banded rods surrounded by a peripheral cloud of partially dispersed fibers. Single fibers in these and in extensively dispersed preparations appear as smooth threads of uniform diameter (55–65 Å). The chromosome fibers are contrasted by positive-group-specific stains, indicating the presence of cationic moieties associated with the DNA. Occasionally Y-shaped chromosomes are seen; these may be replicating structures. These observations are in general agreement with studies of dinoflagellate chromosomes by other techniques, and provide support for the suggestion that these organisms possess a genome organization whose structure is typical of neither prokaryotes nor eukaryotes, and hence may be intermediate forms.     

Vesicle and granule content of sympathetic ganglion cells during limb regeneration of the newt Triturus

Summary Fine structural observations were made on the vesicle and granule content of ganglion cells in the posterior subclavian ganglion and peripheral nerve fibers of the upper forelimb of the newt Triturus. The populations of vesicles and granules in normal ganglion cells and nerve fibers were compared with those observed after limb transection. In normal neurons, clear vesicles range in size from 250 to 1000 Å in diameter, but are most frequently 400–500 Å. Vesicles with dense contents (granules) also vary greatly in size, but most are 450–550 Å in diameter and correspond to dense-core vesicles. Large granules that contain acid phosphatase activity are thought to be lysosomes. During limb regeneration, in both the ganglion cells and peripheral nerves, the ratio of dense vesicles to clear vesicles increases. There is a large increase in number of dense granules with a diameter over 800 Å, particularly in the peripheral regenerating fibers. This study shows that regenerating neurons differ from normal in their content of vesicular structures, especially large, membrane-bounded granules.This work was supported by grants from the National Science Foundation (GB 7912) and from the National Cancer Institute (TICA-5055), National Institutes of Health, United States Public Health Service.     

CORRELATION BETWEEN FIBER LENGTH, ULTRASTRUCTURE, AND THE LENGTH-TENSION RELATIONSHIP OF MAMMALIAN SMOOTH MUSCLE

The length-tension relationship was determined for strips of guinea pig taenia coli and correlated with the length and ultrastructural organization of the component fibers. The mean fiber length in "stretched" strips (passive ≥ active tension) was 30% greater than that for fibers in "unstretched" strips (active >> passive tension). In stretched fibers the dense bodies and 100 A diameter myofilaments were consolidated into a mass near the center of fibers in cross-sectional profile. The thick myofilaments were segregated into the periphery of the fiber profiles. In unstretched fibers the dense bodies-100 A diameter filaments and the thick myofilaments were uniformly distributed throughout cross-sectional profiles. A tentative model is proposed to account for the change in fiber length and ultrastructural organization that accompanies stretch. The basic features of the model require the dense bodies to be linked together into a network by the 100 A diameter filaments. The functional consequences of stretching the fibers are discussed in relation to the model proposed for this network.     

Intracytoplasmic filaments in pulmonary lymphatic endothelial cells

Summary The cytochemistry and ultrastructure of intracytoplasmic filaments of pulmonary lymphatic endothelial cells of neonatal rabbits were studied by comparison with myofilaments of the peribronchial and pulmonary vascular smooth muscle cells. Two types of endothelial filaments were observed: thin filaments (diameter: 50 Å) which lie close to the abluminal cell membrane; and thick filaments (diameter: 90 Å) which are dispersed throughout the cell cytoplasm.Following heavy meromyosin (HMM) treatment, characteristic arrowhead complexes formed in the thin lymphatic endothelial filaments as well as in the actin filaments of the smooth muscle cells. There was no detectable reaction of HMM with the thick filaments.After incubation with EDTA, the thin filaments were labile, and the thick filaments became the major filamentous component in the endothelial cells. In smooth muscle cells, the actin myofilaments were also labile while the 100 Å filaments were stable.These observations support the hypothesis that the actin-like thin endothelial lymphatic filaments form part of a contractile system, while the thick filaments constitute a plastic cell skeleton. The significance of the contractile system in lymphatic endothelial cells might lie in a mechanism for the active regulation of the endothelial intercellular junctions and gaps and hence the permeability of the lymphatic endothelial cell lining.This study was supported by The Council for Tobacco Research—U.S.A. The authors thank Professor Robert C. Rosan, M.D. (Saint-Louis University—U.S.A.) for expert advice. R. Renwart, B. Emanuel and R. Jullet for technical, G. Pison and St. Ons for photographical and N. Tyberghien for secretarial assistance.