The contractile ring

Summary Techniques of individual cell selection and precise ultramicrotomy have been employed to demonstrate that the contractile ring of cleaving HeLa cells is a transitory cytoplasmic organelle of distinctive fine structure and location. The contractile ring is an uninterrupted annulus encircling the equator of dividing cells exactly where the cleavage furrow forms. It is about 10 microns wide, up to 0.2 microns in thickness, and is composed exclusively of circumferentially aligned thin filaments 40–70 Å in diameter. Contractile ring filaments appear to be associated with the overlying plasma membrane.Controlled experiments with a mold metabolite (cytochalasin B) reveals that within a few minutes the drug abolishes the ability of HeLa cells to undergo cytokinesis. Cytochalasin B seems to decompose the contractile ring. It has no other clearly identifiable effects on other cell structures, notably the mitotic apparatus. Cytochalasin B is the only drug known which selectively inhibits cytokinesis in animal cells.In conclusion, the contractile ring is the most likely organelle responsible for cytokinesis in HeLa cells. Similar organelles probably occur in all cleaving animal cells. Successful cleavage depends upon the structural and functional integrity of the contractile ring.     

Ultrastructural observations on membranous structures in developing mouse oocytes

Summary Ultrastructural studies have revealed the presence of unusual membrane complexes within developing mouse oocytes. These structures, most obvious 18 days post fertilization, are found in the nucleus or cytoplasm of cells in meiotic prophase. The complexes, usually found in small groups, are characterized by a slightly bowed appearance, and a thin middle section that is vesiculated at each end. At high magnification the middle section exhibits a pentalaminar structure similar to tight junctional complexes, while the looped membranes of the vesiculated ends are trilaminar in appearance. In addition to being free in the nucleoplasm or cytoplasm, the complexes are also seen in continuity with the inner and outer leaflets of the nuclear envelope, and with typical membranes forming cytoplasmic tubular systems. The possible formation of these complexes from blebs or vesicles derived from the nuclear envelope is presented and the role that these structures may play in developing oocytes is discussed.Supported by Louisiana State University Medical School Institutional Grant.Dr. Skalko's current address is Birth Defects Institute, New York State Health Department, Albany, New York. The authors wish to thank Mr. Garbis Kerimian for his excellent photographic work, Mrs. Janell Buck, Mrs. Edna Burgess and Mrs. Eunice Schwartz for their excellent technical and secretarial assistance.     

Structure and behavior of contractile vacuoles inChlamydomonas reinhardtii

Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.Abbrevations CV contractile vacuole - PM plasma membrane     

Immunocytochemistry of contractile and cytoskeletal proteins in smooth muscle: Lowicryl,LR White,and polyvinylalcohol compared

Summary Three embedding media have been compared with respect to post-embedding immunolabeling of contractile and cytoskeletal antigens in aldehyde-fixed smooth muscle tissue: the methacrylate derivates lowicryl K4M (cured at –35 or 60°C) and LR White (cured at 0 or 60°C) and the water soluble resin, polyvinylalcohol (dried at 60°C). Measurements of intensity of labeling of ultrathin sections in the fluorescence microscope showed that five antigens (actin, myosin light chain, tropomyosin, filamin and vinculin) reacted more or less equally with their respective antibodies in all the embedding media, including those cured at 60°C. One antibody (anti-light meromyosin) reacted well only with polyvinylalcohol-embedded tissue. In contrast to the relative invariance of antibody reactivity between media clear differences in the preservation of ultrastructural integrity were observed. Embedding in polyvinylalcohol (dried at 60°C) and in Lowicryl (cured at –35°C) resulted in superior preservation as compared to Lowicryl or LR White cured at 60°C. Examples of uitrastructural immunocytochemistry with the antibodies against filamin and myosin light chain, using the immunogold staining procedure are presented: the sites of localization by these antibodies were the same with all the media tried. The relative merits of the different methods are discussed.Abbreviations EGTA Ethyleneglycol-bis(-amino ethyl ether)N,N,N,N-tetra acetic acid - PIPES 1,4-Piperazinediethanesulfonic acid - LR London Resin     

Ultrastructural observations on the pars descendens of the proximal tubule in the kidney of the male rat

Summary The pars descendens (pars recta) of the proximal tubule in the male rat kidney, consisting of the terminal part of the second proximal segment (P₂) and of the third proximal segment (P₃), was studied with the electron microscope. A technique of tissue orientation and trimming was used which permitted precise topographic definition of the tubules studied in the electron microscope. The terminal descending part of the P₂ showed some minor differences from the convoluted part of this segment, and ultrastructure also changed along the course of the P₃. In the beginning of the latter segment numerous, shallow interdigitations were observed between adjacent cells; along the course of the segment they decreased in number or disappeared. In the initial part of the P₃ mitochondria were more abundant than in the terminal portion of the segment and at least as numerous as in the straight part of the P₂. Also, the dense, acid phosphatase-positive cytoplasmic bodies decreased somewhat in size along the course of the P₃. The smooth surfaced endoplasmic reticulum reached a higher development in the P₃ than anywhere else in the proximal tubules.Investigation supported by grants from: Fonden til Lægevidenskabens Fremme and the Danish Medical Research Council. — The authors are indebted to Mrs. J. Barslund and Mrs. M. Jacobsen for excellent technical assistance.     

Surface multivesicular structures associated with maturing erythrocytes in rats

Summary Surface multivesicular structures associated with the plasmalemma of erythrocytes were observed in the peripheral blood of rats which have a significant number of circulating reticulocytes. These surface structures appear as ovoid evaginations (0.2 to 0.7 in diameter) of the plasma membrane and contain numerous small vesicles ranging from 0.05 to 0.1 in diameter. The structures were present during the final stages of maturation of erythrocytes, after nuclei and mitochondria had been extruded and only a few polysomes and small vesicles remained. They appear quite distinct from the autophagic vacuoles which have been described in association with degeneration and extrusion of mitochondria from erythrocytes. The exact origin of the small internal vesicles of these surface multivesicular structures is unknown; however, similar vesicles have been observed in the cytoplasm of the maturing erythrocyte especially in the vicinity of the Golgi body. These structures suggest a process by which Golgi elements and other small cytoplasmic vesicles are extruded during the late stages of maturation of rat erythrocytes.Supported by U.S. Public Health Service Research Grant AM 12950.The author is indebted to Dr. Edward G. Rennels and Dr. William B. Winborn for their guidance.     

Osmoregulatory mutants that affect the function of the contractile vacuole inChlamydomonas reinhardtii

Summary Four independent osmoregulatory mutants,osml, osm3,osm4, and osm7, were isolated on the basis of their requirement for growth medium of high osmotic strength. In normal low-osmoticstrength medium, in contrast to wild-type cells, the mutants grow poorly or not at all; in distilled water mutant cells are immobilized and eventually swell and burst. The mutants were examined by ordinary brightfield and phase-contrast microscopy, videomicroscopy, and electron microscopy. The four mutants showed different defects in the contractile vacuole (CV) cycle. Timing of various stages of the CV cycle showed thatosm1 was affected primarily in the early stage of the cycle when the CV begins to grow,osm3 primarily in midcycle when vacuoles fuse to form the CV proper,osm7 at a late stage of the cycle at docking and fusion of the CV with the plasma membrane, andosm4 during contraction of the CV. At the electron microscopic level, in dilute medium, mutant cells by comparison with wild-type cells had large autophagosomes, swollen mitochondria, and dilated ER cisternae. Although electron microscopy showed general abnormalities of the contractile vacuoles consistent with the videomicroscopic observations of living cells, no obvious vacuole membrane abnormalities were seen which would explain the mutational defects. The mutations help define the separate processes that contribute to the coordinated CV cycle inChlamydomonas, and open the way to eventual isolation of some of the genes responsible for CV function.Abbreviations CV contractile vacuole - TAP Tris-acetate-phosphate medium - TAP+L medium supplemented with lactose - TAP+S medium supplemented with sucrose or other sugar     

Ultrastructural alterations in skeletal muscle fibers of streptozotocin-diabetic rats

Summary The ultrastructure of fast-twitch-oxidative-glycolytic (FOG), fasttwitch-glycolytic (FG) and slow-twitch-oxidative (SO) fibers in plantaris and soleus muscles of normal and streptozotocin-diabetic rats was studied. In the diabetic animals, the mitochondria of FOG and SO fibers showed a loss of cristae and an increase in electron-dense granules. There was also an increased number of lipid droplets in close proximity to the mitochondria and the nuclei, and a separation of individual muscle nuclei to form satellite cells. Higher incidences of surface projections and sarcoplasmic splittings at the nuclear region were noticed in SO fibers. The FG fibers showed some disorientation of the T-tubular system. It is concluded that streptozotocin-diabetes has differential effects on the fine structure of the three fiber types of rat skeletal muscle.Supported by USPHS Grant AM 18280-04, Boston University Grant GRS-405-BI, and a grant-in-aid award from Sigma Xi Society     

Three Modes of Melanosome Transfers in Caucasian Facial Skin: Hypothesis Based on an Ultrastructural Study

The transfer mechanism of melanosome from the melanocyte into the keratinocyte was investigated in mildly photodamaged Caucasian facial skin by electron microscopy. Three ways of transfer are suggested by our observations. The first mechanism probably occurs through the following process: 1) protrusion and insertion of the thick dendrite of the melanocyte into the basal keratinocyte, 2) formation of sac-dendrite complex in the subnuclear region, 3) digestion and segregation of the enclosed dendrite, 4) formation of the cistern in the paranuclear region, and 5) pinching-off of the melanosomes in single or aggregated form from the tip of the cistern. The second mechanism probably takes place through a membrane fusion between the melanocyte and the keratinocyte. Such a membrane fusion possibly forms a passage way for release of the melanosome from the former cell to the latter. The third mechanism is considered to include exocytosis of the single melanosome from the melanocyte followed by the endocytosis through the formation of coated-pit in the keratinocyte.     

Ultrastructural study of experimental muscle degeneration and regeneration in the adult rat

Summary Methyl-bupivacaine is a local anaesthetic with a selective myotoxic action. A single subcutaneous injection of the drug into the hind leg of adult rats produces a uniform, complete and irreversible destruction of superficial layers of fibres in the underlying extensor digitorum longus muscle. The degeneration of muscle fibres is followed by phagocytosis and a rapid and complete regeneration.The first stage in the regeneration process is the appearance of presumptive myoblasts within the original basement membrane of the sarcolemmal tube. On the second day after injury aggregates of myoblasts are present and fusion is observed between the cells. The myotubes thus formed increase in size by fusing with additional myoblasts. Myotubes are also observed to fuse with one another. On the fifth day after injury the regeneration process has proceeded to the stage of early muscle fibres with fully differentiated myofibrils with typical sarcomere structures. By ten days only mature muscle fibres of about normal size are present and regeneration appears complete.In previously denervated and methyl-bupivacaine treated muscles the stages of regeneration are similar to those observed in innervated muscles, the only apparent difference being a slowing of cell differentiation and incomplete maturation.An electrophysiological study shows that the motor nerve at the third day after injury forms synaptic contacts with regenerating muscle cells. At that stage of myogenesis the myotubes are highly sensitive to applied acetylcholine.1 (1-n-butyl-DL-piperidine-2-carboxylic acid-2,6-dimethyl-anilide-hydrochloride); Marcaine®, manufactured by AB Bofors, Nobel-Pharma, Mölndal, Sweden.The study was carried out under the auspicies of The Czechoslovak Academy of Sciences and the Royal Academy of Sciences in Sweden.     

Ultrastructural study of periodic lamellar granules in human neutrophils

Granules consisting of periodically arranged membranous lamellae and amorphous electron-opaque material, i.e., periodic lamellar granules, are present in human neutrophils. To date, no extensive ultrastructural studies have been carried out on these granules because of their infrequent presence in neutrophils. The bone marrow of 18 cases of chronic myeloproliferative disorders, including one case of chronic neutrophilic leukemia in which periodic lamellar granules were frequently seen in neutrophils, was investigated by electron microscopy. Periodic lamellar granules were seen in neutrophils in 12 of the 18 cases at varying frequencies. They were preferentially seen in immature neutrophils. The transverse profiles of these granules revealed concentric complete/incomplete rings or periodic parallel straight lines, i.e., various patterns of lamellar arrangement were present. Periodic lamellar granules were positive for myeloperoxidase and lysozyme at the electron-microscopic level. These results suggest that these granules represent a primary neutrophil granule subtype. However, their functional and pathologic significance remains unknown.     

Ultrastructural and phylogenetic studies on Blastocystis isolates from cockroaches

Four Blastocystis isolates from cockroaches were established and these isolates were morphologically confirmed as Blastocystis organisms by light and/or electron microscopy. As these isolates were morphologically indistinguishable from Blastocystis isolated from other animals, phylogenetic analyses were conducted using their small subunit ribosomal RNA genes. A analyses of these sequences with previously reported ones that had been classified into nine Blastocystis clades indicated the presence of a new clade that comprised only Blastocystis organisms from cockroaches (clade X). A clade comprised of amphibian and reptilian Blastocystis organisms (clade IX) was located at the basal position of the Blastocystis tree together with the common ancestor of Proteromonas and Protoopalina, clade X emerged after the divergences of these two basal clades and its branching position was clearly supported by bootstrap analysis.     

Ultrastructural study of change from free-nuclear to cellular endosperm in Ipomoea purpurea Roth and Cytinus hypocistis L.

Abstract

The process of cellularization of the endosperm was studied in Ipomoea purpurea Roth and Cytinus hypocistis L. In both the examined species normal cytochinesis, involving the formation of the phragmoplast, characterizes the change from the nuclear to cellular condition. Nevertheless, some ultrastructural aspects of the cell wall initiation seem to indicate that the modality of freely-growing walls cannot be excluded. The hypothesis of a unique method of wall initiation for the endosperm of the nuclear type, formulated by some Authors, is discussed.     

Ultrastructural studies on the secretory process in the epithelioid cells of the juxtaglomerular apparatus

The epithelioid cells of the juxtaglomerular apparatus have been studied with respect to the release mechanism of the secretory granules. Invaginations of the plasma membrane into the interior of the epithelioid cells are interpreted as stages before or after an exocytotic process. Granules are sometimes observed in close contact with the plasma membrane, and material with electron density similar to that of the granules can also be observed in the invaginations. These morphological features suggest that the granular material of the epithelioid cells is extruded into the texture of the basal lamina. Furthermore, a dense network of microtubules and microfilaments is described and the functional role of this system in exocytosis is discussed.     

Ultrastructural characterization of the tegument in protoscoleces of Echinococcus ortleppi

Cystic echinococcosis is a globally distributed zoonosis caused by cestodes of the Echinococcus granulosus sensu lato (s.l.) complex, with Echinococcus ortleppi mainly involved in cattle infection. Protoscoleces show high developmental plasticity, being able to differentiate into either adult worms or metacestodes within definitive or intermediate hosts, respectively. Their outermost cellular layer is called the tegument, which is important in determining the infection outcome through its immunomodulating activities. Herein, we report an in-depth characterization of the tegument of E. ortleppi protoscoleces performed through a combination of scanning and transmission electron microscopy techniques. Using electron tomography, a three-dimensional reconstruction of the tegumental cellular territories was obtained, revealing a novel structure termed the ‘tegumental vesicular body’ (TVB). Vesicle-like structures, possibly involved in endocytic/exocytic routes, were found within the TVB as well as in the parasite glycocalyx, distal cytoplasm and close inner structures. Furthermore, parasite antigens (GST-1 and AgB) were unevenly localised within tegumental structures, with both being detected in vesicles found within the TBV. Finally, the presence of host (bovine) IgG was also assessed, suggesting a possible endocytic route in protoscoleces. Our data forms the basis for a better understanding of E. ortleppi and E. granulosus s.l. structural biology.     

Ultrastructural study of echinocytes induced by poly (ethylene glycol)-cholesterol

Poly (ethylene glycol)-cholesterol (PEG-Chol) consists of a hydrophilic PEG and hydrophobic cholesterol moiety. When PEG-Chol was applied to erythrocytes, the reagent quantitatively induced protrusions by exclusively distributing in the outer monolayer of the membrane. This kind of response has been regarded as a general response that reduces the stress of expansion of the outer monolayer. However, the relationship between the membrane architecture and the distribution of such molecules is unknown. In this study, we examined the distribution of tagged PEG-Chol along the shape change pathway. The echinocytic shape was initiated by the initial formation of bumps on the rim of the discoid, which subsequently elongated as protrusions. These protrusions contained aggregates of granular structures, which appeared to accommodate the increase in the outer monolayer area. At higher concentrations, PEG-Chol further induced sphero-echinocytosis that resulted in numerous branched protrusion processes. We found that PEG-Chol was exclusively distributed in these protrusions and, in particular, accumulated at the tips. These results suggested that externally intercalated PEG-Chol was sequestrated from erythrocytes as membrane protrusions through an as-yet-unknown mechanism.     

Ultrastructural morphometry of blastogenesis

Summary As a companion to an earlier study, the morphometric attributes of stimulated (blast-derived) lymphocytes in mouse axillary lymph nodes have been established using stereological and autoradiographic methods. Blast transformation was induced in vivo with dinitrochlorobenzene (DNCB) and stimulated cells were labelled with tritiated thymidine. Random samples of cells were taken for light and electron microscopic morphometry.In comparison to the unstimulated lymphocyte, the stimulated cell increased in size and possessed a greater plasma membrane surface area. Increase in cell size was the result of increases in the volumes of all measured subcellular compartments both in the nucleus and the cytoplasm. Heterogeneity of the stimulated cell population precludes firm conclusions regarding the significance of all these ultrastructural changes, though alterations in cell surface are discussed in the context of known biochemical differences accompanying blastogenesis.M.M. Al-Hamdani was supported by a grant from the Ministry of Higher Education and Scientific Research, Republic of Iraq     

The mature mesonephric nephron of the rabbit embryo

Summary In freeze-fracture replicas of the entire cross-fractured mesonephros of 18 day rabbit embryos the basolateral and luminal cell faces of the different nephron segments were studied and compared with their metanephric counterparts. In the proximal tubule, the shallow zonula occludens exhibited only 1–2 strands and resembled the corresponding metanephric zonula, a very leaky type, which was found with a considerable paracellular flow component in sites of isotonic reabsorption. Gap junctions were restricted to the proximal tubule and were seen more frequently in its terminal segment. The distal tubule harboured two types of tight junctions. The most common type, a band of 5–8 closely parallel strands, matched the zonula occludens of the metanephric straight distal tubule. The observed particle density of the basolateral membrane (2,500±328/m²) was less than that of the proximal tubule (2,642±306). In addition, the collecting tubule exhibited a zonula occludens of the tight variety similar to that which occurred in the metanephric collecting duct. Rod-shaped particles of the luminal membrane were mainly concentrated in some of the intercalated cells but also had developed on principal cells, and occasionally, in the distal tubule. The Wolffian duct, with a deep tight zonula occludens, had an obviously rather inactive epithelium with no conspicuous transport-linked membrane specializations.     

Ultrastructural studies on neurophysine-containing vesicles of the neurosecretory system of vertebrates

Summary A new method described in the present paper allows identification of neurophysine-containing (NP) vesicles of the magnocellular neurosecretory system of vertebrates. After oxidation of ultrathin sections, the content of NP vesicles can be dissolved specifically in an alkaline medium. This procedure marks NP vesicles selectively in all portions of the classical neurosecretory system.Dedicated to Professor Dr. Dres h.c. W. Bargmann on the occasion of his 70^th birthday.Granted by the Ministerium für Wissenschaft und Technik der DDR.     

Functional states and fine structure of the contractile apparatus of the penis retractor muscle (PRM) of Helix pomatia L.

Summary The ultrastructure of the isolated glycerinated penis retractor muscle (PRM) of Helix pomatia was investigated. The diameter distributions of thick myofilaments from fibre cross sections in the relaxed, phasic contracted, tonic contracted, and in the catch states show that a characteristic filament spectrum is formed in the catch state and its preceding active state. The significant structural differences are discussed in relation to earlier hypotheses related to the catch state.I wish to thank Prof. Dr. Fr.-W. Schlote for helpful discussion. I also thank M. Jenninger and E. Grafahrend for their technical assistance.