Cytoplasmic structure in rapid-frozen axons

Turtle optic nerves were rapid-frozen from the living state, fractured, etched, and rotary shadowed. Stereo views of fractured axons show that axoplasm consists of three types of longitudinally oriented domains. One type consists of neurofilament bundles in which individual filaments are interconnected by a cross-bridging network. Contiguous to neurofilament domains are domains containing microtubules suspended in a loose, granular matrix. A third domain is confined to a zone, 80-100 nm wide, next to the axonal membrane and consists of a dense filamentous network connecting the longitudinal elements of the axonal cytoskeleton to particles on the inner surface of the axolemma. Three classes of membrane-limited organelles are distinguished: axoplasmic reticulum, mitochondria, and discrete vesicular organelles. The vesicular organelles must include lysosomes, multivesicular bodies, and vesicles which are retrogradely transported in axons, though some vesicular organelles may be components of the axoplasmic reticulum. Organelles in each class have a characteristic relationship to the axonal cytoskeleton. The axoplasmic reticulum enters all three domains of axoplasm, but mitochondria and vesicular organelles are excluded from the neurofilament bundles, a distribution confirmed in thin sections of cryoembedded axons. Vesicular organelles differ from mitochondria in at least three ways with respect to their relationships to adjacent axoplasm: (a) one, or sometimes both, of their ends are associated with a gap in the surrounding granular axoplasm; (b) an appendage is typically associated with one of their ends; and (c) they are not attached or closely apposed to microtubules. Mitochondria, on the other hand, are only rarely associated with gaps in the axoplasm, do not have an appendage, and are virtually always attached to one or more microtubules by an irregular array of side-arms. We propose that the longitudinally oriented microtubule domains are channels within which organelles are transported. We also propose that the granular material in these channels may constitute the myriad enzymes and other nonfibrous components that slowly move down the axon.     

ULTRASTRUCTURE OF THE FEEDING APPARATUS AND MYONEMAL SYSTEM OF THE HETEROTROPHIC DINOFLAGELLATE PROTOPERIDINIUM SPINULOSUM1

The feeding veil or pallium of the thecate heterotrophic dinoflagellate Protoperidinium spinulosum Schiller is a highly vesiculate membranous sac containing several arched, sometimes bifurcated microtubular ribbons. It originates from an internal microtubular basket, passes through a sphincter-like osmiophilic ring located inside the posterior flagellar pore, and emerges from the cell at that pore. The osmiophilic ring is part of an interconnected myonemal system (composed of two striated collars and several striated connectives) that is anchored to the pore plate and to two inward protrusions composed of minute sulcal plates. A related species, Protoperidinium punctulatum (Paulsen) Balech, also possesses a microtubular basket/osmiophilic ring complex. Elongate electron-dense bodies within the basket resemble digestive secretory granules found in other protists. Granular, electron-lucent microbodies clustered at the anterior end of the basket may also have a role in prey digestion. Dense membranous whorls observed within a P. spinulosum cell presented as it was preparing to initiate feeding indicate a condensed storage site for pallium membranes. A narrow microtubule-strengthened pseudopodal appendage found in two non-feeding cells constitutes the tow filament that serves as the initial linkage between the dinoflagellate and its food. The structures that constitute the pallium and pallium precursors, described here for the first time, are unlike those of other known protists, although some similarities with the dinoflagellate peduncle are evident. The existence of this unique system of organelles may have important ramifications in the search for evolutionary relationships among protists.     

Striated flagellar roots: isolation and partial characterization of a calcium-modulated contractile organelle

We report the isolation of striated flagellar roots from the Prasinophycean green alga Tetraselmis striata using sedimentation in gradients of sucrose and flotation on gradients of colloidal silica. PAGE in the presence of 0.1% SDS demonstrates that striated flagellar roots are composed of a number of polypeptides, the most predominant one being a protein of 20,000 Mr. The 20,000 Mr protein band represents approximately 63% of the Coomassie Brilliant Blue staining of gels of isolated flagellar roots. Two-dimensional gel electrophoresis (isoelectric focusing and SDS PAGE) resolves the major 20,000 Mr flagellar root protein into two components of nearly identical Mr, but of differing isoelectric points (i.e., pl's of 4.9 and 4.8), which we have designated 20,000-Mr-alpha and 20,000-Mr-beta, respectively. Densitometric scans of two-dimensional gels of cell extracts indicate that the 20,000-Mr-alpha and -beta polypeptides vary, in their stoichiometry, between 2:1 and 1:1. This variability appears to be related to the state of contraction or extension of the striated flagellar roots at the time of cell lysis. Incubation of cells with 32PO4 followed by analysis of cell extracts by two-dimensional gel electrophoresis and autoradiography reveals that the more acidic 20,000-Mr-beta component is phosphorylated and the 20,000-Mr-alpha component contains no detectable label. These results suggest that the 20,000-Mr-alpha component is converted to the more acidic 20,000-Mr-beta form by phosphorylation. Both the 20,000-Mr-alpha and -beta flagellar root components exhibit a calcium-induced reduction in relative electrophoretic mobilities in two-dimensional alkaline urea gels. Antiserum raised in rabbits against the 20,000-Mr protein binds to both the 20,000-Mr-alpha and 20,000-Mr-beta forms of the flagellar root protein when analyzed by electrophoretic immunoblot techniques. Indirect immunofluorescence on vegetative or interphase cells demonstrate that the antibodies bind to two cyclindrical organelles located in the anterior region of the cell. Immunocytochemical investigations at ultrastructural resolution using this antiserum and a colloidal gold-conjugated antirabbit-IgG reveals immunospecific labeling of striated flagellar roots and their extensions. We conclude that striated flagellar roots are simple ion-sensitive contractile organelles composed predominantly of a 20,000 Mr calcium-binding phosphoprotein, and that this protein is largely responsible for the motile behavior of these organelles.     

Morphology and Taxonomy of Two New Otoplanids (Turbellaria, Proseriata) from the Ligurian Sea

Two new species of Otoplanidae belonging to the subfamily Parotoplaninae are described: Parotoplana macrostyla sp.n. and P. uncinate sp.n. In both species the anterior body end bears a swelling. The rhabdites are grouped into bundles of several elements (up to 20 or more). The caudal plate is fan-shaped. The cuticular apparatus has a stylet and two types of spines. The germovitelloducts do not form the common duct and the bursa stalk has a peculiar structural pattern.     

Evidence that the neck appendages are adsorption organelles in Bacillus subtilis bacteriophage phi29.

A mutant of Bacillus subtilis unable to adsorb phage phi29 efficiently has been isolated. This mutant can be infected by host range mutants of the phage. Since the host range mutations map in cistron 12, which codes for neck appendage protein, this would tend to confirm that these organelles are involved in viral adsorption.     

II. Ultrastructure of duct cell granules in mammalian submaxillary glands

Summary Discrete, PAS-positive granules of relatively uniform electron-density and size characterise the intercalated duct cells of mammalian submaxillary glands. Smaller, electron-dense organelles are seen in the cells at the junction of the intercalary-striated duct region in the guinea-pig. The large granules of variable electron-density which are observed in the proximal, modified intercalary cells in the rabbit closely resemble the granules in the acinar cells of the guinea-pig. Several populations of granules differing in size are found in the striated granular tubules of the rat and hamster; the organelles in the rat show two grades of electron-density whereas those in the hamster are uniformly dense. Numerous small granules with compactly arranged intragranular material occupy the apical part of the striated ducts of the cat, dog and rabbit. The chemical composition of each population of duct cell granules is unknown. The question whether granules containing kallikrein, trypsin-like enzymes and amylase are stored in the duct cells is discussed.We wish to thank The Wellcome Trust for a travel grant to attend the International Symposium on Vasopeptides (Florence, July 1971) where this work was briefly reported.     

Somatic Musculature of Trichodorus porosus and Criconemoides similis

The somatic musculature of Trichodorus porosus is transversely striated, and that of Criconemoides similis is obliquely striated. The species also differ in configuration of the myofibrils, arrangement of the filaments within the myofibrils, and abundance of sarcoplasmic reticulum. Both species are platymyarian and meromyarian. The muscle cells are composed of myofibrils, sarcoplasm, sarcoplasmic reticulum, and various organelles. The myofibrils of both species contain actin and myosin filaments.     

Ultrastructure of the obligate halophilic bacterium Halobacterium halobium

The fine structure of Halobacterium halobium was studied by means of a modified double-fixation technique. The cell envelope is shown to consist of both a “wall” and a plasma membrane. Some electron-dense strands were seen inside the cytoplasm running parallel to the cell envelope. An unusual organelle (or organelles) appeared inside the cytoplasm in the form of parallel striated strands.     

How to roll an endothelial cigar: the biogenesis of Weibel-Palade bodies

Weibel-Palade bodies (WPB) are the regulated secretory organelles of endothelial cells. These cigar-shaped membrane-bound structures function in both hemostasis and inflammation but their biogenesis is poorly understood. Here, we review what is currently known about their formation. The content of WPBs is dominated by the hemostatic factor von Willebrand factor (VWF), whose complex biogenesis ends in the formation of high molecular weight multimers. VWF is also organized into proteinaceous tubules which underlie the striated interior of WPBs as seen in the EM. VWF expression is necessary for formation of WPBs, and its heterologous expression can even lead to the specific recruitment of WPB membrane proteins, including the leukocyte receptor P-selectin, the tetraspanin CD63, and Rab27a. Unusually, the VWF propeptide is implicated in the biogenesis of WPBs, being essential for formation of the storage compartment. The elongation of the cigars and the formation of the tubules are determined by non-covalent interactions between pro- and mature VWF proteins. Surprisingly, high molecular weight multimers seem neither necessary nor sufficient to trigger formation of a storage compartment, and do not seem to have any role in WPB biogenesis. Von Willebrand's disease, usually caused by mutations within VWF, has provided many of the insights into the way in which VWF drives the formation of these organelles.     

Cytokeratin filament organization in the ciliated cells of the quail oviduct

With the exception of keratinocytes and some types of cultured cells, ciliated cells appear to be the major cell type which contains the most developed cytokeratin meshwork. We report, here, on the intermediate filament (IF) organization in ciliated cells of the quail oviduct using ultrastructural and immunocytochemical techniques. Special attention was focused on the relationships between IF and other cell organelles. The meshwork of IFs appears as a subapical disk constituted of separate bundles mainly composed of interwoven 10-nm filaments. From this subapical region, a descending bundle connects the array of IFs occupying the basal part of the cell. The nucleus is maintained in a loose network of IFs. In ciliated cells there are no free centrioles, but IFs are related to centriolar appendages (striated rootlets).     

Mitochondrial network of skeletal muscle fiber

Highly specialized muscle fibers require a unique spatial organization of the mitochondrial network. Mitochondrial morphology is largely determined by the fusion and fission of these organelles. This review summarizes the current concepts of mitochondrial dynamics mechanisms and structural features of the mitochondrial network in striated muscle tissue. The role of mitochondria and their dynamics in muscle fiber physiology are also discussed.     

Cucullosporella mangrovei, ultrastructure of ascospores and their appendages

The ultrastructure ofCucullosporella mangrovei ascospores is described. Mature ascospores possess two wall layers, an outer electron-dense episporium and an innermost tripartite mesosporium. Episporial elaborations form electrondense spore wall ornamentations from which extend fibrils that may constitute a highly hydrated exosporium which was not visualised at either the scanning electron microscope or light microscope level. Ascospores possess a hamate appendage at each pole which unfolds in seawater to form a long thread. Ultrastructurally the polar appendage comprises folded fibro-granular electron-dense material and fine fibrils. The fibrils form a matrix around and within the fibro-granular appendage and around the entire unreleased ascospore. These fibrils have not been observed associated with the ascospore appendages in other species of the Halosphaeriales and are a discrete and new appendage component. The fibro-granular appendage and fibrils are bounded by the outer delimiting membrane which is absent around released ascospores. The nature of the spore appendage is compared with that of other marine and freshwater ascomycetes and the taxonomic assignment of the species is discussed.     

THE FINE STRUCTURE OF BLASTOCLADIELLA EMERSONII ZOOSPORES

The zoospore of Blastocladiella emersonii has been re-examined with the electron microscope. The following new findings were made. A double unit-membrane system surrounds all cell organelles except γ-bodies, vacuoles and a few fragments of membranes. Lipid granules on one side of the large mitochondrion alternate with vesicles. The kinetosome of the posterior flagellum does not have any central fibrils as previously reported; a small, cylindrical structure is found within its anterior end. An associated centriole is located next to the kinetosome. Three striated rootlets pass from the kinetosome by separate channels through the mitochondrion. There appears to be no connection between the striated rootlets and the mitochondrion. Microtubules originating at the anterior end of the kinetosome pass into the cytoplasm between the mitochondrion and the nuclear cap. Long, dense strands were observed in some nuclei. The axoneme is taken up into the spore during encystment and is found in the freshly encysted spore. No trace of the flagellar sheath has been found in the encysted spore.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

Effects of 12-O-tetradecanoyl-phorbol- 13-acetate on myofibril integrity and Ca2+ content in developing myotubes

The cocarcinogen 12-O-tetradecanoyl-phorbol-13-acetate (TPA) has a prompt and selective effect on striated myofibrils in well-formed, multinucleated myotubes. The myofibrillar apparatus is totally disrupted and largely degraded after 3 days in TPA. Fluorescent-tagged antibodies to muscle-specific light meromyosin and electron microscopy document this catabolic effect of TPA. This selective degradation of fully assembled striated myofibrils is readily reversible. This reassembly of myofibrils requires de novo protein synthesis. The TPA-treated myotubes are unusually rich in autophagosomes, organelles rarely observed in normal myotubes. TPA has no discernable effect on the morphology of the subsarcolemmal microfilaments, mitochondria, microtubules, or sarcoplasmic reticulum (SR). The programmed disappearance of the fibroblast-type, intermediate 10-nm filaments (FIF) that occurs as normal myotubes mature is not altered by TPA. However, the TPA-treated myotubes depleted of myofibrils are filled with an extensive meshwork of the muscle-type intermediate 10-nm filaments (MIF). The drug has no obvious effect on the constitutive contractile proteins comprising the submembranous microfilaments, or the FIF in presumptive myoblasts or fibroblasts, nor does it affect the motility associated with these replicating cells. The striking increase in total calcium content which occurs as normal myotubes mature is absent in myotubes treated with TPA.     

Ultrastructure of thymic myoid cells

Mature myoid cells in the parenchyma of reptilian thymus contain all the organelles typical of striated muscle. The presence of both immature and degenerating stages indicates a turnover of myoid cells in the adult thymus. In the earlier stages of differentiation myoid cells resemble thymic epithelial cells. A close parallel exists between developing myoid cells, skeletal muscle differentiating in vitro, rhabdomyoma and rhabdomyosarcoma. Elaborate lattice-like structures are formed by transverse tubules. These structures are compared with similar configurations which have been described in muscle and mitochondrial cristae.     

Morphologic changes in the thymus and lymphoid nodules of the appendix of the white rat subjected to increasing physical loads

By means of histological and morphometrical methods the thymus and appendage have been studied in male white rats subjected to a dosed physical loading (swimming). The physical loading is accompanied with essential changes in structure and cell composition of the immunogenic organs. The rate and character of the changes depend on the adaptability level of the animal's organism to the physical loading. At adaptation to the loading, the process of age involution of the thymus decelerates, amount of lymphoid nodules in the appendage increases, comparing the control parameters, contents of lymphocytes noticeably increase in all zones of the lymphoid nodules. When adaptation to the physical loading is not sufficient, the rate of the thymus involution sharply increases, while in the appendage the number of the lymphoid nodules decreases. However, in some animals at a sharp involution of the thymus, the changes in the appendage do not differ from the control ones.     

Co-operative dynamics in organelles

Some organelles produce elementary life phenomena which are characterized by the spontaneous formation and/or maintenance of ordered macroscopic dynamics like e.g. the shortening of sarcomeres in striated muscle and the transmission of electrical impulses in an axon. It has been widely accepted that such organelles are organized molecular systems where molecular elements work independently under constraint of a more or less rigid and regular structure of the system. On the other hand, such organelles should be regarded as self-organizing systems if the ordered macroscopic dynamics are self-organized. As the macroscopic dynamics gradually emerge, the microscopic dynamics of its elements become linked to each other through a feedback loop. It is crucial for the feedback loop to operate that the macroscopic dynamics are "free" in their behavior. In the present paper, it is pointed out that the traditional view of independent molecular elements has been obtained from experiments in which, by means of external constraint, the macroscopic dynamics is "clamped". Under such conditions, the self-organizing system may behave as an organized one. Based on synergetics we propose criterions for proving self-organizing systems, and, by applying the criterions, we conclude that skeletal muscle actomysin is a co-operative element in the sense of self-organization.     

Occurrence of unusual tubular invaginations of the plasma membrane in smooth muscle cells of the lamprey,Lampetra japonica

Numerous tubular structures were observed in the surface region of smooth muscle cells making up the vascular walls in the lamprey, Lampetra japonica; they were designated as surface tubules. The limiting membrane of the surface tubules was connected to the plasma membrane, allowing communication of the lumen of the tubule with the extracellular space. Tannic acid reacted with osmium, serving as an extracellular marker, penetrated into the tubules but not into the intracellular organelles, such as the endoplasmic reticulum and the Golgi complex. The surface tubules were grouped in longitudinal parallel rows, separated from each other by tubule-free areas where dense plaques were present. Each tubule was fairly cylindrical (approximately 60 nm in diameter) and often ramified into two or three branches with a blind end. Occasionally, these tubules were encircled by the sarcoplasmic reticulum which was located immediately beneath the plasma membrane. Similar tubules were also observed in the surface region of vascular endothelial cells and fibroblasts in the adventitial connective tissue. The possibility that the surface tubules in the present observations are analogous to the smooth muscle caveolae or the striated muscle T-tubule is discussed.