The hemoglobin of the lizard Tarentola annularis has been studied within erythrocytes being digested in the gut of a parasitic pentastomide, Raillietiella sp. The hemoglobin is crystallized in the form of bundles comprised of numerous tubules (up to 2000). These tubules are simple or complex. Simple tubules are 50 nm in diameter; their wall is made up of two electron-opaque rings, separated by a clear ring. Complex tubules are up to 100 nm in diameter and show as many as 13 concentric walls. High magnification of transverse sections of simple tubules show 96 granules; each opaque ring is made up of 48 granules. Human hemoglobin is known to crystallize as 18-nm tubules, the wall of which is made up of six molecules; comparison of these data with our observations indicates that transverse sections of tubules of crystallized lizard hemoglobin should contain 24 molecules. Thus, each molecule of crystallized lizard hemoglobin consists of four granules; these granules may be considered as globin molecules. Erythrocytes in fresh lizard blood do not show crystallized hemoglobin; however, in blood treated with sodium bisulfite, they show tubules similar to that found in the parasite.
Paracrystalline aggregates of F-actin spontaneously assemble at the surface of positively charged liposomes. This single-layered paracrystalline array is made up of parallel and juxtaposed actin filaments aligned in register and showing the typical 36-nm periodicity which corresponds to the half-pitch of the double helix strand. This crystallization of pure actin results from a direct interaction between actin and positively charged lipids and does not occur with negative or neutral lipids.
The structure of two-dimensional crystals of membrane-bound Na,K-ATPase from rabbit kidney has been analyzed with a correlation averaging procedure. Two principally different crystal forms are observed with p1 and p21 symmetry, respectively. In the p1 form the averaged projection structure shows a triangular shaped protein domain interpreted as a protomer (alpha beta-unit) of Na,K-ATPase. In the p21-form the stain-deficient area is extended toward a twofold symmetry axis. The results are in good agreement with a previous analysis where Fourier methods were applied to well ordered crystals of pig kidney Na,K-ATPase and illustrate that the correlation averaging procedure can be used for the analysis of membrane crystals of Na,K-ATPase showing curved lattice lines.
We examinedin vivo the effect of pilocarpine (a cholinergic agent) and cycloheximide (an inhibitor of protein synthesis) on the “bar-like structures” in alveolar type II cells of rat lung to clarify their origin and significance in pulmonary surfactant production and secretion. Lungs were examined with an electron microscope using ultrathin sectioning, freeze-fracture technique, and morphometry. The bar-like structures in type II cells consisted of a concentrically arrangeed endoplasmic reticulum containing some amount of osmiophilic periodic material similar to the lamellae of lamellar bodies. Pilocarpine induced the accumulation of lamellar bodies of normal size which paralleled the increase in the number of bar-like structures in the cytoplasm of the type II cells. Cycloheximide induced a decrease in size of the lamellar bodies and an enlargement of the bar-like structures. Our morphological findings suggest that: (1) The phospholipid that would normally be incorporated into the lamellar bodies might be sequested instead in the concentrically arranged endoplasmic reticulum, forming the bar-like structures, and (2) The enlargement and the increased number of bar-like structures may be responsible in part for the changed metabolic process of surfactant production by alveolar type II cells.
The ciliated cells of tracheal epithelium were mechanically fragmented to remove the cytoplasmic soluble contents, and the apical zone was examined to clarify the three-dimensional structures of basal body and cytoskeletal filaments using freeze-fracture-etch approaches. The basal body was connected to the apical plasma membrane by definite laminae, formerly called alar sheets. The distal one-half of the basal foot was composed of several smooth-surfaced 12-nm fibrils. Intermediate filament networks extended to the lower half plane of the basal body, and enmeshed the basal body tightly by tiny 5- to 8-nm fibrils. Actin core bundles of microvilli also had tiny crosslinking fibrils. Some actin filaments were seen to run horizontally at the upper half plane of the basal body. Tracheal cilated cells also had circular actin filament bundles just inside the zonula adherens as many other epithelial cells. These cytoskeletal networks which enmeshed both basal bodies and core filaments of microvilli may function as a coordinator of ciliary beating.
The investigation of the degree of interpenetration between the two component phases of whewellite kidney stones, the protein matrix and calcium oxalate monohydrate crystallites, is extended by a technique of microchemical analysis, employing X-ray photoelectron spectroscopy (XPS). The technique involves determining the surface elemental (hence chemical) composition of fractured whewellite stones. Comparison with the bulk composition of about 95% COM and 5% matrix yielded information on the fracture path, hence on the spatial distribution of the two phases. The results showed large increases in matrix signal upon fracture, confirming that the minor phase, matrix, which is expected to provide an easy path for fracture, pervades the structure down to the level of individual crystallites.
The validity of a method for localization of potential Ca2+ binding sites was analyzed in pollen tubes ofLilium longiflorum and calyptra cells ofZea mays. The Ca2+-enriched fixation produced electron opaque deposits mainly on the plasma membrane, endoplasmic reticulum, and mitochondria, with details of localization differing somewhat between pollen tubes and maize calyptra cells. Proton microprobe analysis indicated a relative enrichment of Ca in the sections. The newly introduced technique of electron spectroscopic imaging confirmed the presence of Ca (and possibly P) in the induced electron opaque structures.
We have examined morphological events in RBL-2H3 basophilic leukocyte-derived cells following stimulation to secrete with specific antigen. Following stimulation, the cell surface undergoes a rapid and pronounced ruffling concomitant with an overall flattening. Secretory granules which are located in one area before stimulation becomes rapidly dispersed throughout the cytoplasm. Since secretion has been shown to be a relatively slow process in the RBL-2H3 cell line our observations suggest that ruffling and flattening precede the secretion process.
Emiliania huxleyi is a marine unicellular alga that is covered by one or several layers of coccoliths (discs of crystalline calcium carbonate) or of organic scales. Immediately proximal to these layers are several membranous layers that surround the protoplast. In this study an ultrastructural analysis of this membranous envelope is presented using ultrathin-section and freeze-fracture electron microscopy. We found that the protoplast of E. huxleyi is surrounded in a proximal-to-distal direction by a membrane studded with intramembranous particles (IMPs), an intermediate structure consisting of several smooth layers, and an outermost membrane studded with IMPs. On freeze-fracturing the intermediate structure reveals two or three closely apposed smooth fracture faces. It is possible that this structure consists of two or three membranes that are devoid of IMPs. However, the possibility is also discussed that this structure is composed of three or four lipid monolayers so closely stacked that water is hardly or not present between them.
The ultrastructure, in particular, the cell junctions and the electrophysiological properties of the larval midgut ofTenebrio molitor (Insecta, Coleoptera) are described. Gap junctions were not detected by thin-section or freeze-fracture in this tissue. Neither were low-resistance ion pathways between the cells, found in other epithelia, detected electrophysiologically or by intracellular injection of fluorescent tracers under several physiological conditionsin vitro. However, the hindgut epithelium which is continuous with the midgut epithelium is ionically coupled and the tracers pass from cell to cell. In comparison, the midgut of a weevil larva (a related beetle) has gap junctions, is ionically coupled, and tracer movement occurs under the same physiological conditions. The absence of ionic coupling in some insect midgut epithelia appears to be unique to this tissue type.
During mid- and late gestation, the uterus of sandbar sharks possesses specialized sites for exchange of metabolites between the mother and fetus. Attachment sites are highly vascular, rugose elevations of the maternal uterine lining that interdigitate with the fetal placenta. The maternal epithelium remains intact and there is no erosion. The attachment site consists of a simple, low columnar juxtaluminal epithelium underlain by an extensive vascular network. Juxtaluminal epithelial cells possess branched microvilli, saccular invaginations of the apical surface, and coated pits. They contain numerous coated vesicles, lipid-like inclusions, a prominent rough endoplasmic reticulum, and many free ribosomes. Tight junctions join the luminal aspect of adjacent cells. Lateral cell boundaries are highly folded and interdigitated. Capillaries are closely apposed to the basal cell surfaces. The endothelium is pinocytotically active. Comparison with the uterine epithelium of non-placental sharks, mammalian epitheliochorial placentae, and selected transporting epithelia reveals that the structure of the maternal shark placenta is consistent with its putative multiple functions, viz: (1) nutrient transfer; (2) transport of macromolecules, e.g., immunoglobulins; (3) respiration; and (4) osmotic and ionic regulation.
Detailed reconstructions of the flagellar tip (end-piece) in rodent spermatozoa have shown patterns of displacement between the termination points of the axonemal doublets (judging the terminations by the loss of electron density from the A-tubule). The patterns are in good agreement with those derived from sliding microtubule theory. In the hamster at least, the axis of major displacement passes approximately through doublet 1 and between doublets 5 and 6, though there may be some skewness in the clockwise direction. Microtubules derived from the plane at right angles to this (the central pair and presumably one or both of doublets 3 and 8) continue beyond the rest to the extreme tip, where they appear to be linked together at the cell membrane. This arrangement suggests that the tapering form of the end-piece, and of flagellar terminal filaments and ciliary tips in general, may be an adaptation to contain the sliding microtubules and prevent them impinging on the membrane overlying the tip.
Of the three pairs of complementary replicas mentioned in the previous paper (1985, J. Ultrastruct. Res. 91, 38-50) one pair consisted of fracture faces exposing the cytoplasmic surface of the outer surface membrane while the complementary face exposed the cytosol at the membrane surface. The latter face was particulate with randomly distributed particles in the size range of 100 to 200 A. These particles could be shown to be located in the cytosol at the membrane surface. They qualify as particles that are loosely bound to this surface, and it is proposed that at least part of these particles consist of glycolytic enzymes.
The cell wall and plasma membrane of four strains of Butyrivibrio (anaerobic rumen bacteria) have been studied by freeze-etching and thin sectioning. Some strains have a wall of gram-positive ultrastructure, which is vulnerable to lysozyme, while others have a wall which resembles the outer envelope of a gram-negative bacterium and is less susceptible to lysozyme. The plasma membrane of all strains is rather rigid, and its lipids have a very high phase-transition temperature.
Cytochemical investigations have associated acid inorganic trimetaphosphatase (TMPase) activity with the lysosomes of certain cell types. We have used the modified staining technique of Berg to show that this enzyme activity is present in normal mononuclear phagocytes and macrophage cell lines. We have found this enzyme activity to be present in murine RAW264 macrophages, in human U937 macrophages, in normal human blood monocytes, and in guinea pig peritoneal macrophages. All of the RAW264 and U937 macrophages showed intense TMPase activity. Many of the human monocytes and most of the guinea pig macrophages were labeled by this method. The reaction product was associated with the lysosomes of these cell types. The lysosomal staining-pattern was similar to that of acid phosphatase. Differences with regard to Golgi staining were noted. This indicates that TMPase is a lysosomal enzyme of mammalian macrophages. The distinction between TMPase and acid phosphatase activity has been demonstrated by measuring the pH optimum of each enzyme. Using substrates identical to those of the ultrastructural cytochemistry, we show that the pH optimum of TMPase is 4.0 and that of acid phosphatase is 5.0. The enzymatic activities are therefore ultrastructurally and biochemically distinct. Following phagocytosis of latex, yeast (Saccharomyces cerevisiae), or Corynebacterium parvum, TMPase has been found to be associated with phagosomes. This enzyme may take part in the degradation of phagocytosed materials, particularly microorganisms which contain inorganic polyphosphates and metaphosphates.
Anterior and posterior centrioles of Physarum amoebae are indistinguishable by their size during interphase but there is a correlation between the size of the two centrioles in the same amoeba. The interphase length of centrioles in diploid amoebae possessing only one pair of centrioles was 11% longer than in the case of the haploid strain. Treatment with taxol led to a 23 and 32% increase of the mean length in interphase and blocked mitosis, respectively. Conversely, during control mitosis the parental centrioles showed a 12% decrease of their mean length while the size of the daughter centrioles increased progressively. Neither nocodazole nor cold treatment induce a decrease of centriole length. The mean length of the cartwheel structure (internal proximal part) although constant during mitosis could be increased 24% in the presence of taxol. Similarly there was a correlation between the number of anterior satellites and the centriole length.
The mechanical processes occurring around a cutting edge when a thin slice is formed are related to those effective when metal or plastic is lathed. Critically assigning to thin slicing of resins the force vectors proved or assumed to be true for shapping by chipping, the attempt is made to clarify qualitatively and quantitatively the complex interplay after having measured the effective weight force and the order of magnitude of the radius of cutting edges of glass and diamond knives. Practically, only five force vectors in a polygon determine shearing and shortening of chips or thin slices, shortening obeying a broken rational function. By computing the relative values of shortening, all other force vectors can be computed. The results obtained for the normal force on the edge surface and the tangential force in this surface shed light on the effect of systematical variation of setting angle, bevel angle, and chip angle for thin slicing; thus possibilities of improvement in conventional “ultra” microtomy and in unadulterated cryotomy are shown in concluding recommendations.
Transmission electron microscopic investigations revealed extracellular three-lamellar and tubuli-like structures formed by the white rot fungusSporotrichum pulverulentum, which was grown with glucose on a rotary shaker. These structures, originating from the outermost fungal cell wall layer, were isolated by digesting the purified cell walls with snail enzyme. The resistant structures contained in the outer cell wall layer were harvested and chemically analyzed. They were composed of 80–90% carbohydrates, primarily glucose monomeres; 5–10% proteins, including five fractions with molecular weights between 30 000 and 200 000; and, finally, 5–10% lipids, none of which were phospholipids. Chemical analysis of the components after treatment with 2-mercaptoethanol/EDTA, followed by TEM observations, however, suggests that these structures consist of almost equal amounts of carbohydrate, protein, and lipid and that the additional carbohydrate is attached to them without any structural function. Several hypothesis concerning the biological function of the structures are made.
The acrosomal membrane system of bovine spermatozoa was examined by thin-section, freeze-fracture, surface-replica, and negative staining techniques in order to identify structural differentiations of specific acrosomal membrane domains. The outer acrosomal membrane of the apical and principal segments is characterized by a prominent electron-dense complex associated with its luminal face and a random intramembranous particle distribution. In the equatorial segment, the two-dimensional organization of bridging elements extending between the outer and inner acrosomal membrane was determined and correlated to freeze-fracture images. The inner acrosomal membrane lacked the electron-dense assembly noted on the outer acrosomal membrane and in freeze-fracture it appears crystalline. Further studies identified the distribution of the electron-dense subacrosomal material in the space between the inner acrosomal membrane and other nuclear membrane. Finally, new observations on the structural organization of the acrosomal matrix are presented.
