Showing papers in "Journal of Cell Biology in 1970"
TL;DR: Acetone does not cause any detectable change in the structure of the zonula occludens, but the occluding junction becomes leaky to lanthanum following acetone treatment, and the effects of other reagents on the junctions are reported.
Abstract: Zonulae occludentes and gap junctions were examined both in the intact mouse liver and in a junction-rich membrane fraction from homogenized mouse liver. These preparations were visualized with the techniques of uranyl acetate staining en bloc, staining with colloidal lanthanum, negative staining with phosphotungstate, and freeze-cleaving. The zonula occludens is arranged as a meshwork of branching and anastomosing threadlike contacts sealing the lumen of the bile canaliculus from the liver intercellular space. The gap junction is characterized in section by a 20 A gap between the apposed junctional membrane outer leaflets, and permeation of this space with lanthanum or phosphotungstate reveals a polygonal lattice of subunits with a center-to-center spacing of 90–100 A. Freeze-cleaved gap junctions show a similar lattice. Extraction of junction-rich fractions with 60% aqueous acetone results in a disappearance of the 20 A gap in sectioned pellets and an inability to demonstrate the polygonal lattice with either the freeze-cleave or negative staining techniques. Extraction of the membranes with 50% acetone does not produce this effect. Thin-layer chromatography of the acetone extracts reveals a group of phospholipids in the 60% extract that are not detectable in the 50% extract. Acetone does not cause any detectable change in the structure of the zonula occludens, but the occluding junction becomes leaky to lanthanum following acetone treatment. The effects of other reagents on the junctions are reported.
592 citations
TL;DR: The freeze-etch technique was used to observe red blood cell ghosts labeled on both surfaces with covalently bound ferritin, indicating that fracture does not show membrane-surface detail, and results were consistent with the concept that membranes split during the fracture process of Freeze-etching.
Abstract: The freeze-etch technique was used to observe red blood cell ghosts labeled on both surfaces with covalently bound ferritin. Ferritin molecules were never observed on fracture faces, thus indicating that fracture does not show membrane-surface detail. Subliming away the surrounding ice did expose the ferritin on the membrane surface. These results were consistent with the concept that membranes split during the fracture process of freeze-etching.
517 citations
TL;DR: It is postulated that central hydrophilic channels may extend through the subunit assembly to provide a direct route for intercellular communication.
Abstract: A correlation is made between the appearances of the nexus ("gap junction") as revealed by thin-section and by freeze-cleave electron microscopy techniques. These methods reveal different aspects of a complex subunit assembly forming the nexus membranes. In thin sections, the nexus is formed by the very close apposition of two "unit" membranes. The electron-opaque tracer, colloidal lanthanum hydroxide, outlines an aspect of electron-lucent subunits that project into the central region of the nexus. The freeze-cleave technique demonstrates novel membrane faces that are generated from within the interior of plasma membranes by splitting them into two lamellae (Lm): Lm 1 adjacent to the cytoplasm, and Lm 2 adjacent to the extracellular space. Each of the two membranes forming the nexus can be split into these two lamellae. On the new face of Lm 1, particles approximately 50 A in diameter are closely packed in an array which is often hexagonal with a 90–100 A center-to-center spacing. The two apposed lamellae (Lm 2-Lm 2) of the nexus are constructed of sheets of subunits in a similar array. The Lm 1 particles appear to extend into the Lm 2 subunits to form macromolecular complexes. The Lm 2 subunits extend to the center of the nexus to form the contacts outlined by lanthanum in sections. It is postulated that central hydrophilic channels may extend through the subunit assembly to provide a direct route for intercellular communication.
482 citations
TL;DR: Evidence is presented suggesting that nucleotide pyrophosphatase and phosphodiesterase I of rat liver may be manifestations of the same enzyme and the preparative method developed appears to give the best combination of yield, purity, and reproducibility available.
Abstract: Nucleotide pyrophosphatase and phosphodiesterase I of rat liver have been found to be localized primarily in cell particulates highly enriched with respect to the most commonly accepted plasma membrane marker, 5'-nucleotidase, and therefore should themselves be assigned a plasma membrane localization. The observation that plasma membranes sediment in isotonic sucrose with both nuclear and microsomal fractions was exploited to obtain plasma membrane preparations from each fraction. Both preparations are similar in chemical and enzymic composition. Moreover, the preparative method developed in this study appears to give the best combination of yield, purity, and reproducibility available. The question of the possible identity of nucleotide pyrophosphatase and phosphodiesterase I is considered, and evidence is presented suggesting that these activities may be manifestations of the same enzyme.
467 citations
TL;DR: The structure of the junction between sarcoplasmic reticulum (SR) and transverse tubular (T) system at the triad has been studied in twitch fibers of the frog and the junctional area between the feet is apparently empty.
Abstract: The structure of the junction between sarcoplasmic reticulum (SR) and transverse tubular (T) system at the triad has been studied in twitch fibers of the frog. The junction is formed by flattened surfaces of the SR lateral sacs and the T-system tubule, which face each other at a distance of 120–140 A. At periodic intervals of about 300 A, the SR membrane forms small projections, whose tips are joined to the T system membrane by some amorphous material. The SR projections and the amorphous material are here called SR feet. The feet are disposed in two parallel rows, two such rows being present on either side of the T-system tubule. The junctional area between the feet is apparently empty. The feet cover no more than 30% of the T system surface area and 3% of the total SR area. The functional significance of this interpretation of the junctional structure is discussed.
404 citations
TL;DR: An effort is made to gain some perspective from a synthesis of these fragmentary accounts supplemented by the author's own observations based on electron microscopic examination of nearly two hundred insect species.
Abstract: The thorough descriptive studies by the early cytologists Meves (1901, 1907), Retzius (1904, 1909), Bowen (1920, 1922 a-c, 1924), Pollister (1930), and Johnson (1931) provided later workers with a remarkably accurate picture of the microscopic anatomy of insect spermatozoa and of spermiogenesis in several insect species . Their light microscope observations were necessarily lacking in detail, however, and since the advent of the electron microscope, an increasing number of cell biologists have undertaken to extend our understanding of the structure of insect germ cells. Some have confined their attention to spermiogenesis or the morphology of mature spermatozoa of a particular species, while others have been concerned with the comparative morphology of single sperm organelles. Although there are by now many relevant papers of limited scope widely dispersed in the biological literature, there seems to be no single reference to which one can turn for an overview of the structure and development of the insect spermatozoon. In this review an effort is made to gain some perspective from a synthesis of these fragmentary accounts supplemented by the author's own observations based on electron microscopic examination of nearly two hundred insect species.'
394 citations
TL;DR: Results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure, as suggested by the acrylamide gel electrophoresis pattern.
Abstract: Rough microsomes were incubated in an in vitro amino acid-incorporating system for labeling the nascent polypeptide chains on the membrane-bound ribosomes. Sucrose density gradient analysis showed that ribosomes did not detach from the membranes during incorporation in vitro. Trypsin and chymotrypsin treatment of microsomes at 0° led to the detachment of ribosomes from the membranes; furthermore, trypsin produced the dissociation of released, messenger RNA-free ribosomes into subunits. Electron microscopic observations indicated that the membranes remained as closed vesicles. In contrast to the situation with free polysomes, nascent chains contained in rough microsomes were extensively protected from proteolytic attach. By separating the microsomal membranes from the released subunits after proteolysis, it was found that nascent chains are split into two size classes of fragments when the ribosomes are detached. These were shown by column chromatography on Sephadex G-50 to be: (a) small (39 amino acid residues) ribosome-associated fragments and (b) a mixture of larger membrane-associated fragments excluded from the column. The small fragments correspond to the carboxy-terminal segments which are protected by the large subunits of free polysomes. The larger fragments associated with the microsomal membranes depend for their protection on membrane integrity. These fragments are completely digested if the microsomes are subjected to proteolysis in the presence of detergents. These results indicate that when the nascent polypeptides growing in the large subunits of membrane-bound ribosomes emerge from the ribosomes they enter directly into a close association with the microsomal membrane.
385 citations
TL;DR: A modification of the freeze-fracturing technique to permit observation of replicas of both sides of the fracture is described, used to study mouse liver cell membrane structure.
Abstract: A modification of the freeze-fracturing technique to permit observation of replicas of both sides of the fracture is described. It has been used to study mouse liver cell membrane structure. Membranes break to give two faces with three-dimensional complementarity, although there is some small-scale mismatching which is discussed. Since the two distinctive sets of membrane faces are complementary sets, they cannot be the two outside surfaces. In particular, structures (such as particles) seen on these faces are within the membrane. It is not possible from this work to say precisely where the fracture plane goes with respect to a plasma membrane, only that it must be close to the interface between membrane and cytoplasm, or at that interface. Models, consistent with the appearance of the matching replicas, are derived for three regions of the plasma membrane: (a) The nonjunctional plasma membrane, which contains many scattered particles. Except for these particles, the otherwise flat fracture face is not at variance with a bimolecular leaflet structure. (b) Gap junctions. Each of the two membranes comprising a gap junction contains a close-packed array of particles. (c) Tight junctions. Here membranes have ridges within them.
382 citations
TL;DR: A rapid method of preparing plasma membranes from isolated fat cells is described and electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.
Abstract: A rapid method of preparing plasma membranes from isolated fat cells is described. After homogenization of the cells, various fractions were isolated by differential centrifugation and linear gradients. Ficoll gradients were preferred because total preparation time was under 3 hr. The density of the plasma membranes was 1.14 in sucrose. The plasma membrane fraction was virtually uncontaminated by nuclei but contained 10% of the mitochondrial succinic dehydrogenase activity and 25–30% of the RNA and reduced nicotinamide adenine dinucleotide cytochrome c reductase activity of the microsomal fraction. Part of the RNA and NADH-cytochrome c reductase activity was believed to be native to the plasma membrane or to the attached endoplasmic reticulum membranes demonstrated by electron microscopy. The adenyl cyclase activity of the plasma membrane fraction was five times that of Rodbell's "ghost" preparation and retained sensitivity to epinephrine. The plasma membrane ATPase activity was five times that of the homogenate and microsomal fractions. Electron microscopic evidence suggested contamination of the plasma membrane fraction by other subcellular components to be less than the biochemical data indicated.
372 citations
TL;DR: The centrally directed neurite of the dorsal root neuroblast has been described from the period of its initial entrance into the neural tube until a well-defined dorsal root is formed.
Abstract: The centrally directed neurite of the dorsal root neuroblast has been described from the period of its initial entrance into the neural tube until a well-defined dorsal root is formed. Large numbers of microtubules, channels of agranular reticulum, and clusters of ribosomes are found throughout the length of the early axons. The filopodia of the growth cone appear as long thin processes or as broad flanges of cytoplasm having a finely filamentous matrix material and occasionally small ovoid or elongate vesicles. At first the varicosity is a small expansion of cytoplasm, usually containing channels of agranular reticulum and a few other organelles. The widely dilated cisternae of agranular reticulum frequently found within the growth cone probably correspond to the pinocytotic vacuoles seen in neurites in tissue culture. The varicosities enlarge to form bulbous masses of cytoplasm, which may measure up to 5 µ in width and 13 µ in length. They contain channels of agranular reticulum, microtubules, neurofilaments, mitochondria, heterogeneous dense bodies, and a few clusters of ribosomes. Large ovoid mitochondria having ribonucleoprotein particles in their matrix are common. Dense membrane specializations are found at the basal surface of the neuro-epithelial cell close to the area where the early neurites first enter the neural tube.
328 citations
TL;DR: It is concluded that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.
Abstract: Several established clonal strains of rat pituitary cells which produce growth hormone in culture have been shown to secrete a second protein hormone, prolactin. Prolactin was measured immunologically in culture medium and within cells by complement fixation. Rates of prolactin production varied from 6.6 to 12 µg/mg cell protein per 24 hr in four different cell strains. In these cultures ratios of production of prolactin to growth hormone varied from 1.0 to 4.1. A fifth clonal strain produced growth hormone but no detectable prolactin. Intracellular prolactin was equivalent to the amount secreted into medium in a period of about 1–2 hr. Both cycloheximide and puromycin suppressed prolactin production by at least 94%. Hydrocortisone (3 x 10-6 M), which stimulated the production of growth hormone 4- to 8-fold in most of the cell strains, reduced the rate of prolactin production to less than 25% of that in control cultures. Conversely, addition of simple acid extracts of several tissues, including hypothalamus, to the medium of all strains increased the rate of production of prolactin six to nine times and decreased growth hormone production by about 50%. We conclude that multifunctional rat pituitary cells in culture show unusual promise for further studies of the control of expression of organ-specific activities in mammalian cells.
TL;DR: It was suggested that the muscle nuclei which take up thymidine 3H and undergo mitosis are not true muscleuclei but belong to "muscle satellite cells", which was concluded that satellite cell nuclei are eaily distinguished from true Muscle nuclei in the electron microscope.
Abstract: Much is known about the early stages of muscle development-the proliferation of myoblasts, their fusion into myotubes, and the transformation of myotubes into young muscle fibers . However, the mode of growth of muscle in the young animal remains obscure . It is known that muscle growth is extensive (e .g . in the chicken the pectoralis muscle enlarges from 0.7 g at hatching to 300 g in the adult) and is mainly the result of hypertrophy of the fibers (1), with little, if any, increase in their number (2) . Moreover, it is generally assumed that, unlike what happens in most other tissues, there is no division of the nuclei and, consequently, no increase in their number (3) . This view is supported by experiments showing that, while myoblasts can divide at early stages of development, they lose this ability once they have been incorporated into myotubes (4) . Furthermore, during muscle regeneration following injury, myoblasts appear which take up thymidine-'H, indicating their ability to divide, whereas the nuclei of muscle fibers do not (5) . Hence the prevalent opinion has been that new nuclei are not produced in skeletal muscle fibers during growth . This view was questioned when DNA determinations revealed a considerable increase in the number of fiber nuclei during growth in rats (6) and chickens (1), and mitotic figures were observed within the muscle fibers of growing rats (7) . Furthermore, after an injection of thymidine3H, radioautography revealed labeled nuclei within the confines of the basement membrane of muscle fibers (7, 8). In the hope of reconciling these apparently conflicting observations, it was suggested that the muscle nuclei which take up thymidine 3H and undergo mitosis are not true muscle nuclei but belong to \"muscle satellite cells\" (7). Such cells, which had been observed only in the electron microscope (9), consist of a single nucleus surrounded by scanty cytoplasm and are located between the basement membrane and the plasmalemma of the fibers, whereas true muscle nuclei are within the plasmalemma . Since satellite cell nuclei are eaily distinguished from true muscle nuclei in the electron microscope, it was decided to examine radioautographs in this instrument following thymidine 3H injection . Six male rats aged 17 days and weighing 25-30 g were given a single injection of 25-60 )uCi of thymidine 3H; g body weight and sacrificed at intervals varying from I to 72 hr later . The tibialis anterior muscle was fixed by immersion in glutaraldehyde paraformaldehyde (10), postfixed in osmium tetroxide, and embedded in Epon . Thin (silver to gold) sections were prepared for radioautography in the electron microscope with Ilford L4 emulsion (11-13) and poststained with lead citrate (14) . Electron microscopy revealed the existence of two types of nuclei within the basement membrane of the fiber . The nuclei of the first type (85-90% of the total) are in direct contact with the myofibril-containing cytoplasm ; they are the true muscle nuclei. The nuclei of the second type are surrounded by a small amount of cytoplasm, which is separated from the myofibril-containing cytoplasm by an intercellular space; these are the satellite cell nuclei ; they are usually smaller and darker than true muscle nuclei . In radioautographs obtained I hr after injection of thymidine 3H, approximately 3% of the nuclei within the basement membrane were labeled . These invariably belonged to satellite cells (Fig. 1) . Not one of the true muscle nuclei was labeled at that time. The results were similar at 6 and 10 hr after injection (Table I) . It was, therefore, concluded that satellite cell nuclei, but not true muscle nuclei, were able to synthesize DNA . Moreover, since it is known that DNA synthesis precedes mitosis (8), it may be concluded that the mitotic figures previously observed in the muscle fibers of growing rats (7) must belong to satellite cells . This conclusion is in accord with a recent report that, after colchicine treatment of 30-g rats, arrested mitoses of the nuclei of satellite cells, but not of true muscle nuclei, may be observed (15) . By 24 hr, a few true muscle nuclei were also labeled (Table I). At 48 hr, the labeling frequency of true muscle nuclei was increased, and by 72
TL;DR: The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division and has but a minor effect on cell viability and reproductive integrity.
Abstract: The thymidine analogue 5-bromodeoxyuridine (BUdR) has a differential effect on the synthesis of tissue-specific products and molecules required for growth and division. Proliferating myogenic cells cultured in BUdR fail to fuse and fail to initiate the synthesis of contractile protein filaments. Conversely, BUdR has but a minor effect on cell viability and reproductive integrity. Low concentrations of BUdR result in an enhancement of cell number relative to the controls; higher concentrations are cytotoxic. Suppression of myogenesis is reversible after at least 10 cell generations of growth in the analogue. Cells that do not synthesize DNA, such as postmitotic myoblasts and myotubes, are not affected by BUdR. Incorporation of BUdR for one round of DNA synthesis was accomplished by first incubating myogenic cells, prior to fusion, in 5-fluorodeoxyuridine (FUdR) to block DNA synthesis and collect cells in the presynthetic phase. The cells were then allowed to synthesize either normal DNA or BU-DNA for one S period by circumventing the FUdR block with BUdR or BUdR plus thymidine (TdR). The cultures were continued in FUdR to prevent dilution of the incorporated analogue by further division. After 3 days, the cultures from the FUdR-BUdR series showed the typical BUdR effect; the cells were excessively flattened and few multinucleated myotubes formed. Cells in the control cultures were of normal morphology, and multinucleated myotubes were present. These results were confirmed in another experiment in which BUdR-3H was added to 2-day cultures in which myotubes were forming. Fusion of thymidine-3H-labeled cells begins at 8 hr after the preceding S phase. In contrast, cells which incorporate BUdR-3H for one S period do not fuse with normal myotubes.
TL;DR: In this paper, the growing end of the nascent polypeptide is protected from proteolytic attack by the ribosomal structure and the protected segment is located at the carboxy-terminal end.
Abstract: Free ribosomes containing nascent polypeptide chains labeled in vitro were submitted to proteolysis at 0° by a mixture of trypsin and chymotrypsin. Sucrose gradient analysis showed that polysome patterns are retained even after 24 hr of proteolysis in the cold, while messenger RNA-free ribosomes (generated progressively during in vitro incorporation) are, within 2 hr, completely dissociated into subunits by trypsin. Although ribosomes and subunits are not extensively degraded into smaller fragments during low temperature proteolysis, changes in the acrylamide gel electrophoresis pattern showed that most ribosomal proteins are accessible to and are partially degraded by the proteases. Ribosome-bound nascent polypeptides are partially resistant to proteolysis at 0°, although they are totally digested at 37° or when the ribosomal subunit structure is disrupted by other means. Radioactivity incorporated into nascent chains during incubation times shorter than 3 min was mostly resistant to digestion at 0°. A larger fraction of the initial radioactivity became degraded in ribosomes which incorporated for longer times. In these ribosomes, the amount of radioactivity which was resistant to proteolysis was constant and independent of the initial value, which reflects the labeled length of the nascent chains. These results suggest that the growing end of the nascent polypeptide is resistant to digestion and is protected from proteolytic attack by the ribosomal structure. A pulse and chase experiment confirmed this suggestion, showing that the protected segment is located at the carboxy-terminal end of the nascent chain. The protected segment was contained in the large ribosomal subunit and had a length of ∼39 amino acid residues, as estimated by chromatography on Sephadex G-50.
TL;DR: Suspension cultures of Chinese hamster cells, which had stopped dividing and were arrested in G1 following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine.
Abstract: Suspension cultures of Chinese hamster cells (line CHO), which had stopped dividing and were arrested in G1 following growth to high cell concentrations in F-10 medium, could be induced to reinitiate DNA synthesis and to divide in synchrony upon addition of the appropriate amounts of isoleucine and glutamine. Both amino acids were required to initiate resumption of cell-cycle traverse. Deficiencies in other amino acids contained in F-10 medium did not result in accumulation of cells in G1, indicating a specific action produced by limiting quantities of isoleucine and glutamine. In the presence of sufficient glutamine, approximately 2 x 10-6 M isoleucine was required for all cells to initiate DNA synthesis in a population initially containing 1.5 x 105 cells/ml. Under similar conditions, about 4 x 10-6 M isoleucine was required for all G1-arrested cells to progress through cell division. In contrast, 1 x 10-4 M glutamine was necessary for maximum initiation of DNA synthesis in G1 cells, along with sufficient isoleucine. A technique for rapid production of G1-arrested cells is described in which cells from an exponentially growing population placed in F-10 medium deficient in both isoleucine and glutamine or isoleucine alone accumulated in G1 after 30 hr.
TL;DR: In stationary phase cells that had entered the developmental program leading to fruiting body construction, the rRNA turned over rapidly so that by the end of development at least 75% of the ribosomes fabricated during exponential growth had disappeared and had been replaced by new ones synthesized during the morphogenetic sequence.
Abstract: A method is described for the rapid separation of cellular slime mold (Dictyostelium discoideum) cells into nuclear and cytoplasmic fractions. Sucrose density sedimentation profiles of radioactivity from cells that had been grown for long or short periods in the presence of uridine-3H indicate very low levels of cross-contamination between the fractions. The nuclear fraction contains few, if any, ribosomes. In exponentially growing cells, at least 80% of the ribosomes were associated in polysomal complexes. No loss of counts from pre-labeled rRNA was observed during 2 generations (24 hr) of logarithmic growth and, within the polysomal complexes, the distributions of the preformed material and of rRNA synthesized during the 2 generations were identical. In stationary phase cells that had entered the developmental program leading to fruiting body construction, the rRNA turned over rapidly so that by the end of development at least 75% of the ribosomes fabricated during exponential growth had disappeared and had been replaced by new ones synthesized during the morphogenetic sequence. The preformed ribosomes disappeared preferentially from the monosomal contingent; the newly synthesized ribosomes appeared exclusively in the polysomal contingent and did not appear as monosomes in appreciable numbers for at least 6 hr. The possible significance of this wholesale replacement of ribosomes is discussed.
TL;DR: It is demonstrated that in the eosinophil the stacked Golgi cisternae participate in the segregation of secretory proteins and that the entire rough ER and all the Golgi bisternae are involved in the simultaneous segregation and packaging of several proteins.
Abstract: During their differentiation in the bone marrow, eosinophilic leukocytes synthesize a number of enzymes and package them into secretory granules. The pathway by which three enzymes (peroxidase, acid phosphatase, and arylsulfatase) are segregated and packaged into specific granules of eosinophils was investigated by cytochemistry and electron microscopy. During the myelocyte stage, peroxidase is present within (a) all rough ER cisternae, including transitional elements and the perinuclear cisterna; (b) clusters of smooth vesicles at the periphery of the Golgi complex; (c) all Golgi cisternae; and (d) all immature and mature specific granules. At later stages, after granule formation has ceased, peroxidase is not seen in ER or Golgi elements and is demonstrable only in granules. The distribution of acid phosphatase and arylsulfatase was similar, except that the reaction was more variable and fully condensed (mature) granules were not reactive. These results are in accord with the general pathway for intracellular transport of secretory proteins demonstrated in the pancreas exocrine cell by Palade and coworkers. The findings also demonstrate (a) that in the eosinophil the stacked Golgi cisternae participate in the segregation of secretory proteins and (b) that the entire rough ER and all the Golgi cisternae are involved in the simultaneous segregation and packaging of several proteins.
TL;DR: The directions of the microtubules in the cytoplasm of HeLa cells found by electron microscopy are consistent with the aster-like configuration predicted from study of LSM.
Abstract: Particulate structures in the cytoplasm of HeLa and other cultured cells in interphase undergo rapid individual linear displacements (long saltatory movements, LSM). By the use of time-lapse microscopy to locate saltating particles prior to fixation and histochemical examination of the cells, structures of several kinds have been shown to move in this manner. Elements that show LSM include lysosomes, pinosomes, ingested carbon particles, lipoidal granules, and unidentified particles that appear as bright objects in positive phase contrast. The pattern of movement of the particles suggests the presence of linear guiding elements radially disposed from the cytocenter (centriole region). The participation of microtubules in these movements is inferred from the observation that LSM cease after treatment with drugs which depolymerize microtubules, i.e., colchicine, Vinblastine, and podophyllin. The directions of the microtubules in the cytoplasm of HeLa cells found by electron microscopy are consistent with the aster-like configuration predicted from study of LSM. Further support for this arrangement of cytoplasmic microtubules is provided by light microscope observations of colchicine-sensitive radial arrays of acid phosphatase granules in the cytoplasm of some cell lines.
TL;DR: The present investigation has been made possible in part by the use of a fixative particularly effective in preserving cytoplasmic membranes and lipids, and giving them high stainability and differential contrast.
Abstract: The synthesis of "very low" density lipoprotein in liver cells is characterized by the fact that the synthesized products, mostly triglycerides, are processed in the form of discrete, size-limited granules or globules, about 400 A in diameter. The present investigation has been made possible in part by the use of a fixative (OsO4 in bidistilled H2O at pH 6.0, in the absence of electrolytes) particularly effective in preserving cytoplasmic membranes and lipids, and giving them high stainability and differential contrast. Under these technical conditions, the lipoprotein granules retain their morphology and high density to electrons practically unaltered, and may serve as tracers in determining their route of transport from the sites of synthesis, starting at the rough-smooth ER junctions, to the lumen of Golgi concentrating vesicles. From the observations, it may be deduced that, along with lipoprotein granule synthesis and transport, there are also production and transfer of new membranes in the form of tubular extensions of smooth ER network which, by progressive fusion and coalescence, participate in the elaboration of fenestrated plates and solid Golgi sacs. In contradistinction to the entire process of liver lipoprotein granule synthesis, transport, and segregation, as reported in the present paper, appears to constitute a developmental sequence which includes the following communicating compartments, in consecutive order: cisternae of rough ER where proteins and possibly phospholipids are synthesized, smooth ER network where triglycerides are synthesized and transported in the form of dense granules, fusion of smooth ER tubular extensions into Golgi fenestrated plates, and further coalescence into solid Golgi sacs, ending in the segregation of the granules in appended concentrating vesicles, or detached "secretory vesicles." It seems that it is this progressive evolution in growth and configuration of membranes which is reflected in the so called polarity, from forming to mature faces, of the Golgi apparatus.
TL;DR: Observations provide further evidence that wound fibroblasts do not arise from hematogenous precursors and, therefore, must arise from adjacent connective tissue cells.
Abstract: Healing skin wounds were studied in a series of parabiotic rats. The femurs of one parabiont of each pair were shielded whilst both animals were given 800 r from a Co60 source. The animals were wounded 3 days after irradiation. Each animal with partially shielded marrow was then given tritiated thymidine intraperitoneally daily while the cross-circulation was arrested by clamping. After the thymidine-3H had cleared the blood, the clamp was released. Animals were sacrificed, and wounds were prepared for radioautography 1, 2, and 6 days after wounding. In the wounds of the shielded animals thymidine-3H was observed in epidermis, endothelium, leukocytes, fibroblasts, and mast cells. Only neutrophilic leukocytes, monocytes, and lymphocytes were labeled, as determined by light and electron microscope radioautography, in the wounds of each nonshielded parabiont. None of the many fibroblasts present were found to contain label in the wounds of the nonshielded parabionts through the 6 day period. These observations provide further evidence that wound fibroblasts do not arise from hematogenous precursors and, therefore, must arise from adjacent connective tissue cells.
TL;DR: The usefulness of the freeze-etching technique in the study of membranes clearly requires that the controversy over the location of the cleavage plane in membranes be resolved.
Abstract: Freeze-etching promises to be a valuable technique for the ultrastructural study of cells and subcellular components. The tissue specimen is cleaved while frozen by liquid nitrogen, and the exposed faces are replicated by platinum-carbon shadowing while under high vacuum. Thus, this technique eliminates the artifacts of thin-section microscopy produced by fixation, dehydration, embedding, and heavy metal staining. Through the use of this technique, a new feature of cell membrane ultrastructure has been revealed. Cleavage of cell membranes results in the appearance of regularly spaced globular units approximately 85 A in diameter on the exposed membrane face (1, 2). The chemical composition and possible functional properties of the globular units are as yet unknown, and there is also some controversy as to the anatomical location of these structures within the membrane. This controversy is based on a fundamental disagreement as to which face of the membrane is exposed by the cleavage process. Moor and M/ihlethaler (1) and others (3, 4) have suggested that membranes are cleaved along planes which expose either their true outer or cytoplasmic surfaces. According to this view, the 85-A particles are located on the outside surface of the cell membrane; some are also present on the cytoplasmic side of the membrane. It has even been suggested that the particles extend completely through the thickness of the membrane (5). However, studies by Branton (2, 6) question the above interpretations. An analysis of the appearance of membranes in freeze-etched preparations and freeze-cleaving experiments with lipid bilayers has led Branton to conclude that the freeze-fracture process actually splits membranes along a plane within the membrane itself rather than cleaving along its outer or cytoplasmic surface. If this is the case, then the globular particles seen on freeze-cleaved membrane faces would be located somewhere within the interior of the membrane rather than directly on the surface. The usefulness of the freeze-etching technique in the study of membranes clearly requires that the controversy over the location of the cleavage plane in membranes be resolved. One solution would be to label the outer or inner surface of tile membrane with molecules of known structure which can be identified in the freeze-etch prepara-
TL;DR: In 25–40% of sinusoidal cells, an electron-opaque reaction product is localized in segments of the endoplasmic reticulum, including the perinuclear cisternae, a few Golgi vesicles and saccules and in some large membrane-bounded granules.
Abstract: Endogenous peroxidase activity has been demonstrated in sections of rat liver fixed briefly by glutaraldehyde perfusion and incubated in Graham and Karnovsky's medium for cytochemical demonstration of peroxidase activity (29). In 25-40% of sinusoidal cells, an electron-opaque reaction product is localized in segments of the endoplasmic reticulum, including the perinuclear cisternae, a few Golgi vesicles and saccules and in some large membrane-bounded granules. This staining is abolished after prolonged fixation or boiling of tissue sections in glutaraldehyde, and in the absence of H(2)O(2) or DAB from the incubation medium. Furthermore, the reaction is inhibited completely by sodium azide and high concentrations of H(2)O(2), and partially by KCN and aminotriazole. Among the different cells in hepatic sinusoids, the nonphagocytic "fat-storing" cells (39) are always peroxidase negative, whereas the lining cells in process of erythrophagocytosis are consistently peroxidase positive. The possible biological significance of endogenous peroxidase in Kupffer cells is discussed. In addition, the uptake of exogenous horseradish peroxidase by Kupffer cells has been investigated. The exogenous tracer protein, which in contrast to endogenous peroxidase of Kupffer cells is not inhibited by prolonged aldehyde fixation, is taken up by micropinocytosis and remains confined to the lysosomal system of Kupffer cells. The significance of these observations in respect to some recent studies suggesting localization of exogenous peroxidases in the endoplasmic reticulum of Kupffer cells and peritoneal macrophages (22, 23) is briefly discussed.
TL;DR: Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm).
Abstract: Distinct ultrastructural differences exist at the neuromuscular junctions of red, white, and intermediate fibers of a mammalian twitch skeletal muscle (albino rat diaphragm). The primary criteria for recognizing the three fiber types are differences in fiber diameter, mitochondrial content, and width of the Z line. In the red fiber the neuromuscular relationship presents the least sarcoplasmic and axoplasmic surface at each contact. Points of contact are relatively discrete and separate, and axonal terminals are small and elliptical. The junctional folds are relatively shallow, sparse, and irregular in arrangement. Axoplasmic vesicles are moderate in number, and sarcoplasmic vesicles are sparse. In the white fiber long, flat axonal terminals present considerable axoplasmic surface. Vast sarcoplasmic surface area is created by long, branching, closely spaced junctional folds that may merge with folds at adjacent contacts to occupy a more continuous and widespread area. Axoplasmic and sarcoplasmic vesicles are numerous. Both axoplasmic and sarcoplasmic mitochondria of the white fiber usually contain intramitochondrial granules. The intermediate fiber has large axonal terminals that are associated with the most widely spaced and deepest junctional folds. In all three fiber types, the junctional sarcoplasm is rich in free ribosomes, cisternae of granular endoplasmic reticulum, and randomly distributed microtubules.
TL;DR: The techniques described permit the controlled production of large numbers of proliferating somatic cell hybrids in a relatively short period of time through the expedient of varying multiplicities of the parental cells and the total cell density.
Abstract: The techniques described permit the controlled production of large numbers of proliferating somatic cell hybrids in a relatively short period of time. Sendai virus is used to promote cell hybridization. s-propriolactone is employed as the inactivating agent of Sendai virus since it produces complete loss of viral infectivity while preserving viral fusion capacity. Cells are fused in monolayer, instead of in suspension, since fixing cells in two dimensions permits one to control cell contacts during the fusion event through the expedient of varying multiplicities of the parental cells and the total cell density. Under the conditions described, a several hundred fold increase in the number of hybrid clones obtained is seen as compared to the controls.
TL;DR: Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB), which revealed an increase in microbodies, mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna.
Abstract: Structural changes in endosperm cells of germinating castor beans were examined and complemented with a cytochemical analysis of staining with diaminobenzidine (DAB). Deposition of oxidized DAB occurred only in microbodies due to the presence of catalase, and in cell walls associated with peroxidase activity. Seedling development paralleled the disappearance of spherosomes (lipid bodies) and matrix of aleurone grains in endosperm cells. 6 to 7 days after germination, a cross-section through the endosperm contained cells in all stages of development and senescence beginning at the seed coat and progressing inward to the cotyledons. Part of this aging process involved vacuole formation by fusion of aleurone grain membranes. This coincided with an increase in microbodies (glyoxsomes), mitochondria, plastids with an elaborate tubular network, and the formation of a new protein body referred to as a dilated cisterna, which is structurally and biochemically distinct from microbodies although both apparently develop from rough endoplasmic reticulum (ER). In vacuolate cells microbodies are the most numerous organelle and are intimately associated with spherosomes and dilated cisternae. This phenomenon is discussed in relation to the biochemical activities of these organelles. Turnover of microbodies involves sequestration into autophagic vacuoles as intact organelles which still retain catalase activity. Crystalloids present in microbodies develop by condensation of matrix protein and are the principal site of catalase formerly in the matrix.
TL;DR: The technique of extracellular space tracing with horseradish peroxidase is adapted for labeling the transverse tubular system (T system) in rat heart and in atrial muscle the following two types of cells are found: those cells with a well-developed T system in which the tubular diameter is quite uniform and the orientation predominantly longitudinal and, (b) cells with no T system, but with aWell-developed L system.
Abstract: The technique of extracellular space tracing with horseradish peroxidase is adapted for labeling the transverse tubular system (T system) in rat heart. In rat ventricular muscle the T system shows extensive branching and remarkable tortuosity. The T system can only be defined operationally, since it does not display specific morphological features throughout its entire structure. Owing to branching of the T system, a sizable proportion of the apposition between the T system and L system (or closed system) occurs at the level of longitudinal branches of the T system and is not restricted to the Z line region. The regions of apposition between the T system and L system are analyzed in rat ventricular muscle and skeletal muscle (diaphragm) and compared with the intercellular tight junctions (nexuses) of heart muscle by the use of a photometric method. The over-all thickness of the nexus is significantly smaller than that of T-L junctions in both cardiac and skeletal muscles. The thickness of the membranes of the T and L systems are not significantly different in the two muscles, but the gap between both membranes is larger in the heart. In atrial muscle the following two types of cells are found: (a) those cells with a well-developed T system in which the tubular diameter is quite uniform and the orientation predominantly longitudinal and, (b) cells with no T system, but with a well-developed L system. Atrial cells possessing a T system are richly provided with specific granules and show little micropinocytotic activity, whereas cells devoid of T system show intense micropinocytotic activity and few specific granules. The possible functional implications of these findings are discussed.
TL;DR: Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids, consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents.
Abstract: Purified plasma membrane fractions from lactating bovine mammary glands and membranes of milk fat globules from the same source were similar in distribution and fatty acid composition of phospholipids. The sphingomyelin content of the phospholipid fraction of both membranes was higher than in these fractions from other cell components, β-carotene, a constituent characteristic of milk fat, was present in the lipid fraction of the plasma membrane. Cholesterol esters of plasma membrane were similar in fatty acid composition to those of milk fat globule membranes. Disc electrophoresis of either membrane preparation on polyacrylamide gels revealed a single major protein component characteristic of plasma membrane from other sources. Distinct morphological differences between plasma membrane and milk fat globule membranes were observed in both thin sections and in negatively stained material. Plasma membrane was vesicular in appearance while milk fat globule membranes had a platelike aspect. These observations are consistent with derivation of fat globule membrane from plasma membrane accompanied by structural rearrangement of membrane constituents.
TL;DR: The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum membranes, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids.
Abstract: Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium. The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized. The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel. For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied. The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids. Both membranous fractions had many proteins in common including some membrane-bound enzymes. Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude. The content of cytochrome b5 as well as of P-450 was markedly lower in the nuclear membranes. The nuclear membranes were found to have a higher buoyant density and to be richer in protein. The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected. Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments. Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences.
TL;DR: The fine structure of the germarium and the early oocyte is described and between 14 and 17 centrioles are found in the oocyte, by the time the 16 cell cluster of cells is surrounded by follicle cells (Stage 1).
Abstract: A cluster of centrioles has been found in the early Drosophila oocyte Since the oocyte is connected to 15 nurse cells by a system of intercellular bridges or ring canals, the possibility that the cluster of centrioles arose in the germarium from an intercellular migration of centrioles from the nurse cells to the oocyte was analyzed in serial sections for the electron microscope Initially, all of the 16 cells of the future egg chambers possess centrioles, which are located in a juxtanuclear position At the time the 16 cell cluster becomes arranged in a lens-shaped layer laterally across the germarium, the centrioles lose their juxtanuclear position and move towards the oocyte By the time the 16 cell cluster of cells is surrounded by follicle cells (Stage 1), between 14 and 17 centrioles are found in the oocyte Later, these centrioles become located between the oocyte nucleus and the follicle cell border and become aggregated into a cluster less than 15 µ in its largest dimension The fate of these centrioles in the oocyte is not known The fine structure of the germarium and the early oocyte is also described