Showing papers in "Journal of Cell Biology in 1979"

Involvement of histone H1 in the organization of the nucleosome and of the salt-dependent superstructures of chromatin.

Abstract: We describe the results of a systematic study, using electron microscopy, of the effects of ionic strength on the morphology of chromatin and of H1-depleted chromatin. With increasing ionic strength, chromatin folds up progressively from a filament of nucleosomes at approximately 1 mM monovalent salt through some intermediate higher-order helical structures (Thoma, F., and T. Koller, 1977, Cell 12:101-107) with a fairly constant pitch but increasing numbers of nucleosomes per turn, until finally at 60 mM (or else in approximately 0.3 mM Mg++) a thick fiber of 250 A diameter is formed, corresponding to a structurally well-organized but not perfectly regular superhelix or solenoid of pitch approximately 110 A as described by Finch and Klug (1976, Proc. Natl. Acad. Sci. U.S.A. 73:1897-1901). The numbers of nucleosomes per turn of the helical structures agree well with those which can be calculated from the light-scattering data of Campbell et al. (1978, Nucleic Acids Res. 5:1571-1580). H1-depleted chromatin also condenses with increasing ionic strength but not so densely as chromatin and not into a definite structure with a well-defined fiber direction. At very low ionic strengths, nucleosomes are present in chromatin but not in H1-depleted chromatin which has the form of an unravelled filament. At somewhat higher ionic strengths (greater than 5 mM triethanolamine chloride), nucleosomes are visible in both types of specimen but the fine details are different. In chromatin containing H1, the DNA enters and leaves the nucleosome on the same side but in chromatin depleted of H1 the entrance and exit points are much more random and more or less on opposite sides of the nucleosome. We conclude that H1 stabilizes the nucleosome and is located in the region of the exit and entry points of the DNA. This result is correlated with biochemical and x-ray crystallographic results on the internal structure of the nucleosome core to give a picture of a nucleosome in which H1 is bound to the unique region on a complete two-turn, 166 base pair particle (Fig. 15). In the formation of higher-order structures, these regions on neighboring nucleosomes come closer together so that an H1 polymer may be formed in the center of the superhelical structures.

Synaptic vesicle exocytosis captured by quick freezing and correlated with quantal transmitter release.

Abstract: We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.

Epithelioid cell cultures from rat small intestine. Characterization by morphologic and immunologic criteria.

Abstract: Rat small intestinal epithelial cell lines have been established in vitro and subcultured serially for periods up to 6 mo. These cells have an epithelioid morphology, grow as monolayers of closely opposed polygonal cells, and during the logarithmic phase of growth have a population doubling time of 19--22 h. Ultrastructural studies revealed the presence of microvilli, tight junctions, an extensive Golgi complex, and the presence of extracellular amorphous material similar in appearance to isolated basement membrane. These cells exhibit a number of features characteristic of normal cells in culture; namely, a normal rat diploid karyotype, strong density inhibition of growth, lack of growth in soft agar, and a low plating efficiency when seeded at low density. They did not produce tumors when injected in syngeneic animals. Immunochemical studies were performed to determine their origin using antisera prepared against rat small intestinal crypt cell plasma membrane, brush border membrane of villus cells and isolated sucrase-isomaltase complex. Antigenic determinants specific for small intestinal epithelial (crypt and villus) cells were demonstrated on the surface of the epithelioid cells, but they lacked immunological determinants specific for differentiated villus cells. An antiserum specifically staining extracellular material surrounding the cells cultured in vitro demonstrated cross-reactivity to basement membrane in rat intestinal frozen sections. It is concluded that the cultured epithelioid cells have features of undifferentiated small intestinal crypt cells.

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins

Abstract: Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.

Anionic sites in the glomerular basement membrane. In vivo and in vitro localization to the laminae rarae by cationic probes.

Abstract: Cationized ferritin (CF) of narrow pI range (7.3-7.5) and the basic dye ruthenium red (RR) have been used as cationic probes to partially characterize anionic sites previously demonstrated in the glomerular basement membrane (GBM). When CF was given i.v. to normal rats and the left kidney was fixed by perfusion 15 min thereafter, clusters of CF molecules were found throughout the lamina rara interna (LRI), lamina rara externa (LRE), and mesangial matrix distributed at regular (approximately 60 nm) intervals. When kidneys were perfused with aldehyde fixative containing RR, small (20 nm) RR-stained particles were seen in the same locations distributed with the same 60 nm repeating pattern, forming a quasiregular, lattice-like arrangement. Fine (approximately 3 nm) filaments connected the sites and extended between them and the membranes of adjoining endothelial and epithelial cells. When CF was given i.v. followed by perfusion with RR in situ, both probes localized to the same sites. CF remained firmly bound after prolonged perfusion with 0.1-0.2 M KCl or NaCl. It was displaced by perfusion with buffers of high ionic strength (0.4-0.5 M KCl) or pH (less than 3.0 or greater than 10.0). CF also bound (clustered at approximately 60 nm intervals) to isolated GBM's, and binding was lost when such isolated GBM's were treated with buffers of high ionic strength or pH. These experiments demonstrate the existence of a quasi-regular, lattice-like network of anionic sites in the LRI and LRE and the mesangial matrix. The sites are demonstrable in vivo (by CF binding), in fixed kidneys (by RR staining), and in isolated GBM's (by CF binding). The results obtained with CF show that the binding of CF (and probably also RR) to the laminae rarae is electrostatic in nature since it is displaced by treatment with buffers of high ionic strength or pH. With RR the sites resemble in morphology and staining properties the proteoglycan particles found in connective tissue matrices and in association with basement membranes in several other locations.

Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431.

Abstract: We have prepared a conjugate of epidermal growth factor (EGF) and ferritin that retains substantial binding affinity for cell receptors and is biologically active. Glutaraldehyde-activated EGF was covalently linked to ferritin to produce a conjugate that contained EGF and ferritin in a 1:1 molar ratio. The conjugate was separated from free ferritin by affinity chromatography using antibodies to EGF. Monolayers of human epithelioid carcinoma cells (A-431) were incubated with EGF:ferritin at 4 degrees C and processed for transmission electron microscopy. Under these conditions, approximately 6 X 10(5) molecules of EGF:ferritin bound to the plasma membrane of each cell. In the presence of excess native EGF, the number of bound ferritin particles was reduced by 99%, indicating that EGF:ferritin binds specifically to cellular EGF receptors. At 37 degrees C, cell-bound EGF:ferritin rapidly redistributed in the plane of the plasma membrane to form small groups that were subsequently internalized into pinocytic vesicles. By 2.5 min at 37 degrees C, 32% of the cell-bound EGF:ferritin was localized in vesicles. After 2.5 min, there was a decrease in the proportion of conjugate in vesicles with a concomitant accumulation of EGF:ferritin in multivesicular bodies. By 30 min, 84% of the conjugate was located in structures morphologically identified as multivesicular bodies or lysosomes. These results are consistent with other morphological and biochemical studies utilizing 125I-EGF and fluorescein-conjugated EGF.

Microtrabecular lattice of the cytoplasmic ground substance. Artifact or reality.

Abstract: The cytoplasmic ground substance of cultured cells prepared for high voltage transmission electron microscopy (glutaraldehyde/osmium fixed, alcohol or acetone dehydrated, critical-point dried) consists of slender (3-6 nm Diam) strands--the microtrabeculae (55)--that form an irregular three-dimensional lattice (the microtrabecular lattice). The microtrabeculae interconnect the membranous and nonmembranous organelles and are confluent with the cortices of the cytoplast. The lattice is found in all portions of the cytoplast of all cultured cells examined. The possibility that the lattice structure is an artifact of specimen preparation has been tested by (a) subjecting whole cultured cells (WI-38, NRK, chick embryo fibroblasts) to various chemical (aldehydes, osmium tetroxide) and nonchemical (freezing) fixation schedules, (b) examination of model systems (erythrocytes, protein solutions), (c) substantiating the relaibility of critical-point drying, and (d) comparing images of whole cells with conventionally prepared (plastic-embedded) cells. The lattice structure is preserved by chemical and nonchemical fixation, though alterations in ultrastructure can occur especially after prolonged exposure to osmium tetroxide. The critical-point method for drying specimens appears to be reliable as is the freeze-drying method. The discrepancies between images of plastic-embedded and sectioned cells, and images of whole, critical-point dried cells appear to be related, in part, to the electron-scattering properties of the embedding resin. The described observations indicate that the microtrabecular lattice seen in electron micrographs closely represents the nonrandom structure of the cytoplasmic ground substance of living cultured cells.

The periodic association of MAP2 with brain microtubules in vitro.

Abstract: Several high molecular weight polypeptides have been shown to quantitatively copurify with brain tubulin during cycles of in vitro assembly-disassembly. These microtubule-associated proteins (MAPs) have been shown to influence the rate and extent of microtubule assembly in vitro. We report here that a heat-stable fraction highly enriched for one of the MAPs, MAP2 (mol wt approximately 300,000 daltons), devoid of MAP1 (mol wt approximately 350,000 daltons), has been purified from calf neurotubules. This MAP2 fraction stoichiometrically promotes microtubule assembly, lowering the critical concentration for tubulin assembly to 0.05 mg/ml. Microtubules saturated with MAP2 contain MAP2 and tubulin in a molar ratio of approximately 1 mole of MAP2 to 9 moles of tubulin dimer. Electron microscopy of thin sections of the MAP2-saturated microtubules fixed in the presence of tannic acid demonstrates a striking axial periodicity of 32 +/- 8 nm.

Organization of acetylcholine receptors in quick-frozen, deep-etched, and rotary-replicated Torpedo postsynaptic membrane

Abstract: The receptor-rich postsynaptic membrane of the elasmobranch electric organ was fixed by quick-freezing and then viewed by freeze-fracture, deep-etching and rotary-replication. Traditional freeze-fracture revealed a distinct, geometrical pattern of shallow 8.5-nm bumps on the E fracture-face, similar to the lattice which has been seen before in chemically fixed material, but seen less clearly than after quick-freezing. Fracture plus deep-etching brought into view on the true outside of this membrane a similar geometrical pattern of 8.5-nm projections rising out of the membrane surface. The individual projections looked like structures that have been seen in negatively stained or deep-etched membrane fragments and have been identified as individual acetylcholine receptor molecules. The surface protrusions were twice as abundant as the large intramembrane particles that characterize the fracture faces of this membrane, which have also been considered to be receptor molecules. Particle counts have always been too low to match the estimates of postsynaptic receptor density derived from physiological and biochemical studies; counts of surface projections, however, more closely matched these estimates. Rotary-replication of quick-frozen, etched postsynaptic membranes enhanced the visibility of these surface protuberances and illustrated that they often occur in dimers, tetramers, and ordered rows. The variations in these surface patterns suggested that in vivo, receptors in the postsynaptic membrane may tend to pack into "liquid crystals" which constantly appear, flow, and disappear in the fluid environment of the membrane. Additionally, deep-etching revealed a distinct web of cytoplasmic filaments beneath the postsynaptic membrane, and revealed the basal lamina above it; and delineated possible points of contact between these structures and the membrane proper.

Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.

Abstract: The development of clusters of acetylcholine (ACh) receptors at newly formed synapses between embryonic chick spinal cord and muscle cells grown in vitro has been studied by iontophoretic mapping with ACh. A semi-automated technique using on-line computer analysis of ACh responses and a photographic system to record the position of each ACh application permit the rapid construction of extensive and detailed maps of ACh sensitivity. Clusters of receptors, evident as peaks of ACh sensitivity, are present on many uninnervated myotubes. The distribution of ACh sensitivity closely parallels the distribution of 125I-alpha-bungarotoxin binding sites on the same muscle cell. In all cases where individual myotubes were adequately mapped before and after synapse formation, ingrowing axons induced new clusters of receptors rather than seeking out preexisting clusters. Synapses can form at active growth cones within 3 h of nerve-muscle contact. New receptor clusters can appear beneath neurites within a few hours. Many of the uninnervated clusters on innervated myotubes disappear with time. In contrast, receptor clusters on uninnervated myotubes remain in the same location for many hours. Synaptic clusters and clusters on uninervated myotubes are stable even though individual receptors are metabolized rapidly. The morphology of several identified sites of transmitter release was examined. At the scanning EM level, synapses appeared as small, rough-surfaced varicosities with filopodia that radiated outwards over the muscle surface. One synapse was studied by transmission EM. Acetylcholinesterase and a basement lamina were present within the synaptic cleft.

Acetylcholine receptors in regenerating muscle accumulate at original synaptic sites in the absence of the nerve.

Abstract: We examined the role of nerve terminals in organizing acetylcholine receptors on regenerating skeletal-muscle fibers. When muscle fibers are damaged, they degenerate and are phagocytized, but their basal lamina sheaths survive. New myofibers form within the original basal lamina sheaths, and they become innervated precisely at the original synaptic sites on the sheaths. After denervating and damaging muscle, we allowed myofibers to regenerate but deliberately prevented reinnervation. The distribution of acetylcholine receptors on regenerating myofibers was determined by histological methods, using [125I] alpha-bungarotoxin or horseradish peroxidase-alpha-bungarotoxin; original synaptic sites on the basal lamina sheaths were marked by cholinesterase stain. By one month after damage to the muscle, the new myofibers have accumulations of acetylcholine receptors that are selectively localized to the original synaptic sites. The density of the receptors at these sites is the same as at normal neuromuscular junctions. Folds in the myofiber surface resembling junctional folds at normal neuromuscular junctions also occur at original synaptic sites in the absence of nerve terminals. Our results demonstrate that the biochemical and structural organization of the subsynaptic membrane in regenerating muscle is directed by structures that remain at synaptic sites after removal of the nerve.

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction.

Abstract: We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.

Transport of proteins into mitochondria and chloroplasts.

Abstract: It has been well established that mitochondria and chloroplasts are not autonomous organeUes. These organelles are capable of nucleic acid and protein synthesis, but many soluble and membrane proteins that become localized in them are initially synthesized on cytoplasmic ribosomes (cf. references 47 and 122). A fundamental question of considerable current interest is: How is the transport of such proteins through the delimiting membranes of the organelle envelope accomplished? A related concern is whether there are discriminatory mechanisms to ensure the specific incorporation of a product of cytoplasmic protein synthesis into its destined organellar location. Although the problem of transport of proteins into mitochondria and chloroplast has been investigated for more than a decade, most of the early studies employed indirect approaches and yielded results that were equivocal. Only with the recent advances in techniques for rnRNA purification (8, 88), in vitro protein synthesis (92, 110), and isolation of purified mitochondria (89, 121) and chloroplasts (98, 100) with intact outer membranes has it been possible to analyze in vitro transport of proteins into these organelles in a systematic and direct manner. Interest in this area has also been stimulated and encouraged by advances made in the understanding of how secretory proteins are transferred across microsomal membranes (cf. reference 22) and how certain plant and microbial toxins are transported into cells (cf. references 104 and 108). This review will focus on recent papers dealing with the subcellular locations of the sites of synthesis of mitochondrial and chloroplast proteins and with the transport of these proteins into the respective organelles.

Retention of differentiated properties in an established dog kidney epithelial cell line (MDCK).

Abstract: Madin-Darby canine kidney (MDCK) cells grown in tissue culture have the morphological properties of distal tubular epithelial cells, form tight junctions, and lack several proximal tubular enzyme markers. Adenylate cyclase in these cells was stimulated by vasopressin, oxytocin, prostaglandins E1 and E2, glucagon, and cholera toxin. Hormone-stimulated adenylate cyclase activity in isolated membrane preparations was dependent on low concentrations of GTP and had the MgCl2 and pH optima expected for the kidney enzyme. The results, as well as the demonstration of enhanced hemicyst formation induced by cyclic AMP, suggest that the MDCK cell line has retained the differentiated properties of the kidney epithelial cell of origin. When MDCK cells were injected into baby nude mice, continuous nodule growth was observed until adulthood was attained. Histological studies revealed the presence of two cell types: normal mouse fibroblasts which comprise 80--90% of the solid nodule mass, and MDCK cells, which formed epithelial sheets lining internal fluid-filled glands. Electron microscope analysis showed that the mucosal surfaces of the cells were characterized by microvilli which faced the lumen of the glands, that adjacent MDCK cells were joined by tight junctions, and that the serosal surfaces of the epithelial sheets were characterized by smooth plasma membranes which were lined by a continuous basement membrane. These observations lead to the conclusion that the MDCK cells retain regional differentiation of their plasma membranes and the ability to regenerate kidney tubule-like structures in vivo.

Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor.

Abstract: Horseradish peroxidase (HRP) uptake was used to measure fluid-phase pinocytosis in monolayers of human epithelioid carcinoma cells (A-431). Histochemistry confirmed that cell-associated HRP was restricted to intracellular vesicles. Biochemical methods showed that HRP uptake in control cultures was directly proportional to the duration of exposure. The addition of low concentrations of epidermal growth factor (EGF) to the incubation media produced a 10-fold increase in the initial rate of pinocytosis. The EGF effect was rapid (within 30 s) but transient; the rate of pinocytosis returned to control levels within 15 min. Metabolic inhibitors reduced the EGF-stimulated rate of pinocytosis by greater than 90%. A conjugate of EGF and ferritin (F:EGF) was used to simultaneously compare the intracellular locations of EGF and HRP. Much of F:EGF was internalized in approximately 100-nm vesicles, while most of the HRP was located in much larger vesicles (range 0.1--1.2 micrometer) which also contained F:EGF. The tumor-promoter 12-0-tetradecanoyl-phorbol-13-acetate, which shares several biological activities with EGF, was also effective in stimulating an increase in the rate of pinocytosis.

The surface epithelium of teleostean fish gills. Cellular and junctional adaptations of the chloride cell in relation to salt adaptation.

Abstract: Various species of teleostean fishes were adapted to fresh or salt water and their gill surface epithelium was examined using several techniques of electron microscopy. In both fresh and salt water the branchial epithelium is mostly covered by flat respiratory cells. They are characterized by unusual outer membrane fracture faces containing intramembranous particles and pits in various stages of ordered aggregation. Freeze fracture studies showed that the tight junctions between respiratory cells are made of several interconnecting strands, probably representing high resistance junctions. The organization of intramembranous elements and the morphological characteristics of the junctions do not vary in relation to the external salinity. Towards the base of the secondary gill lamellae, the layer of respiratory cells is interrupted by mitochondria-rich cells ("chloride cells"), also linked to respiratory cells by multistranded junctions. There is a fundamental reorganization of the chloride cells associated with salt water adaptation. In salt water young adjacent chloride cells send interdigitations into preexisting chloride cells. The apex of the seawater chloride cell is therefore part of a mosaic of sister cells linked to surrounding respiratory cells by multistranded junctions. The chloride cells are linked to each other by shallow junctions made of only one strand and permeable to lanthanum. It is therefore suggested that salt water adaptation triggers a cellular reorganization of the epithelium in such a way that leaky junctions (a low resistance pathway) appear at the apex of the chloride cells. Chloride cells are characterized by an extensive tubular reticulum which is an extension of the basolateral plasma membrane. It is made of repeating units and is the site of numerous ion pumps. The presence of shallow junctions in sea water-adapted fish makes it possible for the reticulum to contact the external milieu. In contrast in the freshwater-adapted fish the chloride cell's tubular reticulum is separated by deep apical junctions from the external environment. Based on these observations we discuss how solutes could transfer across the epithelium.

Isolation of the pericellular matrix of human fibroblast cultures.

Abstract: The pericellular matrix of human fibroblast cultures was isolated, using sequential extraction with sodium deoxycholate and hypotonic buffer in the presence of protease inhibitor. The matrix attached to the growth substratum had a "sackcloth-like" structure as seen by phase contrast, immunofluorescence, and scanning electron microscopy, and it had a vaguely filamentous ultrastructure similar to that seen in intact cell layers. The matrix consisted of hyaluronic acid and heparan sulfate as the major glycosaminoglycan components and fibronectin and procollagen as major polypeptides as shown by metabolic labeling, gel electrophoresis, immunofluorescence, and collagenase digestion. This pericellular matrix can be regarded as an in vitro equivalent of the loose connective tissue matrix.

Rapid induction of morphological changes in human carcinoma cells A-431 by epidermal growth factors.

Abstract: The morphological effects of epidermal growth factor (EGF) on human carcinoma cells A-431 have been examined by scanning electron microscopy. These flat polygonal cells normally exhibit only small membrane folds, but show extensive ruffling and extension of filopodia within 5 min of exposure to EGF at 37 degrees C. This ruffling activity is transient, subsiding within another 5--15 min, but several other changes in surface morphology follow. Within the first hour of exposure to the hormone, the cell surface becomes exceedingly smooth and the nuclei seem to protrude above the plane of the otherwise thin monolayer, giving the cells a "fried egg" appearance. Cells at the edges of colonies gradually retract from the substrate, leading to reorganization, by 12 h, of the monolayer into multilayered colonies. EGF thus induces both rapid and long-term alterations in the morphology of these epidermoid cells.

Fibroblast cellular and plasma fibronectins are similar but not identical.

Abstract: Fibronectins are multimeric, adhesive glycoproteins present on cell surfaces and circulating in blood. Cellular fibronectin produced by fibroblasts in vitro and fibronectin isolated from plasma are known to be very similar immunologically and biochemically. We investigated whether or not they are identifical. Purified chicken and human cell-surface fibronectins are 150-fold more active in hemagglutination of fixed erythrocytes than plasma fibronectins. Cell-surface fibronectin is also 50-fold more active in restoring a more normal morphology to transformed cells originally missing the protein. However, in two other assays that measure cell attachment to collagen and cell spreading, cell-surface and plasma fibronectins have identical specific activities. In sodium dodecyl sulfate polyacrylamide gels, the subunits of human and chicken plasma fibronectins have significantly smaller apparent subunit molecular weights than cellular fibronectins present on cell surfaces or secreted into culture media. These differences are also present in a characteristic large subfragment of both forms of fibronectin after limited proteolysis by trypsin. We conclude that by both biological and biochemical criteria, cellular and plasma fibronectins are similar but not identical.

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation.

Abstract: Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.

An electron microscope autoradiographic study of the carbohydrate recognition systems in rat liver. I. Distribution of 125I-ligands among the liver cell types.

Abstract: Electron microscope autoradiography was used to study the cellular localization of seven glycoproteins rapidly cleared from the circulating plasma of rats and taken up by the liver. 1 and 15 min after intravenous administration of the 125I-glycoproteins, livers were fixed in situ by perfusion and processed for autoradiography. Autoradiographic grains in the developed sections were found to represent the intact 125I-ligand. A quantitative analysis of the distribution and concentration (density) of autoradiographic grains over the three major cell types of the liver was then performed. Three molecules, asialo-fetuin, asialo-orosomucoid, and lactosaminated RNase A dimer, the oligosaccharide chains of which terminate in galactose residues, were bound and internalized almost exclusively (greater than 90%) by hepatocytes. Conversely, four molecules, the oligosaccharide chains of which terminate in either N-acetyl-glucosamine (agalacto-orosomucoid) or mannose (ahexosamino-orosomucoid, preputial beta-glucuronidase, and mannobiosaminated RNase A dimer), were specifically bound and internalized by cells lining the blood sinusoids--that is, by Kupffer cells and endothelial cells. Endothelial cells were two to six times more active (on a cell volume basis) than were Kupffer cells in the internalization of these four 125I-ligands. Mannose and N-acetylglucosamine-terminated glycoproteins competed with each other for uptake into either endothelial cells or Kupffer cells, indicating that a single system recognized mannose or N-acetyl-glucosamine residues. Finally, agalacto-orosomucoid and ahexosamino-orosomucoid were also associated with hepatocytes, but competition experiments utilizing excess asialo-orosomucoid demonstrated that residual galactosyl residues were responsible for this association.

Cytochalasin inhibits the rate of elongation of actin filament fragments

Abstract: Submicromolar concentrations of cytochalasin inhibit the rate of assembly of highly purified dictyostelium discoideum actin, using a cytochalasin concentration range in which the final extent of assembly is minimally affected. Cytochalasin D is a more effective inhibitor than cytochalasin B, which is in keeping with the effects that have been reported on cell motility and with binding to a class of high-affinity binding sites from human erythrocyte membranes (Lin and Lin. 1978. J. Biol. CHem. 253:1415; Lin and Lin. 1979. Proc. Natl. Acad. Sci. U.S.A. 76:2345); 5x10(-7) M cytochalasin B lowers it to 70 percent of the control value, whereas 10(-7) M cytochalasin B lowers the rate to 25 percent. Fragments of F-actin were used to increase the rate of assembly fivefold by providing more filament ends on to which monomers could add. Under these conditions, cytochalasin has an even more dramatic effect on the assembly rate; the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 2x10(-7) M and 10(-8) M, respectively. The assembly rate is most sensitive to cytochalasin when actin assembly is carried out in the absence of ATP (with 3 mM ADP present to stabilize the actin). In this case, the concentrations of cytochalasin B and cytochalasin D required for half-maximal inhibition are 4x10(-8) M and 1x10(-9) M, respectively. A scatchard plot has been obtained using [(3)H]cytochalasin B binding to F-actin in the absence of ATP. The K(d) from this plot (approximately 4x10(-8) M) agrees well with the concentration of cytochalasin B required for half-maximal inhibition of the rate of assembly under these conditions. The number of cytochalasin binding sites is roughly one per F-actin filament, suggesting that cytochalasin has a specific action on actin filament ends.

Identification and organization of the components in the isolated microvillus cytoskeleton.

Abstract: We have examined the effects of ATP and deoxycholate (DOC) on the cytoskeletal organization of Triton-demembranated microvilli (MV) isolated from chicken intestine brush borders. Isolated MV are composed of a core of tightly bundled microfilaments from which arms project laterally to the plasma membrane with a 33-nm periodicity. These lateral arms spiral around the core microfilaments as a helix with a 25 degrees pitch. Demembranated MV consist of four polypeptides with mol wt of 110,000, 95,000, 68,000, and 42,000, present in molar ratios of 1.1:1.6:1.3:10.0. After addition of 50 microM ATP and 0.1 mM Mg++, the cytoskeletons are organized as a tight bundle of microfilaments from which lateral arms are missing. In these ATP-treated cytoskeletons, the 110-kdalton polypeptide is reduced in amount and the 95,000, 68,000, and 42,000 polypeptides are present in a 1.3:1.2:10.0 ratio. In contrast, after incubation with 0.5% DOC, the core microfilaments are no longer tightly bundled yet the lateral arms remain attached with a distinct 33-nm periodicity. These DOC-treated cytoskeletons are depleted of the 95,000 and 68,000 polypeptides and are composed of the 110,000 and 42,000 polypeptides in a 2:10 molar ratio. These results suggest that the microfilaments are associated into a core bundle by the 95- and 68-kdalton polypeptides and from this core bundle project the lateral arms composed of the 110-kdalton polypeptide.

Autoradiographic identification of acetylcholine in the rabbit retina.

Abstract: Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of both [3H]phosphorylcholine and [3H]acetylcholine. With time, [3H]phosphorylcholine proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15-min exposure to 0.3 microM [3H]choline were incubated for a subsequent hour under chase conditions designed either to retain newly synthesized acetylcholine within synapses or to promote its release. At the end of this time the two groups of retinas were found to contain equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed silver grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, silver grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of the cells of the ganglion cell layer, and in two bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water, and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections were found to faithfully reflect those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine to have been synthesized only by cells of the inner retina. Taken together, these results indicate that the photoreceptor and ganglion cells are distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. They imply that the only neurons to release acetylcholine within the rabbit retina are a small group of probable amacrine cells.

Calcium-induced asymmetrical beating of triton-demembranated sea urchin sperm flagella.

Abstract: Asymmetrical bending waves can be obtained by reactivating demembranated sea urchin spermatozoa at high Ca2+ concentrations Moving-film flash photography shows that asymmetrical flagellar bending waves are associated with premature termination of the growth of the bends in one direction (the reverse bends) while the bends in the opposite direction (the principal bends) grow for one full beat cycle, and with unequal rates of growth of principal and reverse bends The relative proportions of these two components of asymmetry are highly variable The increased angle in the principal bend is compensated by a decreased angle in the reverse bend, so that there is no change in mean bend angle; the wavelength and beat frequency are also independent of the degree of asymmetry This new information is still insufficient to identify a particular mechanism for Ca2+-induced asymmetry When a developing bend stops growing before initiation of growth of a new bend in the same direction, a modification of the sliding between tubules in the distal portion of the flagellum is required This modification can be described as a superposition of synchronous sliding on the metachronous sliding associated with propagating bending waves Synchronous sliding is particularly evident in highly asymmetrical flagella, but is probably not the cause of asymmetry The control of metachronous sliding appears to be unaffected by the superposition of synchronous sliding

Electron probe analysis of vascular smooth muscle. Composition of mitochondria, nuclei, and cytoplasm.

Abstract: Electron probe analysis of dry cryosections was used to determine the composition of the cytoplasm and organelles of rabbit portal-anterior mesenteric vein (PAMV) smooth muscle. All analytical values given are in mmol/kg wt +/- SEM. Cytoplasmic concentrations in normal, resting muscles were: K, 611 +/- 1.7; Na, 167 +/- 2.7; Cl, 278 +/- 1.0; Mg, 36 +/- 1.1; Ca, 1.9 +/- 0.5; and P, 247 +/- 1.1. Hence, the sum of intracellular Na + K exceeded cytoplasmic Cl by 500 mmol/kg dry wt, while the calculated total, nondiffusible solute was approximately 50 mmol/kg. Cytoplasmic K and Cl were increased in smooth muscles incubated in solutions containing an excess (80 mM) of KCl. Nuclear and cytoplasmic Na and Ca concentrations were not significantly different. The mitochondrial Ca content in normal fibers was low, 0.8 +/- 0.5, and there was no evidence of mitochondrial Ca sequestration in muscles frozen after a K contracture lasint 30 min. Transmitochondrial gradients of K, Na, and Cl were small (0.9--1.2). In damaged fibers, massive mitochondrial Ca accumulation of up to 2 mol/kg dry wt in granule form and associated with P could be demonstrated. Our findings suggest (a) that the nonDonnan distribution of Cl in smooth muscle is not caused by sequestration in organelles, and that considerations of osmotic equilibrium and electroneutrality suggest the existence of unidentified nondiffusible anions in smooth muscle, (b) that nuclei do not contain concentrations of Na or Ca in excess of cytoplasmic levels, (c) that mitochondria in PAMV smooth muscle do not play a major role in regulating cytoplasmic Ca during physiological levels of contraction but can be massively Ca loaded in damaged cells, and (d) that the in situ transmitochondrial gradients of K, Na, and Cl do not show these ions to be distributed according to a large electromotive Donnan force.

Agonist-induced myopathy at the neuromuscular junction is mediated by calcium.

Abstract: Inactivation of cholinesterases at mammalian neuromuscular junctions (nmj) produces extensive muscle "necrosis." Fine-structurally, this myopathy begins near the nmj with an increase in large-diameter vesicles in the soleplasm, the dissolution of Z-disks, dilation of mitochondria, destruction of sarcoplasmic reticulum, and often a highly specific contracture of the muscle under the endplate. Since a Ca++-activated protease which specifically removes Z-disks is known to exist in mammalian skeletal muscle, we tested the possibility that the myopathy after esterase inactivation is due to the prolongation of acetylcholine lifetime and thus of Ca++ influx. We first produced the myopathy near endplates by inactivating esterases with diisopropylfluorophosphate (DFP) followed by nerve stimulation for 1--2 h in vitro. The myopathy was later mimicked by bath application of carbamylcholine without esterase inhibitors. This myopathy could be prevented by inactivating the acetylcholine receptors (AChR) with alpha-bungarotoxin (alpha-BGT) or by removing Ca++ from the bath with EGTA. These results favor the hypothesis that esterase inhibition leads to an agonist-induced myopathy, which is mediated by Ca++ and requires an intact AChR.

Distribution of fetal bovine serum fibronectin and endogenous rat cell fibronectin in extracellular matrix.

Abstract: Normal rat kidney cells were cultured in medium supplemented with normal fetal bovine serum (FBS) or FBS depleted of fibronectin. The cell surface fibronectin of these cultures was visualized by indirect immunofluorescence using species-specific antisera for either rat fibronectin or bovine fibronectin. Anti-rat-fibronectin revealed fibrillar structures on the cells grown in either normal medium or fibronectin-depleted medium. Anti-bovine fibronectin revealed similar fibrillar networks, but only on the cells grown in medium containing bovine fibronectin. Staining in each case was abolished by absorption with the homologous antigen. It appears that exogenous fibronectin was incorporated into the same structures as endogenous fibronectin. This finding suggests that circulating fibronectin may serve as a building block for the assembly of extracellular matrix, possibly by cells which are incapable of synthesizing it.

Epidermal cells adhere preferentially to type IV (basement membrane) collagen.

Abstract: Epidermal cells from adult guinea pig skin attach and differentiate preferentially on substrates of type IV (basement membrane) collagen, compared to those of types I--III collagen. In contrast, guinea pig dermal fibroblasts attach equally well to all four collagen substrates. Fibronectin mediates the attachment of fibroblasts but not of epidermal cells to collagen.

Organization of neuronal microtubules in the nematode Caenorhabditis elegans.

Abstract: We have studied the organization of microtubules in neurons of the nematode Caenorhabditis elegans. Six neurons, which we call the microtubule cells, contain bundles of darkly staining microtubules which can be followed easily in serial-section electron micrographs. Reconstruction of individual microtubules in these cells indicate that most, if not all, microtubules are short compared with the length of the cell process. Average microtubule length varies characteristically with cell type. The arrangement of microtubules gives an overall polarity to each bundle: the distal ends of the microtubles are on the outside of the bundle, whereas the proximal ends are preferentially inside. The distal and proximal ends each have a characteristic appearance indicating that these microtubules may have a polarity of their own. Short microtubules in processes of other neurons in C. elegans have also been observed.