Showing papers in "Journal of Cell Biology in 1976"

125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts.

Abstract: 125I-labeled human epidermal growth factor (hEGF) binds in a specific and saturable manner to human fibroblasts. At 37 degrees C, the cell-bound 125I-hEGF initially may be recovered in a native form by acid extraction; upon subsequent incubation, the cell-bound 125I-hEGF is degraded very rapidly, with the appearance in the medium of 125I-monoiodotyrosine. At 0 degrees C, cell-bound 125I-hEGF is not degraded but slowly dissociates from the cell. The data are consistent with a mechanism in which 125I-hEGF initially is bound to the cell surface and subsequently is internlized before degradation. The degradation is blocked by inhibitors of metabolic energy production (azide, cyanide, dinitrophenol), some protease inhibitors (Tos-Lys-CH2Cl, benzyl guanidobenzoate), a lysosomotropic agent (chloroquine) various local anesthetics (cocaine, lidocaine, procaine), and ammonium chloride. After the binding and degradation of 125I-hEGF the fibroblasts are no longer able to rebind fresh hormone. The binding capacity of these cells is restored by incubation in a serum-containing medium; this restoration is inhibited by cycloheximide or actinomycin D.

Gap junctions between the oocyte and companion follicle cells in the mammalian ovary.

Abstract: Tracer and freeze-fracture electron microscopy of the ovaries of neonatal rat and adult mouse, rat, rabbit, and primate have revealed the presence of gap junctions between follicle cells and oocytes. The junctional connections are found at the ends of follicle cell projections which traverse the zona pellucida and terminate upon microvilli and evenly contoured nonmicrovillar regions of the oolemma. Gap junctions are often seen associated with a macula adherens type of junction. The gap junctions occasionally consist of minute ovoid plaques, but nore frequently appear as rectilinear single- or multiple-row aggregates of particles on the P-face or pits on the E-face. The functional significance of follicle cell-oocyte gap junctions is discussed with respect to the regulation of meiosis and luteinization.

Membrane flow during pinocytosis. A stereologic analysis.

Abstract: HRP has been used as a cytochemical marker for a sterelogic analysis of pinocytic vesicles and secondary lysosomes in cultivated macrophages and L cells. Evidence is presented that the diaminobenzidine technique (a) detects all vaculoes containing encyme and (b) distinguishes between incoming pinocytic vesicles and those which have fused with pre-existing lysosomes to form secondary lososomes. The HRP reactive pinocytic vesicle spaces fills completely within 5 min after exposure to enzyme, while the secondary lysosome compartment is saturated in 45--60 min. The size distribution of sectioned (profile) vaculoe diameters was measured at equilibrium and converted to actual (spherical) dimensions using a technique modified from Dr. S. D. Wicksell. The most important findings in this study have to do with the rate at which pinocytosed fluid and surface membrane move into the cell and on their subsequent fate. Each minute macrophages form at least 125 pinocytic vesicles having a fractional vol of 0.43% of the cell's volume and a fractional area of 3.1% of the cell's surface area. The fractional volume and surface area flux rates for L cells were 0.05% and 0.8% per minute respectively. Macrophages and L cells thus interiorize the equivalent of their cell surface area every 33 and 125 min. During a 3-period, the size of the secondary lysosome compartment remains constant and represents 2.5% of the cell volume and 18% of the surface area. Each hour, therefore, the volume and surface area of incoming vesicles is 10 times greater than the dimensions of the secondary lysosomes in both macrophages and L cells. This implies a rapid reduction in vesicle size during the formation of the secondary lysosome and the egress of pinocytosed fluid from the vacuole and the cell. In addition, we postulate that membrane components of the vacuole are subsequently recycled back to the cell surface.

Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect.

Abstract: Gallotannin, consisting mainly of low molecular weight esters such as penta- and hexagalloylglucoses (commercially available as tannic acid produced from Turkish nutgall), can be used for increasing and diversifying tissue contrast in electron microscopy. When applied on tissue specimens previously fixed by conventional methods (aldehydes and OsO4), the low molecular weight galloylglucoses (LMGG) penetrate satisfactorily the cells and induce general high contrast with fine delineation of extra- and intracellular structures, especially membranes. In some features, additional details of their intimate configuration are revealed. Various experimental conditions tested indicate that the LMGG display a complex effect on fixed tissues: they act primarily as a mordant between osmium-treated structures and lead, and concomitantly stabilize some tissue components against extraction incurred during dehydration and subsequent processing. Experiments with aldehyde blocking reagents (sodium borohydride and glycine) suggested that the LMGG mordanting effect is not dependent on residual aldehydes groups in tissues.

Membrane receptors for hormones and neurotransmitters.

Abstract: Receptors for peptide hormones and neurotransmitters are integral components of the plasma membrane of cells which serve to couple the external milieu to the intracellular regulators of metabolism. These macromolecules are usually high molecular weight glycoproteins, and in many cases appear to have more than one subunit capable of binding the hormone. The interaction of the hormone or neurotransmitter with its receptor is rapid, reversible, and of high affinity and specificity. Many receptors exhibit cooperative properties in hormone binding or biological function. The concentration of receptors on the membrane is a function of continued synthesis and degradation, and may be altered by a variety of factors including the hormone itself. The fluid mosaic nature of the membrane may allow hormone receptors and effectors to exist in free floating states. Further investigations of the hormone-receptor interaction will no doubt yield new insights into both the mechanism of hormone action and membrane structure and function.

Fluorescent antibody localization of myosin in the cytoplasm, cleavage furrow, and mitotic spindle of human cells.

Abstract: We have studied the distribution of myosin molecules in human cells using myosin-specific antibody coupled with fluorescent dyes. Rabbits were immunized with platelet myosin or myosin rod. They produced antisera which precipitated only myosin among all the components in crude platelet extracts. From these antisera we isolated immunoglobulin-G (IgG) and conjugated it with tetramethylrhodamine or fluorescein. We separated IgG with 2-5 fluorochromes per molecule from both under- and over-conjugated IgG by ion exchange chromatography and used it to stain acetone-treated cells. The following controls established the specificity of the staining patterns: (a) staining with labeled preimmune IgG; (b) staining with labeled immune IgG adsorbed with purified myosin; (c) staining with labeled immune IgG mixed with either unlabeled preimmune or immune serum; and (d) staining with labeled antibody purified by affinity chromatography. In blood smears, only the cytoplasm of platelets and leukocytes stained. In spread Enson and HeLa cells, stress fibers stained strongly in closely spaced 0.5 mum spots. The cytoplasm stained uniformly in those cells presumed to be motile before acetone treatment. In dividing HeLa cells there was a high concentration of myosin-specific staining in the vicinity of the contractole ring and in the mitotic spindle, especially the region between the chromosomes and the poles. We detected no staining of erythrocytes, or nuclei of leukocytes and cultured cells, or the surface of platelets and cultured cells.

Actin, alpha-actinin, and tropomyosin interaction in the structural organization of actin filaments in nonmuscle cells.

Abstract: During the spreading of a population of rat embryo cells, approximately 40% of the cells develop a strikingly regular network which precedes the formation of the straight actin filament bundles seen in the fully spread out cells. Immunofluorescence studies with antibodies specific for the skeletal muscle structural proteins actin, alpha-actinin, and tropomyosin indicate that this network is composed of foci containing actin and alpha-actinin, connected by tropomyosin-associated actin filaments. Actin filaments, having both tropomyosin and alpha-actinin associated with them, are also seen to extend from the vertices of this network to the edges of the cell. These results demonstrate a specific interaction of alpha-actinin and tropomyosin with actin filaments during the assembly and organization of the actin filament bundles of tissue culture cells. The three-dimensional network they form may be regarded as the structural precursor and the vertices of this network as the organization centers of the ultimately formed actin filament bundles of the fully spread out cells.

A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei.

Abstract: A modified procedure for the isolation of a nuclear pore complex-lamina fraction from rat liver nuclei is described. Evidence is provided that the isolated lamina, a 150-A thick, proteinaceous structure, apposes the inner nuclear envelope membrane, connecting nuclear pore complexes and surrounding the entire nucleus.

Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions

Abstract: The distribution and quantitation of 125I-alpha-bungarotoxin (alpha-BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetanic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, alpha-BTX was bound to approximately 90% of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of alpha-BTX relative to this membrane was determined to be 46,000 +/- 27% sites/mum2.

A highly ordered ring of membrane-associated filaments in budding yeast.

Abstract: In Saccharomyces cerevisiae, a highly ordered ring of 10-nm filaments is intimately associated with the plasma membrane within the neck of the bud. The ring is formed during early bud emergence and disappears when cytokinesis begins.

Platelet factors stimulate fibroblasts and smooth muscle cells quiescent in plasma serum to proliferate

Abstract: Whole blood serum is widely recognized as essential for the growth of diploid cells in culture. Dermal fibroblasts and arterial smooth muscle cells fail to proliferate in culture in the presence of serum derived from platelet-poor plasma. Platelet-poor plasma serum is capable of maintaining monkey arterial smooth muscle cells quiescent in culture at either low (1.5 x 10(3)) or high (2.0 x 10(4)) population densities. The proportion of cell traversing the cell cycle under these conditions was approximately 3%. Equal numbers of quiescent smooth muscle cells initiated DNA synthesis and cell division when treated with whole blood serum or with an equivalent quantity of platelet-poor plasma serum supplemented with a factor(s) derived from a supernate obtained after exposure of human platelets to purified thrombin in vitro.

Subcellular localization of B apoprotein of plasma lipoproteins in rat liver.

Abstract: Multispecific antigen-binding fragments (Fab) from rabbit antisera against rat very low density lipoproteins (VLDL) and Fab against rat low density lipoproteins that were monospecific for the B apoprotein were conjugated to horseradish peroxidase. Conjugates were incubated with 6-mum frozen sections from fresh and perfusion-fixed livers and with tissue chopper sections (40 mum thick) from perfusion-fixed livers. In the light microscope, specific reaction product was present in all hepatocytes of experimental sections as intense brown to black spots whose locations corresponded to the distribution of the Golgi apparatus: along the bile canaliculi, near the nuclei, and between the nuclei and bile canaliculi. Perfusion fixation with formaldehyde produced satisfactory ultrastructural preservation with retention of lipoprotein antigenic determinants. In the electron microscope, patches of cisternae and ribosomes of the rough endoplasmic reticulum (ER) and particularly its smooth-surfaced ends, vesicles located between the rough ER and the Golgi apparatus, the Golgi apparatus and its secretory vesicles and VLDL particles in the space of Disse all bore reaction product. The tubules and vesicles of typical hepatocyte smooth ER did not contain reaction product, nor did the osmiophilic particles contained therin. The localization obtained in this study together with other evidence suggests a sequence for the biosynthesis of VLDL that differs in some respects from that proposed by others: (a) the triglyceride-rich particle originates in smooth ER where triglycerides are synthesized; (b) at the junction of the smooth and rough ER the particle receives apoproteins synthesized in the rough ER; (c) specialized tubules transport the particle, now a nascent lipoprotein, to the Golgi apparatus where concentration occurs in secretory vesicles; (d) secretory vesicles move to the sinusoidal surface where the particles are secreted into the space of Disse by fusion of the vesicular membrane with the plasma membrane of the hepatocyte.

Teleost chloride cell. II. Autoradiographic localization of gill Na,K-ATPase in killifish Fundulus heteroclitus adapted to low and high salinity environments.

Abstract: The specific binding and inhibitory action of (3H)ouabain were employed to localize transport Na,K-ATPase in the euryhaline teleost gill, a NaCl-transporting osmoregulatory tissue in which both enzyme activity and transepithelial transport vary with environmental salinity. In killifish fully adapted to 10%, 100%, or 200% seawater, the gills were internally perfused and externally irrigated in situ. After suitable internal or external exposure to (3H)ouabain, individual gill arches were excised for Na,K-ATPase assay, measurement of radiolabel binding, or quantitative high-resolution autoradiography. Internal exposure to 50 muM ouabain resulted in essentially complete enzyme inhibition, and binding paralleled the increases in enzyme activity at higher salinities; in contrast, external exposure gave minimal and erratic results consistent with leakage of external ouabain into interstitial fluid. (3H)Ouabain autoradiographs demonstrated that, irrespective of exposure or salinity, most of the gill binding was associated with chloride cell. These cells increased in size and number with salinity and, at the subcellular level, the distribution pattern for bound ouabain was always identical to that for the amplified basal-lateral (tubular system) membrane. The combined physiologicmorphologic results constitute final direct proof that chloride cells are the primary site of gill Na,K-ATPase. More important, they provide convincing evidence for unexpected increases in basal-lateral enzyme at higher salinities and thus raise a fundamental objection to the long-postulated role of the Na pump in secretory NaCl transport.

Collagenous bone matrix-induced endochondral ossification hemopoiesis.

Abstract: Transplantation of collagenous matrix from the rat diaphyseal bone to subcutaneous sites resulted in new bone formation by an endochondral sequence. Functional bone marrow develops within the newly formed ossicle. On day 1, the implanted matrix was a discrete conglomerate with fibrin clot and polymorphonuclear leukocytes. By day 3, the leukocytes disappeared, and this event was followed by migration and close apposition of fibroblast cell surface to the collagenous matrix. This initial matrix-membrane interaction culminated in differentiation of fibroblasts to chondroblasts and osteoblasts. The calcification of the hypertrophied chondrocytes and new bone formation were correlated with increased alkaline phosphatase activity and 45Ca incorporation. The ingrowth of capillaries on day 9 resulted in chondrolysis and osteogenesis. Further remodelling of bony trabeculae by osteoclasts resulted in an ossicle of cancellous bone. This was followed by emergence of extravascular islands of hemocytoblasts and their differentiation into functional bone marrow with erythropoietic and granulopoietic elements and megakaryocytes in the ossicle. The onset and maintenance of erythropoiesis in the induced bone marrow were monitored by 59Fe incorporation into protein-bound heme. These findings imply a role for extracellular collagenous matrix in cell differentiation.

Segmental differentiations of cell junctions in the vascular endothelium. Arteries and veins.

Abstract: A systematic survey of endothelial junctions in elastic (aorta) and muscular (mesenteric) arteries and in medium (renal and mesenteric) and large (cava inferior) size veins has been carried out in the rat using freeze-cleaved preparations. The arterial endothelium is provided with a complex of occluding and communicating junctions (gap junctions) comparable to, though less elaborate than, that described in arterioles. The particles of the occluding junctions behave like "single unit" particles and have the tendency to remain on B faces upon membrane cleavage. In the venous endothelium the junctions take the form of long occluding junctions with few associated communicating junctions (maculae communicantes). As in arterial endothelium, the junctional particles appear preferentially on B faces in cleaved preparations. These structures, although continuous over long distances, are interrupted focally by areas in which the junctional elements are similar to those found in venules: the ridges and grooves are short, discontinuous, randomly distributed along the general line of cell contact, and often particle-free. In muscular arteries two unusual types of junctions are encountered. Both are disposed in loops over short distances along the perimeter of the cell. One type appears to be a strectched-out version of the usual combination of occluding and communcating junctions of the arterial endothelium (this type is also occasionally encountered in the venous endothelium). The other type is reminiscent of the septate junctions found in the epithelia of invertebrates but the apparent similarity remains to be checked by further work.

Improved procedures for immunoferritin labeling of ultrathin frozen sections.

Abstract: In employing fixed frozen ultrathin sections as substrates for immunoferritin labeling of intracellular antigens, we have found that conventional glutaraldehyde fixation sometimes permits very little specific staining of the sections, either because it inactivates certain protein antigens, or because it renders them inaccessible to the antibody stains. We have developed several fixation procedures that are chemically milder and allow a uniform but less extensive cross-linking of the specimen. With these procedures and precautions in the handling of the more fragile frozen sections, excellent structural preservation and specific immunoferritin labeling has been achieved with several systems.

Reversible particle movements associated with unstacking and restacking of chloroplast membranes in vitro.

Abstract: Freeze-fracture and freeze-etch techniques have been employed to study the supramolecular structure of isolated spinach chloroplast membranes and to monitor structural changes associated with in vitro unstacking and restacking of these membranes. High-resolution particle size histograms prepared from the four fracture faces of normal chloroplast membranes reveal the presence of four distinct categories of intramembranous particles that are nonrandomly distributed between grana and stroma membranes. The large surface particles show a one to one relationship with the EF-face particles. Since the distribution of these particles between grana and stroma membranes coincides with the distribution of photosystem II (PS II) activity, it is argued that they could be structural equivalents of PS II complexes. An interpretative model depicting the structural relationship between all categories of particles is presented. Experimental unstacking of chloroplast membranes in low-salt medium for at least 45 min leads to a reorganization of the lamellae and to a concomitant intermixing of the different categories of membrane particles by means of translational movements in the plane of the membrane. In vitro restacking of such experimentally unstacked chloroplast membranes can be achieved by adding 2-20 mM MgCl2 or 100-200 mM NaCl to the membrane suspension. Membranes allowed to restack for at least 1 h at room temperature demonstrate a resegregation of the EF-face particles into the newly formed stacked membrane regions to yield a pattern and a size distribution nearly indistinguishable from the normally stacked controls. Restacking occurs in two steps: a rapid adhesion of adjoining stromal membrane surfaces with little particle movement, and a slower diffusion of additional large intramembranous particles into the stacked regions where they become trapped. Chlorophyll a:chlorophyll b ratios of membrane fraction obtained from normal, unstacked, and restacked membranes show that the particle movements are paralleled by movements of pigment molecules. The directed and reversible movements of membrane particles in isolated chloroplasts are compared with those reported for particles of plasma membranes.

pH-dependent binding of immunoglobulins to intestinal cells of the neonatal rat.

Abstract: Rat and rabbit IgG immunoglobulins conjugated to horseradiah peroxidase as a histochemical marker bind at 0 degrees C to the luminal surface of absorptive cells in isolated segments of jejunum from 10-12-day old rats. Binding is observed at pH 6.0, near the normal luminal pH of the duodenum and jejunum at this age, but not at pH 7.4. Furthermore, no binding occurs when cells are exposed at pH 6.0 to either free peroxidase or peroxidase conjugated to chicken or sheep IgG immunoglobulins or bovine serum albumin. The sensitivity of binding to pH suggests a means whereby immunoglobulins which are selectively absorbed by the cells can be released efficiently at the abluminal surface.

Interactions of actin, myosin, and a new actin-binding protein of rabbit pulmonary macrophages. II. Role in cytoplasmic movement and phagocytosis.

Abstract: Actin and myosin of rabbit pulmonary macrophages are influenced by two other proteins. A protein cofactor is required for the actin activation of macrophage myosin Mg2 ATPase activity, and a high molecular weight actin-binding protein aggregates actin filaments (Stossel T.P., and J.H. Hartwig. 1975. J. Biol. Chem. 250:5706-5711)9 When warmed in 0.34 M sucrose solution containing Mg2-ATP and dithiothreitol, these four proteins interact cooperatively. Acin-binding protein in the presence of actin causes the actin to form a gel, which liquifies when cooled. The myosin contracts the gel into an aggregate, and the rate of aggregation is accelerated by the cofactor. Therefore, we believe that these four proteins also effec the temperature-dependent gelation and aggregation of crude sucrose extracts pulmonary macrophages containing Mg2-ATP and dithiothreitol. The gelled extracts are composed of tangled filaments. Relative to homogenates of resting macrophages, the distribution of actin-binding protein in homogenates of phagocytizing macrophages is altered such that 2-6 times more actin-binding protein is soluble. Sucrose extracts of phagocytizing macrophages gel more rapidly than extracts of resting macrophages. Phagocytosis by pulmonary macrophages involves the formation of peripheral pseudopods containing filaments. The findings suggest that the actin-binding protein initiates a cooperative interaction of contractile proteins to generate cytoplasmic gelation, and that phagocytosis influences the behavior of the actin-binding protein.

Ultrastructural differences during embryonic cell death in normal and peripherally deprived ciliary ganglia.

Abstract: Normally occurring neuron death and that brought about by prior removal of the peripheral target organ was studied ultrastructurally in embryonic chick ciliary ganglion in order to better understand the mechanism of cell death in this system. Before the period of cell death, all neurons in the normal ganglion developed a well-organized rough endoplasmic reticulum (RER) which coincided with peripheral synapse formation. None of the peripherally deprived neurons underwent this change, suggesting that some interaction with the periphery, possibly synapse formation, triggered them into the secretory state. Cell death in peripherally deprived neurons was signalled by nuclear changes followed by freeing of ribosomes from polysomes and RER and presumably cessation of protein synthesis. In contrast, normal cell death was brought about by dilation of the RER with eventual cytoplasmic disruption, nuclear changes appearing only secondarily. It is suggested that failure to form or maintain peripheral synapses could result in the accumulation of transmission-related proteins with consequent cisternal dilation, and eventual cell death.

Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers

Abstract: Improved media have reduced the amount of serum protein required for clonal growth of normal human and chicken fibroblast-like cells. In the presence of limiting amounts of serum protein, attachment of colonies to tissue culture plastic surfaces is weak. Treatment of the culture surface with polylysine or other basic polymers causes the cells to adhere much more tightly. Growth is also improved on the surfaces treated with basic polymers, and further reductions in the concentration of serum as possible. At sufficiently low protein concentrations, growth of some types of cells is totally dependent on the use of a treated surface. Several different types of normal human and chicken fibroblast-like cells show improved growth on polylysine-coated surfaces, but no improvement was obtained in growth of a line of SV-40 transformed WI-38 cells. Acidic and neutral polymers are generally inactive. Collagen and gelatin improve growth slightly, but the effect is much less than that obtained with basic polymers. Both natural and synthetic polymers with an excess of basic groups are active, including histone, polyarginine, polyhistidine, polylysine, polyornithine, and protamine. The only critical requirement appears to be a polymer that carries a positive charge at a physiological pH.

Morphological changes in the neuritic growth cone and target neuron during synaptic junction development in culture.

Abstract: Our object was to characterize the morphological changes occurring in pre- and postsynaptic elements during their initial contact and subsequent maturation into typical synaptic profiles. Neurons from superior cervical ganglia (SCG) of perinatal rats were freed of their supporting cells and established as isolated cells in culture. To these were added explants of embryonic rat thoracic spinal cord to allow interaction between outgrowing cord neurites and the isolated autonomic neurons. Time of initial contact was assessed by light microscopy; at timed intervals thereafter, cultures were fixed for electron microscopy. Upon contact, growth cone filopodia became extensively applied to the SCG neuronal plasmalemma and manifested numerous punctate regions in which the apposing plasma membranes were separated by only 7-10 nm. The Golgi apparatus of the target neuron hypertrophied, and its production of coated vesicles increased. Similar vesicles were seen in continuity with the SCG plasmalemma near the close contact site; their apparent contribution of a region of postsynaptic membrane with undercoating was considered to be the first definitive sign of synapse formation. Tracer work with peroxidase and ferritin confirmed that the traffic of coated vesicles within the neuronal soma is largely from Golgi region to somal surface. Subsequent to the appearance of postsynaptic density, the form and content of the growth cone was altered by the loss of filopodia and the appearance of synaptic vesicles which gradually became clustered opposite the postsynaptic density. As the synapse matured, synaptic vesicles increased in number, cleft width and content increased, presynaptic density appeared, branched membranous reticulum became greatly diminished, and most lysosomal structures disappeared. Coated vesicles continued to be associated with the postsynaptic membrane at all stages of maturation. The incorporation of Golgi-derived vesicles into discrete regions of the cell membrane could provide the mechanism for confining specific characteristics of the neuronal membrane to the synaptic region.

Studies of excitable membranes. II. A comparison of specializations at neuromuscular junctions and nonjunctional sarcolemmas of mammalian fast and slow twitch muscle fibers.

Abstract: Mammalian fast and slow twitch skeletal muscles are compared by freeze-fracture, thick and thin sectioning, and histochemical techniques using conventional and high voltage electron microscopy. Despite gross morphological differences in endplate structure visualized at relatively low magnifications in this sections, rat extensor digitorum longus (EDL) (fast twitch) and soleus (slow twitch) fibers cannot be distinguished on the basis of size, number, or distribution of molecular specializations of the pre- and postsynaptic junctional membranes exposed by freeze fracturing. Specializations in the cortex of the juxtaneuronal portions of the junctional folds are revealed by high voltage electron stereomicroscopy as a branching, ladder-like filamentous network associated with the putative acetylcholline receptor complexes. These filaments are considered to be involved in restricting the mobility of receptor proteins to the perineuronal aspects of the postynaptic membrane. Although the junctional membranes of both EDL and soleus appear similar, a differential specialization of the secondary synaptic cleft was noted. The extracellular matrix in the bottom of soleus clefts was observed as an ordered system of filamentous "combs," These filamentous arrays have not been detected in EDL junctions. Examination of the extrajunctional sarcolemmas of EDL and soleus reveal additional differences which may be correlated with variations in electrical and contractile properties. For example, particle aggregates termed "square arrays" previously described in the sarcolemmas of some fibers of the rat diaphragm were observed in large numbers in sarcolemmas of EDL fibers but were seldom encountered in soleus fibers. These gross compositional differences in the membranes are discussed in the light of functional differences between fiber types.

Involvement of vesicle coat material in casein secretion and surface regeneration.

Abstract: The ultrastructure of the apical zone of lactating rat mammary epithelial cells was studied with emphasis on vesicle coat structures. Typical 40-60 nm ID "coated vesicles" were abundant, frequently associated with the internal filamentous plasma membrane coat or in direct continuity with secretory vesicles (SV) or plasma membrane proper. Bristle coats partially or totally covered membranes of secretory vesicles identified by their casein micelle content. This coat survived SV isolation. Exocytotic fusion of SV membranes and release of the casein micelles was observed. Frequently, regularly arranged bristle coat structures were identified in those regions of the plasma membrane that were involved in exocytotic processes. Both coated and uncoated surfaces of the casein-containing vesicles, as well as typical "coated vesicles", were frequently associated with microtubules and/or microfilaments. We suggest that coat materials of vesicles are related or identical to components of the internal coat of the surface membrane and that new plasma membrane and associated internal coat is produced concomitantly by fusion and integration of bristle coat moieties. Postexocytotic association of secreted casein micelles with the cell surface, mediated by finely filamentous extensions, provided a marker for the integrated vesicle membrane. An arrangement of SV with the inner surface of the plasma membrane is described which is characterized by regularly spaced, heabily stained membrane to membrane cross-bridges (pre-exocytotic attachment plaques). Such membrane-interconnecting elements may represent a form of coat structure important to recognition and interaction of membrane surfaces.

Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions.

Abstract: The aqueous extract of the venom glands of black widow spiders was fractionated on a column of Sephadex G-200 and then on a column of DEAE-Sephadex A-50 pH 8.2. A protein fraction was obtained that caused a great increase in the frequency of occurrence of miniature end plate potentials at the frog neuromuscular junction, and caused swelling of the nerve terminals and depleted them of their vesicles. The fraction consists of a least four protein components that are similar in their molecular weights (about 130,000) and isoelectric points (ranging from pH 5.2 to 5.5) and are immunologically indistinguishable. It contains no sugar residues and has little or no lipolytic or proteolytic activity. The fraction is toxic to mice and is different from the fractions that act on houseflies, the crayfish stretch receptor and the cockroach heart. It seems pure enough to warrant a detailed study of its site and mode of action.

A filamentous cytoskeleton in vertebrate smooth muscle fibers.

Abstract: There are three classes of myofilaments in vertebrate smooth muscle fibers. The thin filaments correspond to actin and the thick filaments are identified with myosin. The third class of myofilaments (100 A diam) is distinguished from both the actin and the myosin on the basis of fine structure, solubility, and pattern of localization in the muscle fibers. Direct structural evidence is presented to show that the 100A filament constitute an integrated filamentous network with the dense bodies in the sarcoplasm, and that they are not connected to either the actin or myosin filaments. Examination of (a) isolated dense bodies, (b) series of consecutive sections through the dense bodies, and (c) redistributed dense bodies in stretched muscle fibers supports this conclusion. It follows that the 100-A filaments complexes constitute a structrally distinct filamentous network. Analysis of polyacrylamide gels after electrophoresis of cell fractions that are enriched with respect to the 100-A filaments shows the presence of a new muscle protein with a molecular weight of 55,000. This protein can form filamentous segments that closely resemble in structure the native, isolated 100-A filaments. The results indicate that the filamentous network has a structure and composition that distinguish it from the actin and myosin in vertebrate smooth muscle.

Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils.

Abstract: In human peripheral blood polymorphonuclear leukocytes and lymphocytes, GSH-oxidizing agents promote the movement of surface-bound concanavalin A (Con A) into caps and inhibit the assembly of microtubules (MT) that is normally induced by Con A binding. Con A capping and inhibition of MT assembly occur when GSH levels in cell suspensions are decreased by 30-70%, and return to GSH to control levels is accompanied by the appearance of cytoplasmic MT and by inhibition of the capping response with Con A. Oxidation of GSH markedly stimulates the hexose monophosphate shunt, and regeneration of GSH occurs rapidly. The data indicate that MT cannot be assembled or maintained in the face of decreased GSH levels. Thus, GSH homeostasis becomes critical during physiological events such as phagocytosis which simultaneously induce the assembly of MT and the production of agents like H2O2 that can oxidize GSH.

Vitamin-A-induced mucous metaplasia. An in vitro system for modulating tight and gap junction differentiation.

Abstract: Stratified squamous epithelia from 14-day chick embryo shank skin contain rare tight-junctional strands and only small gap junctions. Exposure of this tissue to retinoic acid (vitamin-A) (20 U/ml) in organ culture, however, induces mucous metaplasia, accompanied by tight-junction formation and gap-junction growth; untreated specimens continue to keratinize. To investigate sequential stages of junctional assembly and growth, we examined thin sections and freeze-fracture replicas at daily intervals for 3 days. During the metaplastic process, tight junctions assemble in midepidermal and upper regions, beginning on day 1 and becoming maximal on day 3. Two tight-junctional patterns could be tentatively identified as contributing to the emergence of fully formed zonulae occludentes: (a) the formation of individual ridges along the margins of gap junctions; (b) de novo generation of continuous ramifying strands by fusion of short strand segments and linear particulate aggregates near cellular apices. Gap junction enlargement, already maximal at day 1, occurs primarily three to four cell layers deep. Growth appears to occur by annexation of islands of 20-40 8.5-nm particles into larger lattices of islands separated by particle-free aisles. Eventually, a single gap junction may occupy much of the exposed membrane face in freeze-fractured tissue, but during apical migration of the cells such junctions disappear. The vitamin- A chick-skin system is presented as a responsive model for the controlled study of junction assembly.

Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.

Abstract: In order to better characterize the new rapid staining method for flow cytofluorometry proposed by Krishan, we have tested its stability and several other properties, and have carried out a quantitative comparison of the fluorescence histograms obtained using propidium iodide or the acriflavine-Feulgen staining procedure. Using a human hematopoietic cell line in the logarithmic phase of growth, and analyzing the data by means of a mathematical method we have devised, we found that the fluorescence intentsity of cells stained with propidium iodide remains stable for at least 48 h; it is insensitive to dye concentration between 0.025 and 0.10 mg/ml (37-150 muM); it is not affected by incubation with ribonuclease before staining; propidium iodide in 0.1% sodium citrate remains stable for at least 20 days; and quantitative estimates of the fractions of cells in the different phases of the cell cycle are in good agreement with those obtained from acriflavine-Feulgen staining and from autoradiography after pulse labeling with tritiated thymidine. We conclude that this method is useful for the measurement of relative DNA content by flow cytofluorometry, although modifications in the technique are necessary for some cell types which grow in monolayers.

The role of actin in the temperature-dependent gelation and contraction of extracts of Acanthamoeba.

Abstract: The temperature-dependent assembly and the interaction of Acanthamoeba contractile proteins have been studied in a crude extract. A cold extract of soluble proteins from Acanthamoeba castellanii is prepared by homogenizing the cells in a sucrose-ATP-ethyleneglycol-bis-(beta-aminoethyl ether) N,N'-tetraacetic acid buffer and centrifuging at 136,000 g for 1 h. When this supernate of soluble proteins is warmed to room temperature, it forms a solid gel. Upon standing at room temperature, the gel slowly contracts and squeezes out soluble components. The rates of gelation and contraction are both highly temperature dependent, with activation energies of about 20 kcal per mol. Gel formation is dependent upon the presence of ATP and Mg++. Low concentrations of Ca++ accelerate the contractile phase of this phenomenon. The major protein component of the gel is actin. It is associated with myosin, cofactor, a high molecular weight protein tentatively identfied as actin-binding protein, and several other unidentified proteins. Actin has been purified from these gels and was found to be capable of forming a solid gel when polymerized in the presence of ATP, MgCl3, and KCL. The rate of purified actin polymerication is very temperature dependent and is accelerated by the addition of fragments of muscle actin filaments. These data suggest that Acanthamoeba contractile proteins have a dual role in the cell; they may generate the forces for cellular movements and also act as cytoskeletal elements by controlling the consistency of the cytoplasm.