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At least 883 records · Page 49Linked to original sources
PubMed1990

Neuromodulin (GAP43): a neuronal protein kinase C substrate is also present in 0-2A glial cell lineage. Characterization of neuromodulin in secondary cultures of oligodendrocytes and comparison with the neuronal antigen.

Neuromodulin (also called GAP43, G50, F1, pp46), a neural-specific calmodulin binding protein, is a major protein kinase C substrate found in developing and regenerating neurons. Here, we report the immunocytochemical characterization of neuromodulin in cultured 0-2A bipotential glial precursor cells obtained from newborn rat brain. Neuromodulin is also present in oligodendrocytes and type 2 astrocytes (stellate-shaped astrocytes), which are both derived from the bipotential glial 0-2A progenitor cells, but is absent of type 1 astrocytes (flat protoplasmic astrocytes). These results support the hypothesis of a common cell lineage for neurons and bipotential 0-2A progenitor cells and suggest that neuromodulin plays a more general role in plasticity during development of the central nervous system. The expression of neuromodulin in secondary cultures of newborn rat oligodendrocytes and its absence in type 1 astrocytes was confirmed by Northern blot analysis of isolated total RNA from these different types of cells using a cDNA probe for the neuromodulin mRNA and by Western blot analysis of the cell extracts using polyclonal antibodies against neuromodulin. The properties of the neuromodulin protein in cultured oligodendrocytes and neuronal cells have been compared. Although neuromodulin in oligodendrocytes is soluble in 2.5% perchloric acid like the neuronal counterpart it migrates essentially as a single protein spot on two-dimensional gel electrophoresis whereas the neuronal antigen can be resolved into at least three distinct protein spots. To obtain precise alignments of the different neuromodulin spots from these two cell types, oligodendrocyte and neuronal cell extracts were mixed together and run on the same two-dimensional gel electrophoresis system. Oligodendroglial neuromodulin migrates with a pI identical to the basic forms of the neuronal protein in isoelectric focusing gel. However, the glial neuromodulin shows a slightly lower mobility in the second dimensional lithium dodecyl sulfate-PAGE than its neuronal counterpart. As measured by 32Pi incorporation, neuromodulin phosphorylation in oligodendrocytes is dramatically increased after short-term phorbol ester treatments, which activate protein kinase C, and is totally inhibited by long-term phorbol ester treatments, which downregulates protein kinase C, thus confirming its probable specific in vivo phosphorylation by protein kinase C. In primary cultures of neuronal cells, two of the three neuromodulin spots were observed to be phosphorylated with an apparent preferential phosphorylation of the more acid forms.

Animals↗
PubMed1998

A single gene product, claudin-1 or -2, reconstitutes tight junction strands and recruits occludin in fibroblasts.

Three integral membrane proteins, clau- din-1, -2, and occludin, are known to be components of tight junction (TJ) strands. To examine their ability to form TJ strands, their cDNAs were introduced into mouse L fibroblasts lacking TJs. Immunofluorescence microscopy revealed that both FLAG-tagged claudin-1 and -2 were highly concentrated at cell contact sites as planes through a homophilic interaction. In freeze-fracture replicas of these contact sites, well-developed networks of strands were identified that were similar to TJ strand networks in situ and were specifically labeled with anti-FLAG mAb. In glutaraldehyde-fixed samples, claudin-1-induced strands were largely associated with the protoplasmic (P) face as mostly continuous structures, whereas claudin-2-induced strands were discontinuous at the P face with complementary grooves at the extracellular (E) face which were occupied by chains of particles. Although occludin was also concentrated at cell contact sites as dots through its homophilic interaction, freeze-fracture replicas identified only a small number of short strands that were labeled with anti-occludin mAb. However, when occludin was cotransfected with claudin-1, it was concentrated at cell contact sites as planes to be incorporated into well- developed claudin-1-based strands. These findings suggested that claudin-1 and -2 are mainly responsible for TJ strand formation, and that occludin is an accessory protein in some function of TJ strands.

Animals↗
PubMed1956

Synapses in the central nervous system.

A number of different synapses have been described in the medulla, cerebellar cortex, and cerebral cortex of the rat. All of these possess the same fundamental fine structure as follows: 1. Close apposition of the limiting membranes of presynaptic and postsynaptic cells without any protoplasmic continuity across the synapse. The two apposed membranes are separated by a cleft about 200 A wide, and display localized regions of thickening and increased density. 2. The presynaptic expansion of the axon, the end-foot or bouton terminal, contains a collection of mitochondria and clusters of small vesicles about 200 to 650 A in diameter. Although the significance of these structures in the physiology of the synapse is still unknown, two suggestions are made: that the mitochondria, by means of the relation between their enzymatic activity and ion transport, participate in the electrical phenomena about the synapse; and that the small synaptic vesicles provide the morphological representation of the prejunctional, subcellular units of neurohumoral discharge at the synapse demanded by physiological evidence.

Animals↗
PubMed1956

Electron microscopic observations of the central nervous system.

In order to establish criteria for the identification of the neural and glial cells of the central nervous system, sections of the brains and spinal cords of mice, rabbits, guinea pigs, and rats; and portions of tumors of the human brain have been examined by electron microscopy. Identification of neurons is made possible by the characteristic cytoplasmic picture, in which there is a distinct granular and less constant membranous ergastoplasmic pattern. In no other cell of the central nervous system is such a distinct granular component present in the ergastoplasm. The shape of the neuron in electron microscopic preparations is similar to that seen by light microscopy with several dendrites containing a similar cytoplasm arising from the perikaryon. Synapses are relatively common on the surface of the neuron and its dendrites. Microglial cells are relatively small and dense with few processes, and are arranged as perineuronal and perivascular satellites for the most part. Occasionally phagocytized material is present in their cytoplasm. The oligodendroglial cells are identifiable by their position as perineuronal satellites and in the white matter as cells arranged in rows. They have a uniformly round to ovoid nucleus with a pale cytoplasm, which has a sparse, finely granular component and a few small mitochondria. The processes are few and relatively straight when cut in longitudinal section. The predominant cellular type in an oligodendroglioma was similar, with a pale cytoplasm. The astrocytes are variable in appearance. Their nuclei are moderately large, irregularly ovoid, and the cytoplasm adjacent to the nucleus is finely granular and scant. In the protoplasmic astrocytes the cytoplasm has a complicated infolded arrangement with reduplication of the plasma membrane, numerous processes extending radially from the cell and rebranching. To a certain extent this same folded plasma membrane was noted in the fibrous astrocytes. However, their more distant processes were narrowed, relatively straight, and filled with numerous dense fibrils. The processes of the astrocyte often surrounded axons, and other cellular processes, and surrounded some vessels, while attaching to a part of the wall of another vessel. Proliferating cells in experimentally produced gliosis and in astrocytic neoplasms were similar in structure. The ependymal cells and the epithelium of the choroid plexus have a specialized surface with microvillous projections of the cytoplasm covered by the plasma membrane. Cilia in varying numbers are present in both epithelia.

Animals↗
PubMed1965

FURTHER STUDIES ON CELL DIVISION WITHOUT MITOTIC APPARATUS IN SEA URCHIN EGGS.

A large quantity of paraffin oil, sucrose solution, or sea water was injected into the eggs of the heart urchin Clypeaster japonicus shortly before the onset of the first cleavage. The injected oil became spherical, pushing the mitotic apparatus aside. The sucrose solution mixed with the protoplasm and caused disintegration of the mitotic apparatus, and the sea water formed a vacuole at the center of the cell. In all these cases, cleavage may take place almost normally in spite of the absence of the mitotic apparatus or its displacement within the cell. In some eggs, furrowing may take place when more than fifty per cent of the endoplasm has been replaced with sea water before onset of cleavage.

Animals↗
PubMed1965

FIBRILLAR DIFFERENTIATION IN MYXOMYCETE PLASMODIA.

Myxomycete plasmodia of four different types (not including Physarum polycephalum) were studied in thin sections viewed in the electron microscope. In the cytoplasm of the protoplasmodia of Clastoderma debaryanum and the phaneroplasmodia of Fuligo septica fixed in situ, fibrillar differentiations of three rather distinct kinds were observed. One of these is filamentous and closely resembles the filaments (or "microtubules") of the mitotic apparatus of other species. The larger phaneroplasmodia of two species belonging to the Physarales and the plasmodium of Hemitrichia vesparium showed fewer and less well defined fibrils, and no fibrils were seen in the aphanoplasmodium of Stemonitis fusca. Good stabilization of such fibrils in larger plasmodia may require fixation methods more rigidly controlled than those which succeed with microscopic protoplasmodia. The function of the observed fibrils cannot yet be determined. Their presence in cytoplasm fixed in situ, however, lends support to those theories of protoplasmic movement which are dependent on integral cross-bonding of one or a few molecular species.

Cell Differentiation↗
PubMed1957

Electron microscope studies of early stages of sperm penetration in Hydroides hexagonus (annelida) and Saccoglossus kowalevskii (enteropneusta).

1. The early events of sperm entry in Saccoglossus and Hydroides are described and examined in relation to present knowledge of the acrosome reaction and of egg membrane lysins. In Saccoglossus and several other species these events occur in two phases. First. The acrosome filament of the spermatozoön spans the egg membrane barriers, reaches the reactive egg protoplasm, and causes the egg to begin its fertilization reaction. Second. The filament and its connected sperm head move through the egg membrane barriers and enter the egg proper. The first phase is completed in a matter of seconds but the second phase usually requires several minutes. 2. The peripheral areas of the eggs of the two species differ as seen in sections. In Hydroides, but not in Saccoglossus, the vitelline membrane is bounded by a distinct outer border layer of small concentrically differentiated bodies and penetrated by microvilli from the egg. 3. The acrosome filament, seen in the living condition as a delicate thread in Hydroides and as an exceedingly tenuous thread in Saccoglossus, appears to be tubular in both species when seen in electron micrographs of thin sections. 4. The acrosomal region of Hydroides appears to consist of two components-a peripheral one, which may collapse during the acrosome reaction, and a central one related to the acrosome filament. 5. Deliberately induced polyspermic material was used to increase the probability of finding examples of sperm penetration in thin sections. 6. As seen in sections, areas of low electron density, interpreted as spaces or pits from which the material of the membrane is absent, surround the attached or penetrating spermatozoa. (a) In Hydroides the spaces vary greatly in many characteristics including shape, position in the membrane, and size with relation to the enclosed sperm head. In one specimen a portion of the membrane is missing from border to border; no spermatozoön is seen but immediately beneath the space is the apex of a fertilization cone. (b) In every case in which a determination could be made, the spermatozoön in the membrane has undergone its acrosome reaction. (c) In Saccoglossus some pits are found with which several spermatozoa are associated. Generally, where the spermatozoa are more numerous the pit is larger. (d) Pits similar to those seen in Saccoglossus sections are observed in living eggs. They remain in Membrane I after sperm entry. (e) From the above and other considerations it is suggested that the pits and spaces are formed by local action of a lysin or lysins emanating from the individual spermatozoön at the site of sperm entry. 7. It is considered that the suggested lysin would participate in sperm entry by eroding the membrane barrier in the vicinity of the sperm head, thus permitting the sperm head to pass through the membrane. Since the acrosome filament much earlier stimulates the egg's initial fertilization response, this lysin would facilitate the second phase of the early events of sperm entry.

Acrosome↗
PubMed1968

The incorporation of radioactive proline into cultured cells. Interpretations based on radioautography and electron microscopy.

Cultured carrot explants, stimulated to grow rapidly in a medium containing coconut milk, were labeled with radioactive proline. After an initial period of absorption (8 hr for proline-(3)H; 24 hr for proline-(14)C) the tissue was allowed to grow for a further period of 6 days in a similar medium free from the radioactivity. Samples were prepared for electron microscopy and radioautography at the end of the absorption period and also after the further growth. The distribution of the products from the radioactive proline in the cells is shown by high-resolution radioautography and is rendered quantitative for the different regions of the cells. The results show that the combined label, which was present in the form of proline and the hydroxyproline derived from it, was all in the protoplasm, not in the cell walls. Any combined label that appeared to be over the cell walls is shown to be due to scatter from adjacent cytoplasmic sites. Initially the radioactivity was concentrated in nuclei, even more so in nucleoli, but it subsequently appeared throughout the ground cytoplasm and was also concentrated in the plastids. The significance of these observations for the general concept of a plant cell wall protein and for the special problem of growth induction in otherwise quiescent cells is discussed.

Autoradiography↗
PubMed1958

The contribution of lower oxides of osmium to the density of biological specimens in electron microscopy.

Centrifugally stratified eggs of the sand-dollar, Dendraster excentricus, have cytoplasmic structures segregated into distinct layers. Fat droplets, yolk granules, and mitochondria are separated by "hyaline" layers of protoplasm. Aggregations of particles of 150 to 200 A diameter ("heavy bodies") are found near the mitochondrial layer and a concentration gradient of free 150 to 200 A particles corresponds to a similar gradient of basophilia in thick sections. Eggs fixed in buffered osmium-tetroxide at pH 7.4 and embedded in methacrylate were sectioned and floated on a "bleaching" solution of acidified hydrogen peroxide. "Bleached" sections showed a considerable loss in general density and especially a loss in the sharp images of cellular membranes. It was shown that such loss is not due to sublimation of structure in the electron beam. Assuming that the "bleach" acts principally to reoxidize lower oxides of osmium, it was concluded that reduced and bound lower oxides of osmium play a major role in creation of the electron micrograph image, especially in the delineation of phospholipide components of cellular membranes. Particles of 150 to 200 A diameter showed little or no loss in density, but rather a high intrinsic electron density. Refractometric data were presented to substantiate the tentative conclusions.

Animals↗
PubMed1969

Crystalline inclusions in the nuclear envelope and granular endoplasmic reticulum of the fish spinal cord.

Crystalline inclusions were found in the nuclear envelope and granular endoplasmic reticulum of spinal cord oligodendroglia of the common guppy (Poecilia reticulata) and the lungfish (Polypterus enlicheri). A considerably increased incidence of these inclusions was noted in guppies with congenital and hereditary (sex-linked, recessive) lordosis. Identical crystalline inclusions were observed in protoplasmic and fibrous astrocytes, ependymal cells, and capillary endothelial cells of the spinal cord of the lordotic fish. The oligodendroglia in these fish also revealed a prominent alteration of the endoplasmic reticulum and Golgi apparatus, with the accumulation of dense (secretion?) granules and large amounts of electron-opaque material in markedly dilated sacs of the endoplasmic reticulum. The authors postulate that this alteration is caused by a genetic defect in the control mechanism governing the elaboration of this material in the lordotic guppy, with subsequent stasis and crystallization of this material.

Animals↗
PubMed1959

Spermiogenesis in Lumbricus herculeus; an electron microscope study.

Small pieces of the sperm sacs of Lumbricus herculeus were fixed for 4 hours in chrome-osmium, embedded in methacrylate, sectioned with a Porter-Blum microtome, and studied with a R.C.A. EMU-2C electron microscope. Each spermatid of a group developing synchronously is attached by a cytoplasmic strand to a common nutrient protoplasmic mass. This mass contains mitochondria and yolk bodies but is anucleate. The proximal centriole, that is, the centriole nearer the nucleus, is at first associated with a small peg which becomes firmly attached to the nuclear membrane. Later these two bodies become separated during the development of the middle-piece which is differentiated in the usual manner from a nebenkern formed by the fusion of 6 or 7 mitochondria. The acrosome develops in relation to the dictyosome (Golgi body), itself composed of 8 or more individual flattened sacs and situated in the cytoplasm opposite the point of attachment of the spermatid to the nutrient mass. Soon after its formation, the acrosome becomes incorporated into a cytoplasmic appendage or acrosome carrier. The carrier moves from its original position, along the lateral border of the elongating nucleus, to the distal margin of the nucleus where the acrosome is deposited. No evidence was found of a centriole located at the point of junction between nucleus and acrosome as suggested by earlier workers.

Acrosome↗
PubMed1977

Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells.

We have used thin section and freeze-fracture electron microscopy to study membrane changes occurring during exocytosis in rat peritoneal mast cells. By labeling degranulating mast cells with ferritin-conjugated lectins and anti-immunoglobulin antibodies, we demonstrate that these ligands do not bind to areas of plasma membrane or granule membrane which have fused with, or are interacting with, granule membrane. Moreover, intramembrane particles are also largely absent from both protoplasmic and external fracture faces of plasma and granule membranes in regions where these membranes appear to be interacting. Both the externally applied ligands and intramembrane particles are sometimes concentrated at the edges of fusion sites. The results indicate that membrane proteins are displaced laterally into adjacent membrane regions before the fusion process and that fusion occurs between protein-depleted lipid bilayers. The finding of protein-depleted blebs in regions of plasma and granule membrane interaction raises the interesting possibility that blebbing may be a process for exposing the granule contents to the extracellular space and for the elimination of excess lipid while conserving membrane proteins.

Antibodies↗
PubMed1977

Polarized bundles of actin filaments within microvilli of fertilized sea urchin eggs.

We report on the internal ultrastructure of long, finger-like microvilli which cover the surface of the fertilized sea urchin egg. Eggs were attached to polylysine-coated surfaces; their upper portions were sheared away with a stream of buffer which left behind only their plasma membranes and adjacent cytoplasmic structures. Scanning electron microscopy (EM) of such fragments revealed intact thin protoplasmic projections radiating away from the body of the cortex. By transmission EM of cortices similarly prepared on grids, small bundles of microfilaments appear as cores within the thin cytoplasmic projections. These microfilaments are shown to be composed of actin by their ability to interact with muscle heavy meromyosin (HMM). HMM-decorated microfilaments possess repeating arrowheads which uniformly point toward the cell interior. Actin bundles in the microvilli of sea urchin eggs may mediate microvillus support and elongation.

Actins↗
PubMed1977

Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells.

HeLa cell plasma membranes have been purified after binding cells to polylysine-coated polyacrylamide beads. Cell attachment to beads and membrane recovery were maximal in a sucrose-acetate buffer, pH 5.0, at 25 degrees C. Measurements of ouabain-sensitive NaK-adenosine triphosphatase, membrane-bound 125I-wheat germ agglutinin, and chemical analyses showed that membranes on beads were of comparable or greater purity than membranes isolated by conventional methods. Because the isolation procedure is rapid (approximately 2.5 h), and produces membranes whose protoplasmic surfaces are fully exposed, it should be a useful supplement to standard isolation techniques.

Cell Count↗
PubMed1978

Particle arrays in earthworm postjunctional membranes.

Analysis of freeze-fractured earthworm body wall muscle reveals distinctive trough-shaped concavities in the protoplasmic leaflet of the muscle cell membrane which contain diagonally oriented rows of particles sometimes in highly ordered arrays. The troughs correspond to the concave postjunctional patches of sarcolemma seen previously in thin sections of myoneural junctions identified as cholinergic, and the intramembranous particles within the troughs correspond in concentration and arrangement to granular elements present in the outer dense lamina of the postjunctional membrane which were interpreted as acetylcholine receptors. The freeze-fracture data provide a more accurate picture of the arrangement of these putative receptors within the plane of the membrane, and indicate also that they extend into the membrane at least as far as its hydrophobic layer.

Animals↗
PubMed1978

Integrated stereological and biochemical studies on hepatocytic membranes. III. Relative surface of endoplasmic reticulum membranes in microsomal fractions estimated on freeze-fracture preparations.

New methods are required for identifying membranes in subcellular fractions with respect to their origin, if such preparations are to be evaluated morphometrically. One method is freeze-fracturing which reveals intramembrane particles whose size, pattern, and numerical density differ for various membrane types. The question is examined whether the differences in numerical particle density per square micrometer of membrane (alpha) can be used to differentiate membrane vesicles found in microsomal fractions from liver cells with respect to their origin in the hepatocytes. It is found that the range of alpha for the protoplasmic face (PF) of endoplasmic reticulum (ER) membrane (1,900 less than alpha less than 3,250) is intermediate between those for plasma and mitochondrial membranes. Since PF(ER) should appear in the outer leaflet of microsomal vesicles, alpha was estimated on concave profiles of freeze-fracture preparations; the numerical frequency distribution of vesicles with respect to alpha was trimodal, with a major peak around 2,900/micrometer2 and 66% of the vesicles in the range determined for PF(ER). Using a new stereological method, it was calculated that 63% of the membrane surface in these microsomal fractions was of ER origin by this criterion. On the same preparations, an attempt was made to label the ER-derived membranes cytochemically for glucose-6-phosphatase. A line intersection count revealed 62% of the membrane surface to be of ER origin on the basis of marker enzyme labeling. These findings indicate a smaller part of ER membranes in microsomal fractions than would be predicted from biochemical data (77%). The possible reasons for such discrepancies are discussed; shifts in particle densities due to the preparation procedure could lead to an underestimate by freeze-fracturing, whereas the prediction from biochemical data could be overestimates if marker enzymes were not homogeneously distributed.

Animals↗
PubMed1978

Role of Ca++ in virus-induced membrane fusion. Ca++ accumulation and ultrastructural changes induced by Sendai virus in chicken erythrocytes.

Some of the ultrastructural (freeze-etching technique), morphological, and biochemical effects of Sendai virus interaction with chicken erythrocytes have been studied under fusogenic (in the presence of CaCl2) and nonfusogenic (in the presence of ethyleneglycol-bis-N,N'-tetraacetic acid, [EGTA]) conditions. The following phenomena occur, irrespective of the presence of CaCl2 or EGTA: (a) binding of iodinated virus particles to chicken erythrocytes at 4 degrees C and their partial release from the cells at 37 degrees C; (b) gradual incorporation of the viral envelope and viral M-protein into plasma membrane, as visualized in the protoplasmic and exoplasmic fracture (P and E, respectively) faces of the membrane; and (c) virus-dependent transient clustering of intramembrane particles at 4 degrees C, which is reversible after transferring the cells back to 37 degrees C. The following virus-induced phenomena occur only in the presence of CaCl2: (a) rounding of cells followed by their fusion; (b) transient decrease in the density of intramembrane particles; and (c) the virus induces uptake of 45CaCl2 by chicken erythrocytes. The uptake is specific as it is inhibited by LaCl3, and no accumulation of [14C]glucose-1-phosphate ([14C]G-1-P) could be observed under the 45 CaCl2 uptake conditions. The data show that fusion of virus with plasma membrane is a Ca++-independent process and, as such, it should be distinguished from the virus-induced membrane-membrane and cell fusion processes. The latter is absolutely dependent on the rise of intracellular Ca++, as reflected by the fact that Ca++-induced rounding of chicken erythrocytes always precedes fusion (Volsky, D. and A. Loyter. 1977.Biochim. Biophys. Acta 471:253--259).

Adsorption↗
PubMed1979

Isolation of concanavalin A caps during various stages of formation and their association with actin and myosin.

Regions of plasma membrane of dictyostelium discoideum amoebae that contain concanavalin A (Con A)-receptor complexes are more resistant to disruption by Triton X-100. This resistance makes possible the isolation of Con A-associated membrane fragments in sufficient quantity and homogeneity to permit the direct biochemical and ultrastructural study of receptor-cytoskeletal interactions across the cell membrane. After specific binding of Con A to the cell surface, a large amount of the cell's actin and myosin copurifies with the plasma membrane fragments. Myosin is more loosely bound to the isolated membranes that actin and is efficiently removed by treating membranes with ATP and low ionic strength. If cells are not lysed immediately after lectin binding, all of the Con A that is bound to the cell surface is swept into a cap in a process requiring metabolic energy. When cells are lysed at different stages of cap formation, the amount of actin and myosin that copurifies with the isolated membranes remains the same. Thick and thin filaments that are attached to the protoplasmic surface of the isolated membranes underlie lectin-receptor complexes during all stages of cap formation. Once the cap is complete, the amount of actin and myosin that tightly bound to the plasma membrane is concentrated into the cap along with the Con A-receptor complexes. These results suggest that the ATP-dependent sliding of membrane-associated actin and myosin filaments is responsible for the accumulation of Con A-receptor complexes into a cap on the cell surface.

Actins↗