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M H Ellisman

Publications and source records attributed to M H Ellisman.

At least 145 records · Page 8Linked to original sources
PubMed1982

Electron microscopic visualization of the tetrodotoxin-binding protein from Electrophorus electricus.

Preparations of highly purified tetrodotoxin-binding protein (sodium channel) from the electric organ of the eel Electrophorus electricus were examined in negatively stained preparations. Structures observed in preparations exhibiting the highest tetrodotoxin binding tended to aggregate into ordered clusters with a unique ribbon-like conformation. The individual particles of these aggregates are elongated or rod-shaped, approximately 40 A wide and 170 A long. Stereoscopic imaging of the three-dimensional aspects of the structures revealed that the rod-like image is not an edge view of a flattened disc but represents a cylindrical structure. Individual rods in nonclustered forms were also observed but with greater frequency in preparations with lower specific activity. The dimensions of the particles suggest that they represent a protein core formed by perhaps one copy of the large glycopeptide previously identified as being part of the sodium channel. The structure of the sodium channel component visualized by negative staining is discussed in the context of the excitable properties it contributes to biological membranes.

Animals↗
PubMed1982

Immunocytochemical localization of sodium channel distributions in the excitable membranes of Electrophorus electricus.

The tetrodotoxin binding protein, a major component of the Na+ channel, has been purified from the electric organ of the South American eel Electrophorus electricus. Antibodies to this protein were raised in rabbits and their specificity was demonstrated by a highly sensitive radioimmunoassay and by immunoprecipitation procedures. These antibodies were used to examine the distribution of the binding protein in the eel electroplax membranes and along myelinated nerve axons. The distribution of the antigen was determined by using the peroxidase-antiperoxidase technique at both the light and electron microscopic levels. In the electrocytes of the electric organ, only the innervated face showed staining in experimental material. The stained regions of electroplax plasmalemma included the caveolae of the innervated surface while caveolae of the non-innervated surface did not stain. Thus, the innervated surface including caveolae exclusively contains the Na+ channels. Along myelinated axons, staining was limited to the nodal zone of the node of Ranvier. The paranodal and internodal zones did not stain for the binding protein. Limited diffusion of primary IgG and subsequent reactants into the paranodal and internodal sites was eliminated as a possible source of focal staining at nodes because mechanically demyelinated preparations also exhibited focal nodal staining. Thus, this tetrodotoxin binding protein component of the Na+ channel is located solely within the nodal zone of the node of Ranvier.

Animals↗
PubMed1982

Evidence that all newly synthesized proteins destined for fast axonal transport pass through the Golgi apparatus.

Effects of the sodium ionophore, monensin, were examined on the passage from neuronal cell body to axon of materials undergoing fast intracellular transport. In vitro exposure of bullfrog dorsal root ganglia to concentrations of drug less than 1.0 micron led to a dose-dependent depression in the amount of fast-transported [3H]leucine- or [3H]glycerol-labeled material appearing in the nerve trunk. Incorporation of either precursor was unaffected. Exposure of a desheathed nerve trunk to similar concentrations of monensin, while ganglia were incubated in drug-free medium, had no effect on transport. With [3H]fucose as precursor, fast transport of labeled glycoproteins was depressed to the same extent as with [3H]leucine; synthesis, again, was unaffected. By contrast, with [3H]galactose as precursor, an apparent reduction in transport of labeled glycoproteins was accounted for by a marked depression in incorporation. The inference from these findings, that monensin acts to block fast transport at the level of the Golgi apparatus, was supported by ultrastructural examination of the drug-treated neurons. An extensive and selective disruption of Golgi saccules was observed, accompanied by an accumulation of clumped smooth membranous cisternae. Quantitative analyses of 48 individual fast-transported protein species, after separation by two-dimensional gel electrophoresis, revealed that monensin depresses all proteins to a similar extent. These results indicate that passage through the Golgi apparatus is an obligatory step in the intracellular routing of materials destined for fast axonal transport.

Animals↗
PubMed1981

Myelination-dependent axonal membrane specializations demonstrated in insufficiently myelinated nerves of the dystrophic mouse.

"Dystrophic' mice of the 129/ReJ-Dy strain have a genetic defect affecting Schwann cell proliferation. Spinal nerve roots of these animals contain myelinated and unmyelinated axons in addition to groups of large "amyelinated' axons. In affected regions of the spinal roots, myelinated axons are missing their myelin sheaths. Where the myelination terminates or begins, half-nodes are created. Freeze-fracture analysis of these half-nodes shows that only the myelinated side contains rows of dimeric particles in the axonal P-face of the paranode. The P-face on the amyelinated side of a half-node, and the remainder of the amyelinated axon. contains a dense even distribution of particles, many of which are the size of dimeric-particle subunits, but only a few of which are arranged into short rows. As the long circumferential rows are not found on the unmyelinated side of the myelinated side of the half-node we conclude that the paranodal rows of dimeric particles are dependent upon myelination for their organization.

Animals↗
PubMed1981

The membrane morphology of the neuromuscular junction, sarcolemma, sarcoplasmic reticulum and transverse tubule system in murine muscular dystrophy studied by freeze-fracture electron microscopy.

'Dystrophic' mice of the 129/ReJ-dy strain have a genetic defect in Schwann cell proliferation and neuromuscular junction formation. The presynaptic membrane specializations associated with vesicle fusion and acetylcholine release, as well as the postsynaptic membrane specializations associated with acetylcholine receptivity, appear normal in these animals when visualized with freeze-fracture techniques. However, there is a reduction in the infolding of the postsynaptic membrane, which forms the secondary synaptic cleft at the motor endplate and is the site of acetylcholinesterase activity. The orthogonal arrays of the non-junctional sarcolemma are found on dystrophic muscles, but at lower than normal densities. These observations are made on muscle fibers in which the membrane molecular organization of the sarcoplasmic reticulum and transverse tubule systems appear normal. Several possible linkages between the deficit in myelination and the altered synaptic morphology are discussed in the context of neuromuscular interaction.

Acetylcholine↗
PubMed1981

An increase in smooth endoplasmic reticulum and a decrease in Golgi apparatus occur with ionic conditions that block initiation of fast axonal transport.

The ultrastructure of bullfrog spinal ganglia was analyzed after incubation in media containing concentrations of calcium and cobalt known to inhibit export of proteins from the soma to the axon. Although most somal organelles were morphologically unchanged by the various incubation media, striking changes were seen in the smooth endoplasmic reticulum (SER) and the Golgi apparatus (GA). In order of effect, calcium-free medium (CFM), normal medium supplemented with cobalt (NM--Co), and CFM supplemented with cobalt (CFM--Co) produced increasing amounts of SER coupled with decreasing densities of GA stacks. In the extreme case, CFM--Co incubation resulted in a nearly 10-fold increase in SER volume as well as in a virtually complete depletion of GA stacks. Axons originating within the ganglion were also examined and showed little change after the various incubations. The rank order of the altered incubation media in producing morphological changes was the same as the relative effectiveness of the media in depressing the fast axonal transport of [3H]protein within the dorsal root ganglion neurons. The morphological and biochemical results are discussed with respect to establishing the localization of the calcium-dependent step(s) that has been proposed to occur in the neuronal soma during the initiation of axonal transport.

Animals↗
PubMed1981

The distribution of orthogonal arrays and their relationship to intercellular junctions in neuroglia of the freeze-fractured hypothalamo-neurohypophysial system.

Using freeze-fracture techniques, we have investigated membrane specializations of the glia associated with the hypothalamo-neurohypophysial system of the rat. In the paraventricular (PVN) and supraoptic (SON) nuclei, astrocytes in areas of high neuronal density (i.e., magnocellular regions) display orthogonal arrays of 6--7 nm particles solely near gap junctions, while astrocytes in areas of lower neuronal density (i.e., parvocellular regions), contain additional arrays in membranes not displaying gap junctions. Arrays are especially numerous on astrocytic perivascular end-feet in both nuclei and in the laminations of the pial-glial limitans ventral to the SON. Ependymal cells near the PVN show arrays both on their lateral surfaces (displaying gap junctions) and on their apical surfaces (facing the CSF). Tight junctions are not noted on astrocytes or ependymal cells, but are noted on both the somas and myelin lamellae of oligodendroglia. Both of these latter membranes occasionally contain gap junctions as well; however, orthogonal arrays are never noted on oligodendroglia. The plasma membranes of pituicytes in the neurohypophysis display gap junctions, complex junctions, and tight junctions. Orthogonal arrays are noted near the first two of these, but not near the last. Arrays in the neutral lobe appear most dense on membranes adjacent to subpial or perivascular spaces. Pituicyte membranes containing orthogonal arrays appear infrequently near the neural stalk, increasing towards the distal end of the neural lobe. The distribution of orthogonal arrays in this system, as well as in other systems in which they have been noted, suggests a polarization of membrane activity.

Animals↗
PubMed1981

Enhanced visualization of peripheral nerve and sensory receptors in the scanning electron microscope using cryofracture and osmium-thiocarbohydrazide-osmium impregnation.

Two methods of specimen preparation for the scanning electron microscope (SEM) have been combined for the reliable exposure and examination of nervous system tissue. When the specimen is postfixed with OSO4 prior to aqueous cryofracturing, large internal surfaces of nervous tissue are exposed, with minimal distortion to the cytoarchitecture. All tissue surfaces and interstices are subsequently impregnated with a conductive, metallic layer of osmium using a modified osmium-thiocarbohydrazide-osmium technique (OTOTO). This OTOTO technique permits SEM examination without any additional vacuum evaporated or ion-sputtered metallic layers, and has been found to eliminate specimen charging reliably. Nervous tissue has been examined in the secondary electron mode of the SEM with unrestricted use of beam currents varying from 1.3 to 60 microamperemeter, at accelerating voltages ranging from 2.5 to 80 kV, and at both low (10 X) and high (80 000 X) magnifications. In addition, a differential deposition of osmium in the tissue after the OTOTO technique has been identified using both transmission electron microscopy and energy dispersive X-ray microanalysis. The enhanced mass-density of myelin resulting from the amplification of osmium's natural affinity for unsaturated lipids was best demonstrated by the backscatter electron mode of the SEM. This mode of imaging was found useful in the identification of myelin sheaths.

Animals↗
PubMed1980

The localization of sodium and calcium to schwann cell paranodal loops at nodes of Ranvier and of calcium to compact myelin.

High-voltage electron microscopy (HVEM) has been used to determine the distribution of cationic precipitates in myelinated axons resulting from the application of two cytochemical techniques: a direct osmium pyroantimonate treatment for precipitating Na+, Ca2+ and Mg2+; and a 5 mM Ca2+ inclusion procedure (Oschman & Wall) for imparting electron density to Ca2+ binding sites. Electron probe wavelength spectroscopy was then used on semi-thick tissue sections to identify the species of ions present in the following regions: Schwann cell paranodal loops, axoplasm at the node, compact myelin and extracellular matrix. With these combined procedures we were able to localize elevated concentrations of both Na+ and Ca2+ to cytoplasmic compartments of the Schwann cell paranodal loops, as well as to detect the presence of Ca2+ at elevated levels in compact myelin. The involvement of the Schwann cell paranodal loops in providing a source and/or sink for Na+ involved in impulse conduction is suggested by these results, and the significance of such a role is discussed. A role for Ca2+ in the formation and stabilization of myelin lamellae is also suggested.

Animals↗
PubMed1980

Reassembly of protein-lipid complexes into large bilayer vesicles: perspectives for membrane reconstitution.

Protein-lipid complexes in apolar solvents reassemble into large bilayer protein-lipid vesicles (PLVs) with diameters of several micrometers. PLVs form spontaneously upon hydration of the protein-lipid complex residue after solvent removal. This procedure has been applied to the following membrane proteins: bovine and squid rhodopsin, reaction centers from Rhodopseudomonas sphaeroides, beef heart cytochrome c oxidase, and acetylcholine receptors from Torpedo californica. PLVs have a large internal aqueous space (e.g., 790 mul/mg of lipid for cattle rhodopsin vesicles). Freeze-fracture replicas of PLVs revealed that both internal and external leaflets contained numerous intramembranous particles with diameters between 80 and 120 A, depending on the specific protein incorporated in the membrane. The optical spectral properties of rhodopsin and reaction centers in PLVs were similar to those recorded in the respective natural membrane. Furthermore, bovine rhodopsin in PLVs was chemically regenerable with 9-cis-retinal. Actinic illumination induced proton efflux from reaction center vesicles that was abolished by proton ionophores. Therefore, this method is suitable for the incorporation of some membrane proteins in their functional state. PLVs were penetrated with microelectrodes and visualized by the injection of a fluorescent dye. Preliminary electrical recordings were obtained by sealing PLVs to a hole in a septum separating two aqueous compartments. These studies suggest that PLVs assembled by this procedure permit the simultaneous analysis of reconstituted membranes by chemical, optical, and electrical techniques.

Animals↗
PubMed1980

Rows of dimeric-particles within the axolemma and juxtaposed particles within glia, incorporated into a new model for the paranodal glial-axonal junction at the node of Ranvier.

Using freeze-fracture techniques, we have analyzed the glial-axonal junction (GAJ) between Schwann cells and axons in the peripheral nervous system, and between oligodendrocytes and axons in the central nervous system of the rat. We have identified a new set of dimeric-particles arranged in circumferential rows within the protoplasmic fracture faces (P-faces) of the paranodal axolemma in the region of glial-axonal juxtaposition. These particles, 260 A in length, composed of two 115-A subunits, are observed in both aldehyde-fixed and nonfixed preparations. The rows of dimeric-particles within the axonal P-face are associated with complementary rows of pits within the external fracture face (E-face) of the paranodal axolemma. These axonal particles are positioned between rows of 160-A particles that occur in both fracture faces of the glial loops in the same region. We observed, in addition to these previously described 160-A particles, a new set of 75-A glial particles within the glial P-faces of the GAJ. These 75-A particles form rows that are centered between the rows of 160-A particles and are therefore superimposed over the rows of dimeric-particles within the paranodal axolemma. Our new findings are interpreted with respect to methods of specimen preparation as well as to a potential role for the paranodal organ in saltatory conduction. We conclude that this particle-rich junction between axon and glia could potentially provide an intricate mechanism for ion exchange between these two cell types.

Animals↗
PubMed1980

Microtrabecular structure of the axoplasmic matrix: visualization of cross-linking structures and their distribution.

Axoplasmic transport is a dramatic example of cytoplasmic motility. Constituents of axoplasm migrate as far as 400 mm/d or at approximately 5 micron/s. Thin-section studies have identified the major morphological elements within the axoplasm as being microtubules, neurofilaments (100-A filaments), an interconnected and elongated varicose component of smooth endoplasmic reticulum (SER), more dilated and vesicular organelles resembling portions of SER, multivesicular bodies, mitochondria, and, finally, a matrix of ground substance in which the tubules, filaments, and vesicles are suspended. In the ordinary thin-section image, the ground substance is comprised of wispy fragments which, in not being noticeably tied together, do not give the impression of representing more than a condensation of what might be a homogeneous solution of proteins. With the high-voltage microscope on thick (0.5-micron) sections, we have noticed, however, that the so-called wispy fragments are part of a three-dimensional lattice. We contend that this lattice is not an artifact of aldehyde fixation, and our contention is supported by its visability after rapid-freezing and freeze-substitution. This lattice or microtrabecular matrix of axoplasm was found to consist of an organized system of cross-bridges between microtubules, neurofilaments, cisternae of the SER, and the plasma membrane. We propose that formation and deformation of this system are involved in rapid axonal transport. To facilitate electron microscope visualization of the trabecular connections between elements of axoplasm, the following three techniques were used: first, the addition of tannic acid to the primary fixative, OsO4 postfixation, then en bloc staining in uranyl acetate for conventional transmission electron microscope (TEM); second, embedding tissue in polyethylene glycol for thin sectioning, dissolving out the embedding medium from the sections and blocks, critical-point-drying (J. J. Wolosewick, 1980, J. Cell Biol., 86:675-681.), and then observing the matrix-free sections with TEM or the blocks with a scanning electron microscope; and third, rapid freezing of fixed tissue followed by freeze-etching and rotary-shadowing with replicas observed by TEM. All of these procedures yielded images of cross-linking elements between neurofilaments and organelles of the axoplasm. These improvements in visualization should enable us to examine the distribution of trabecular links on motile axonal organelles.

Animals↗
PubMed1979

Molecular specializations of the axon membrane at nodes of Ranvier are not dependent upon myelination.

Nodes of Ranvier from normal and 'dystrophic' mice have been examined with quantitative freeze-fracture electron microscopy. Regions of nodal, paranodal and interparanodal axolemma of normal fibres are clearly distinguishable on the basis of particle size distributions in electron micrographs of freeze-fractured replicas. Protoplasmic fracture faces of normal nodes of Ranvier, contain approximately 40% 100 A particles and about 25% elongated particles 150 by 250 A. Paranodal and interparanodal membranes contain a more uniform distribution of smaller diameter particles. 'Dystrophic', mice of the 129/ReJ-Dy strain have a genetic defect of Schwann cell development and myelinogenesis. Axons of the sciatic and deep peroneal nerves in dystrophic mice, which appear to be normally myelinated, possess approximately the same distributions of particles as axons in normal mice. However, in affected regions of the ventral and dorsal roots, Schwann cell wrappings may be missing, creating heminodes of Ranvier where the myelination terminates or begins again. At such heminodes, there is a circular band of axonal membrane which bears particles of sizes and packing densities similar to that found at normal nodes. High voltage electron microscopic examination of 0.25--1 micron thick sections from these hemi-nodal regions reveals the presence of a filamentous layer beneath the particle-rich membrane. In addition, completely amyelinated regions of root axons contain particle patches having size-density distributions similar to that of both normal and hemi-nodal membranes. Thus, the nodal membrane displays a characteristic particle-size distribution profile. The occurrence of this particle profile does not appear to be dependent upon the presence or absence of Schwann cells. These observations suggest that the functions subserved by the numerous particles at the node of Ranvier are not dependent upon myelination for their local differentiation within the axonal membrane.

Animals↗