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D W Pumplin

Publications and source records attributed to D W Pumplin.

At least 37 records · Page 2Linked to original sources
PubMed1990

Quick-freeze, deep-etch replication of cells in monolayers.

We have made several technical improvements for quick-freeze, deep-etch replication of monolayers of cells grown on, or attached to, glass coverslips. Cells studied include muscle cells of rat and Xenopus cultured on glass coverslips, and erythrocytes attached to coverslips coated with poly-L-lysine. We describe methods for identifying particular areas of cultures, e.g., clusters of acetylcholine receptors on muscle cells, by light microscopy and then relocating these areas after replication. For good preservation of structure by quick-freezing, it is necessary to ensure that the surface to be frozen is covered by a minimum depth of water (less than 10 microns). Insufficient or excess water left on the sample during freezing causes recognizable artifacts in its replica. We describe two ways to control the water table--one by improving visual control of water removal, the other by blowing excess water off the sample surface with a jet of nitrogen applied during its descent to the freezing block. Finally, we describe a new specimen holder that allows us to etch and replicate six samples at once with good thermal contact between the stage and samples.

Animals↗
PubMed1989

Acetylcholine receptor clusters of rat myotubes have at least three domains with distinctive cytoskeletal and membranous components.

Cultured rat myotubes develop high concentrations of acetylcholine receptors (AChR) in specialized areas of attachment to their substrate. We examined the ultrastructure of identified AChR clusters by quick-freeze, deep-etch, rotary replication or by thin sectioning of whole myotubes fixed in the presence of saponin and tannic acid to preserve the cytoskeleton. Our findings show that AChR clusters are composed of at least three distinct domains, differing in their cytoskeletal, intramembrane, and external components. At contact domains, the myotube's ventral membrane lacked AChR and lay within 10-15 nm of the substrate; electron-dense strands connected the two. The overlying cytoplasm contained bundles of parallel microfilaments passing above and through an irregular network of globular material, resembling the relationship of microfilament bundles to focal contacts already described in fibroblasts. Coated-membrane domains lay between the microfilament bundles and were overlain by cytoplasmic plaques of a regular network of polygons having associated coated pits. These plaques closely resembled the network of polymerized clathrin described in fibroblasts and macrophages. Coated membrane also lacked AChR and adhered to the substrate by electron-dense strands, but did not anchor microfilament bundles. The cytoplasm overlying AChR domains contained a complex network composed of at least two layers. The layer closest to the membrane consisted of protrusions from the cytoplasmic surface, some connected by fine filaments less than 5 nm in diameter. An overlying layer contained larger diameter filaments, some forming an anastomotic network reminiscent of the cortical cytoskeleton of erythrocytes. Longer filaments inserting into this network appeared identical to members of nearby microfilament bundles. The morphology of AChR domains supports the idea that AChR are immobilized by a network containing actin and spectrin.

Actin Cytoskeleton↗
PubMed1988

Molecular events in synaptogenesis: nerve-muscle adhesion and postsynaptic differentiation.

The clustering of acetylcholine receptors (AChR) in the postsynaptic membrane of newly innervated muscle fibers is one of the earliest events in the development of the vertebrate neuromuscular junction. Here, we describe two hypotheses that can account for AChR clustering in response to innervation. The "trophic factor" hypothesis proposes that the neuron releases a soluble factor that interacts with the muscle cell in a specific manner and that this interaction results in the local accumulation of AChR. The "contact and adhesion" hypothesis proposes that the binding of the nerve to the muscle cell surface is itself sufficient to induce AChR clustering, without the participation of soluble factors. We present a model for the molecular assembly of AChR clusters based on the contact and adhesion hypothesis. The model involves the sequential assembly of three distinct membrane domains. The first domain to form serves to attach microfilaments to the cytoplasmic surface of the muscle cell membrane at sites of muscle-nerve adhesion. The second domain to form is clathrin-coated membrane; it serves as a site of insertion of additional membrane elements, including AChR. Upon insertion of AChR into the cell surface, a membrane skeleton assembles by anchoring itself to the AChR. The skeleton, composed in part of actin and spectrin, binds and immobilizes significant numbers of AChR, thereby forming the third membrane domain of the AChR cluster. We make several predictions that should distinguish this model of AChR clustering from one that invokes soluble, trophic factors.

Animals↗
PubMed1988

Membrane domains of AChR clusters of cultured rat myotubes revealed by rapid-freeze, deep-etch, rotary-replication.

Acetylcholine receptor clusters of cultured rat myotubes are examined by thin-section EM and by rapid-freeze, deep-etch, rotary-replication technique to observe the cortical cytoskeleton; AChR are localized by binding of fluorescent toxin. Cluster membrane is composed of three types of membrane domain which interdigitate with each other. The AChR domain is rich in the integral membrane receptor, lies further from the substrate than the other two, and is overlain by an extensive irregular meshwork of anastomosing filaments containing actin and a beta isoform of spectrin. Contact domains resemble focal contacts of fibroblasts, in that membrane lying close to the substrate is overlain by bundles of actin filaments running parallel to the membrane; some finer filaments link the parallel filaments to each other and to the membrane. Coated-membrane domains are overlain by polymerized clathrin and, like contact domains, lie close to the substrate and have external connections to it; these domains are associated with coated vesicles, but not with intracellular filaments. The three domains have few if any connections to each other, and occupy mutually exclusive territories in the cluster.

Actin Cytoskeleton↗
PubMed1987

Disruption and reformation of the acetylcholine receptor clusters of cultured rat myotubes occur in two distinct stages.

We have examined the redistribution of acetylcholine receptor (AChR) intramembrane particles (IMPs) when AChR clusters of cultured rat myotubes are experimentally disrupted and allowed to reform. In control myotubes, the AChR IMPs are evenly distributed within the AChR domains of cluster membrane. Shortly after addition of azide to disrupt clusters, IMPs become unevenly scattered, with some microaggregation. After longer treatment, IMPs are depleted from AChR domains with no further change in IMP distribution. Contact domains of clusters are relatively poor in IMPs both before and after cluster dispersal. Upon visualization with fluorescent alpha-bungarotoxin, some AChR in azide-treated samples appear as small, bright spots. These spots do not correspond to microaggregates seen in freeze-fracture replicas, and probably represent receptors that have been internalized. The internalization rate is insufficient to account completely for the loss of IMPs from clusters, however. During reformation of AChR clusters upon removal of azide, IMP concentration in receptor domains increases. At early stages of reformation, IMPs appear in small groups containing compact microaggregates. At later times, AChR domains enlarge and IMPs within them assume the evenly spaced distribution characteristic of control clusters. These observations suggest that the disruption of clusters is accompanied by mobilization of AChR from a fixed array, allowing AChR IMPs to diffuse away from the clusters, to form microaggregates, and to become internalized. Cluster reformation appears to be the reverse of this process. Our results are thus consistent with a two-step model for AChR clustering, in which the concentration of IMPs into a small membrane region precedes their rearrangement into evenly spaced sites.

Animals↗
PubMed1986

Vitronectin at sites of cell-substrate contact in cultures of rat myotubes.

Affinity-purified antibodies to the serum glycoprotein, vitronectin, were used to study sites of cell-substrate contact in cultures of rat myotubes and fibroblasts. Cells were removed from the substrate by treatment with saponin, leaving fragments of plasma membrane attached to the glass coverslip. When stained for vitronectin by indirect immunofluorescence, large areas of the substrate were brightly labeled. The focal contacts of fibroblasts and the broad adhesion plaques of myotubes appeared black, however, indicating that the antibodies had failed to react with those areas. Contact sites within the adhesion plaque remained unlabeled after saponin-treated samples were extracted with Triton X-100, or after intact cultures were sheared with a stream of fixative. These procedures expose extracellular macromolecules at the cell-substrate interface, which can then be labeled with concanavalin A. In contrast, when samples were sheared and then sonicated to remove all the cellular material from the coverslip, the entire substrate labeled extensively and almost uniformly with anti-vitronectin. Extracellular molecules associated with substrate contacts were also studied after freeze-fracture, using a technique we term "post-release fracture labeling." Platinum replicas of the external membrane were removed from the glass with hydrofluoric acid to expose the extracellular material. Anti-vitronectin, bound to the replicas and visualized by a second antibody conjugated to colloidal gold, labeled the broad areas of close myotube-substrate attachment and the nearby glass equally well. Our results are consistent with the hypothesis that vitronectin is present at all sites of cell-substrate contact, but that its antigenic sites are obscured by material deposited by both myotube and fibroblast cells.

Animals↗
PubMed1983

Normal variations in presynaptic active zones of frog neuromuscular junctions.

Neuromuscular junctions of frog cutaneous pectoris muscles were examined by the complementary-replica freeze-fracture technique. The large number of active zones revealed in this way permitted quantitation of normal and disrupted zones in control muscles and in muscles deprived of external calcium for periods of up to eight hours. Although some 87% of the active zones examined had the usual appearance of two double rows of intramembrane particles flanking a low ridge, several varieties of disrupted active zones appeared in which the rows were interrupted, dispersed, rotated, or reduced to single rows. The frequency of disruptions was not increased by calcium deprivation. Disorganized active zones at tips of neuromuscular junctions may represent normal remodelling of the junctions.

Animals↗
PubMed1983

Distinctions between gap junctions and sites of intermediate filament attachment in the leech C.N.S.

Freeze-fracture studies on the nerve cord of the leech Hirudo medicinalis reveal that the plasma membranes of various cells, including glial and muscle cells, contain at least two distinct types of aggregated intramembrane particles, identified as hemidesmosomes and gap junctions. Hemidesmosomes consist of angular particles irregularly arranged in circular or elongate patches in external leaflets (E-faces), and are associated with a bundle of intermediate filaments extending into the cytoplasm. Hemidesmosomes of specific axons abut on extracellular space at openings in the surrounding glial sheath. Gap junctions are patches of rounder particles in cytoplasmic leaflets (P-faces) and are more uniformly spaced; they have a corresponding array of pits in the complementary E-face. Gap junctions connect processes of adjacent smooth muscle cells, and apparently interconnect glial processes. Thus, different types of cells in the leech C.N.S. have similar intramembrane specializations. Moreover, the hemidesmosomes and gap junctions might, on superficial examination, be confused.

Animals↗
PubMed1983

Lipid domains of acetylcholine receptor clusters detected with saponin and filipin.

The acetylcholine receptor (AChR) clusters of cultured rat myotubes contain two distinct, interdigitating, membrane domains, one enriched in AChR, the other poor in AChR but associated with sites of myotube-substrate contact (Bloch, R.J., and B. Geiger, 1980, Cell, 21:25-35). We have used two cholesterol-specific cytochemical probes, saponin and filipin, to investigate the lipid nature of these membrane domains. When studied with freeze-fracture electron microscopy or fluorescence microscopy, these reagents reacted moderately and preferentially with the AChR-rich domains of AChR clusters. Little or no reaction with the membrane in "contact" domains was seen. In contrast, membrane regions surrounding the AChR clusters reacted extensively with filipin. These results suggest that, in rat myotubes, the composition or the state of the lipids differs between the two membrane domains of the AChR clusters, and between clusters and surrounding membrane. In chick myotubes, AChR clusters do not appear to react with filipin or saponin, although surrounding membrane reacts extensively with these reagents.

Animals↗
PubMed1983

(Na+ + K+)-ATPase correlated with a major group of intramembrane particles in freeze-fracture replicas of cultured chick myotubes.

Immunofluorescence microscopy with a fluorescein-labeled monoclonal antibody was used to map the distribution of sodium- and potassium-ion stimulated ATPase [( Na,K]-ATPase) on the surface of tissue-cultured chick skeletal muscle. At this level of resolution it appeared that the (Na,K)-ATPase molecules were distributed nearly uniformly over the plasma membrane. These molecules could be cross-linked by use of the monoclonal antibody followed by a second antibody directed against the monoclonal antibody; the resulting fluorescent pattern was a set of small dots (patches) on the muscle surface. This pattern was stable over several hours, and there was little evidence of interiorization or of coalescence of the patches. Myotubes labeled with immunofluorescence were fixed in glutaraldehyde, cryoprotected with glycerin, then fractured and replicated by standard methods. Replicas of the immunofluorescence-labeled myotubes revealed clusters of intramembrane particles (IMP) only when the immunofluorescent images indicated a patching of the (Na,K)-ATPase molecules. Double antibody cross-linking of antigenic sites on myotubes with each of three other monoclonal antibodies to plasma membrane antigens likewise resulted in patched patterns of immunofluorescence, but in none of these cases were clusters of intramembrane particles found in freeze-fracture replicas. In each case it was shown that the (Na,K)-ATPase molecules were not patched. Other control experiments showed that patching of (Na,K)-ATPase molecules did not cause co-patching of one of the other plasma membrane proteins defined by a monoclonal antibody and did not cause detectable co-clustering of acetylcholine receptors. Detailed mapping showed that there was a one-to-one correspondence between immunofluorescent patches related to the (Na,K)-ATPase and clusters of IMP in a freeze-fracture replica of the same cell. We conclude that the intramembrane particles patched by double antibody cross-linkage of the (Na,K)-ATPase are caused by (Na,K)-ATPase molecules in the fracture plane. Quantification of the IMP indicated that the (Na,K)-ATPase-related particles account for up to 50% of particles evident in the replicas, or up to about 400 particles/micrometers2 of plasma membrane. Particles related to the (Na,K)-ATPase were similar to the average particle size and were as heterodisperse in size as the total population of IMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1983

Myasthenic patients' IgG causes redistribution of acetylcholine receptors: freeze-fracture studies.

Immunoglobulin from patients with myasthenia gravis (MG) accelerates the degradation of acetylcholine receptors (AChRs) of skeletal muscle by a mechanism dependent on receptor cross-linking. However, the intermediate steps between cross-linking and endocytosis and degradation are not known. We have used freeze-fracture electron microscopy to study the effects of myasthenic immunoglobulin on the distribution of AChRs in the surface membranes of cultured rat skeletal muscle. Large, angular intramembrane particles were identified as AChRs. These particles were present in three configurations: distributed individually; in "hot spots"; and in clusters of two to 60 particles. Large depressions (greater than 100 nm in diameter) were thought to be endocytotic pits; they occurred preferentially in hot spots, and approximately half of the depressions were closely associated with clusters of AChRs. In control myotubes, hot spots contained a high density of AChR particles that were evenly, rather than randomly, distributed. Exposure to myasthenic patients' IgG induced clustering of the particles within such hot spots. In non-hot spot regions, control myotubes had mainly individually distributed AChRs, while clusters were seen only rarely. The addition of myasthenic IgG caused a rapid increase, followed by a gradual decrease, in the incidence of individual clusters as well as an increase in cluster size. This redistribution of particles was dependent on cross-linking by the myasthenic anti-AChR antibodies. Monovalent Fab fragments of myasthenic IgG had no effect on the distribution of AChR particles. However, addition of a second "piggyback" antibody to cross-link Fab-AChR complexes caused a redistribution of AChR particles like that due to the original myasthenic IgG. These findings indicate that cross-linking of AChRs by divalent antibody causes clustering of AChR particles, which may play an important role in their accelerated endocytosis and degradation.

Animals↗
PubMed1981

Are the presynaptic membrane particles the calcium channels?

The number of large intramembrane particles associated with sites of synaptic vesicle release at the squid giant synapse was determined and compared to the average maximal presynaptic calcium current in order to derive an estimate of the conductance each particle would have if it were a calcium channel. This value, 0.21 pS, compares favorably with conductances of calcium channels in other preparations, substantiating the idea that the large intramembrane particles, which are concentrated at "active zones," represent calcium channels.

Animals↗
PubMed1979

Clusters of intramembrane particles associated with binding sites for alpha-bungarotoxin in cultured chick myotubes.

Developing chick myotubes in tissue culture were freeze-fractured to yield complementary replicas of large areas of membrane. Regions of muscle fibers with high concentrations of acetylcholine receptors were identified by binding of fluorescent-labeled alpha-bungarotoxin. Membranes in such regions contained clusters of large (100 A Diam) angular particles, similar in appearance to particles found in postsynaptic membranes of cholinergic synapses. Particles appeared in apposing areas of cytoplasmic and external leaflets but were more prevalent in the cytoplasmic leaflet. The areas of high particle concentration were coextensive with the fluorescence due to bound toxin. Treatment of cultures with tetrodotoxin increased the size of fluorescent spots and areas of high concentration of particles relative to those found in control cultures. In muscle cultures grown in the presence of spinal cord explants, some neurites contacted and innervated nearby myotubes. Intense fluorescence due to binding or alpha-bungarotoxin was present at portions of such neurite-myotube contacts. At these same portions, a high concentration of large angular particles was present in the sarcolemma adjacent to the neurite. In addition, an ordered arrangement of large particles was seen in the cytoplasmic leaflet of the neuronal plasmalemma directly apposing the muscle. The possible significance of these arrangements is discussed. Clusters on myotubes tended to be larger (contain more particles) when they occurred in groups, defined as three or more clusters with an intercluster distance of less than 0.5 micrometers. Clusters were also larger in myotubes treated with tetrodotoxin and in myotubes adjacent to some neurites in nerve-muscle cocultures. Several depressions containing particles similar to those in the clusters were found in the sarcolemma. The implications of these depressions are discussed in light of current theories of incorporation of proteins into cell membranes.

Acetylcholine↗
PubMed1977

Action of brown widow spider venom and botulinum toxin on the frog neuromuscular junction examined with the freeze-fracture technique.

1. Structural changes which normally accompany transmitter release at frog neuromuscular junctions are visualized with the freeze-fracture technique. The effects of brown widow spider venom and botulinum toxin were evaluated in terms of their ability to block or produce these structural changes. Changes produced by these neuropoisons were correlated with their known effects on neurotransmitter release. 3. Fusion of synaptic vesicles with the presynaptic plasmalemma, normally evoked by electrical stimulation, was abolished at neuromuscular junctions from frogs treated with botulinum toxin. 3. The concentration of large intramembranous particles in the presynaptic plasmalemma, an indication of the excess of synaptic vesicle fusion over recovery of synaptic vesicle membrane, was increased by treatment with brown widow spider venom, even in the presence of botulinum toxin. 4. When external calcium was present, sites of vesicle fusion induced by brown widow spider venom, as well as by electrical stimulation, were located mainly in the active zone. In the absence of external calcium, many plasmalemmal deformations, also though to be sites of vesicle fusion, were more evenly dispersed over the presynaptic surface of nerve terminals. 5. Botulinum toxin decreased the number of vesicle fusion sites in the active zone induced by spider venom in the presence of external calcium but had little effect on the number of fusion sites induced by spider venom in the absence of external calcium. 6. Nerve terminals soaked in a sodium-free Ringer solution were partially depleted of vesicles. Addition of spider venom to this Ringer did not cause additional depletion of vesicles. 7. Formation of cation-permeable channels in the presynaptic membrane could account for these effects of spider venom on the frog neuromuscular junction. Botulinum toxin blocks vesicle fusion by some means which is not yet understood.

Animals↗
PubMed1977

The release of acetylcholine elicited by extracts of black widow spider glands: studies using rat superior cervical ganglia and inhibitors of electrically stimulated release.

An extract of the venom glands of black widow spiders (BWGE) induces the release of acetylcholine (ACh) from the superior cervical ganglia of rats. The release of ACh follows first-order kinetics, which suggests that the venom either lowers the ganglionic store of ACh, or continually reduces the rate of release. Since ganglionic ACh did not decrease in the presence of BWGE, it is likely that the venom continually reduces the rate of release. The rate constant for BWGE-induced release of ACh is depressed about 45% by treatment of the ganglion with either botulinum toxin or a low Ca++/high Mg++Ringer's solution. The rate constant is depressed about 30% by treatment of the ganglion with 8.3 microng/ml of cytochalasin-B. Since these agents inhibit the release of ACh which is elicited by electrical stimulation of the ganglion, it is suggested that one action of BWGE is the stimulation of some step in the physiological mechanism involved in the release of neurotransmitters. Treatment of the ganglion with tetrodotoxin had no effect on the rate constant for release induced by BWGE. The action of BWGE on the ganglion was not reversible after 10 minutes. When treated with BWGE, ganglia whose stores of transmitter had been labeled by electrical stimulation in the presence of [3H]choline released ACh having uniform specific activity. The data suggest the presence of more than one activity in the extracts.

Acetylcholine↗