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R Sealock

Publications and source records attributed to R Sealock.

35 records · Page 2Linked to original sources
PubMed1991

Commercial preparations of colloidal gold-antibody complexes frequently contain free active antibody.

Using a simple fluorescence test, we show that commercially prepared colloidal gold complexes with goat second antibodies often contain free active antibody. Because such antibodies will compete with antibody-colloidal gold particles for antigen binding sites, labeling intensity at the ultrastructural level must necessarily be submaximal to an unknown degree with such preparations. A survey of five preparations suggests that the problem may be widespread. We recommend that a test of the sort described be incorporated routinely into protocols with all colloidal gold products.

Animals↗
PubMed1990

Dystrophin as a focal adhesion protein. Collocalization with talin and the Mr 48,000 sarcolemmal protein in cultured Xenopus muscle.

Monoclonal antibodies against dystrophin and the postsynaptic 58 kDa protein from Torpedo electric organ were used to localize homologs of these proteins in cultured skeletal muscle (Xenopus laevis). The Xenopus homolog is an Mr 48,000 protein and, like dystrophin, is a sarcolemmal protein. Both proteins localized precisely to talin-positive sites, hence with each other, on the substrate-apposed sarcolemma. Therefore, the first sites of appearance of dystrophin on cultured muscle cells are focal adhesions, i.e. specific sites of cytoskeleton/extracellular matrix interaction. These data also add to evidence that dystrophin and the 58 kDa act together.

Animals↗
PubMed1990

A protein homologous to the Torpedo postsynaptic 58K protein is present at the myotendinous junction.

The 58K protein is a peripheral membrane protein enriched in the acetylcholine receptor (AChR)-rich postsynaptic membrane of Torpedo electric organ. Because of its coexistence with AChRs in the postsynaptic membrane in both electrocytes and skeletal muscle, it is thought to be involved in the formation and maintenance of AChR clusters. Using an mAb against the 58K protein of Torpedo electric organ, we have identified a single protein band in SDS-PAGE analysis of Xenopus myotomal muscle with an apparent molecular mass of 48 kD. With this antibody, the distribution of this protein was examined in the myotomal muscle fibers with immunofluorescence techniques. We found that the 48K protein is concentrated at the myotendinous junctions (MTJs) of these muscle fibers. The MTJ is also enriched in talin and vinculin. By double labeling muscle fibers with antibodies against talin and the 48K protein, these two proteins were found to colocalize at the membrane invaginations of the MTJ. In cultured myotomal muscle cells, the 48K protein and talin are also colocalized at sites of membrane-myofibril interaction. The 48K protein is, however, not found at focal adhesion sites in nonmuscle cells, which are enriched in talin. These data suggest that the 48K protein is specifically involved in the interaction of myofibrillar actin filaments with the plasma membrane at the MTJ. In addition to the MTJ localization, 48K protein is also present at AChR clusters both in vivo and in vitro. Thus, this protein is shared by both the MTJ and the neuromuscular junction.

Animals↗
PubMed1989

Immunochemical identification of desmin in Torpedo postsynaptic membranes and at the rat neuromuscular junction.

Preparations of acetylcholine receptor-rich (AChR-rich) postsynaptic membranes from electric tissue of electric rays often contain an Mr 55,000 protein (55kD protein) that has not been previously characterized. Using a monoclonal antibody (MAb 1403) against the 55kD protein from Torpedo californica and a pan-specific, anti-intermediate filament antibody (Pruss et al., 1981; Cell 27:419-428), we show that the 55kD protein has the properties expected of Torpedo desmin. By the electron microscope immunogold method applied to perfusion-fixed electric tissue, MAb 1403 labeled only cytoplasmic filaments in the electroplax. These filaments were neither more concentrated nor arranged detectably differently in postsynaptic regions relative to nonpostsynaptic regions. The 55kD protein could also be fractionated away from isolated postsynaptic membranes by gradient centrifugation. The protein is thus a minor component of the postsynaptic membrane in situ and after isolation. On semithin cryosections of rat skeletal muscle, on the other hand, MAb 1403, which recognizes rat desmin but not rat vimentin, gave strong fluorescent labeling of the postsynaptic region, weaker labeling of the Z-line, and still weaker labeling of the cell surface immediately surrounding extra-junctional nuclei. The pattern of postsynaptic labeling suggests that desmin, presumably in the form of intermediate filaments, occurs near the AChR-rich crests of the junctional folds, but is particularly concentrated among and around the ends of the folds. Similar results were obtained with a second monoclonal antibody raised against authentic desmin. These results suggest that desmin intermediate filaments may have an important role in organization of the postsynaptic cytoplasm in rat muscle.

Animals↗
PubMed1987

A postsynaptic Mr 58,000 (58K) protein concentrated at acetylcholine receptor-rich sites in Torpedo electroplaques and skeletal muscle.

In the study of proteins that may participate in the events responsible for organization of macromolecules in the postsynaptic membrane, we have used a mAb to an Mr 58,000 protein (58K protein) found in purified acetylcholine receptor (AChR)-enriched membranes from Torpedo electrocytes. Immunogold labeling with the mAb shows that the 58K protein is located on the cytoplasmic side of Torpedo postsynaptic membranes and is most concentrated near the crests of the postjunctional folds, i.e., at sites of high AChR concentration. The mAb also recognizes a skeletal muscle protein with biochemical characteristics very similar to the electrocyte 58K protein. In immunofluorescence experiments on adult mammalian skeletal muscle, the 58K protein mAb labels endplates very intensely, but staining of extrasynaptic membrane is also seen. Endplate staining is not due entirely to membrane infoldings since a similar pattern is seen in neonatal rat diaphragm in which postjunctional folds are shallow and rudimentary, and in chicken muscle, which lacks folds entirely. Furthermore, clusters of AChR that occur spontaneously on cultured Xenopus myotomal cells and mouse muscle cells of the C2 line are also stained more intensely than the surrounding membrane with the 58K mAb. Denervation of adult rat diaphragm muscle for relatively long times causes a dramatic decrease in the endplate staining intensity. Thus, the concentration of this evolutionarily conserved protein at postsynaptic sites may be regulated by innervation or by muscle activity.

Animals↗
PubMed1986

Talin is a post-synaptic component of the rat neuromuscular junction.

Talin is a protein, recently discovered in chicken gizzard, which occurs at sites of actin-plasma membrane interaction in several cell types. Vinculin also occurs at many of these sites, possibly in association with talin. In this study, three antisera against talin were used to probe the neuromuscular junction of rat skeletal muscle, which is also a site of vinculin accumulation. By immunofluorescence, all three sera stained the junction strongly in frozen sections of rat diaphragm. The extrajunctional periphery was lightly and irregularly stained in some muscle cells; others seemed not to be stained outside the junction. Staining remained at junctions and increased in extrajunctional regions of muscle denervated 6 weeks before sacrifice. The staining in all cases was abolished by competition with purified talin. One serum tested by immunoblotting recognized one protein at Mr 215 000 (identical with the value for chicken gizzard talin) and traces of a second at Mr 190 000 (corresponding to a known proteolytic fragment of talin). We conclude that rat muscle talin is similar in its general protein structure to chicken gizzard talin, and is a post-synaptic component of the neuromuscular junction.

Animals↗
PubMed1985

Immunochemical demonstration that amino acids 360-377 of the acetylcholine receptor gamma-subunit are cytoplasmic.

Two monoclonal antibodies (mabs) previously prepared against Torpedo acetylcholine receptor are shown to recognize a synthetic nonadecapeptide corresponding to lys360-glu377 of the gamma subunit. The reaction was demonstrated by solid-phase enzyme-linked immunoabsorbent assays, by inhibition of binding of the mabs to receptor, and by immunoprecipitation of the peptide conjugated to bovine serum albumin. Immunogold electron microscopy on isolated postsynaptic membranes from Torpedo showed that both mabs bind to intracellular epitopes on the receptor. These results establish that amino acid residues 360-377 of the receptor gamma-subunit, and probably the analogous region of the delta-subunit, reside on the cytoplasmic side of the membrane. Since the primary structures of all four subunits suggest a common transmembrane arrangement, the corresponding domains of the alpha- and beta-subunits are probably also cytoplasmic.

Amino Acid Sequence↗
PubMed1984

Ultrastructural localization of the Mr 43,000 protein and the acetylcholine receptor in Torpedo postsynaptic membranes using monoclonal antibodies.

Four mouse monoclonal antibodies (mabs) were shown by immunoblotting procedures to recognize the major, basic, membrane-bound Mr 43,000 protein (43K protein) of acetylcholine receptor-rich postsynaptic membranes from Torpedo nobiliana . These mabs and a mab against an extracellular determinant on the acetylcholine receptor were used to localize the two proteins in electroplax (Torpedo californica) and on unsealed postsynaptic membrane fragments at the ultrastructural level. Bound mabs were revealed with a rabbit anti-mouse Ig serum and protein A-colloidal gold. The anti-43K mabs bound only to the cytoplasmic surface of the postsynaptic membrane. The distributions of the receptor and the 43K protein along the membrane were found to be coextensive. Distances between the membrane center and gold particles were very similar for anti-receptor and anti-43K mabs (29 +/- 7 nm and 26 to 29 +/- 7 to 10 nm, respectively). These results show that the 43K protein is a receptor-specific protein having a restricted spatial relationship to the membrane. They thus support models in which the 43K protein is associated with the cytoplasmic domains of the receptor molecule.

Animals↗
PubMed1984

Ultrastructural immunocytochemistry of particulate fractions using polyvinyl chloride microculture wells.

A method is described for immunoelectron microscopy of particulate subcellular fractions using polyvinyl chloride (soft) microculture wells as mechanical supports and reaction vessels. Appropriate quantities of particles are centrifuged onto the well bottoms, fixed and permeabilized if necessary, then labeled by standard procedures, fixed in glutaraldehyde and tannic acid, and prepared for thin section electron microscopy. The centrifugation, the fixations, and the embedment in Epon are discussed in detail.

Animals↗
PubMed1982

Cytoplasmic surface structure in postsynaptic membranes from electric tissue visualized by tannic-acid-mediated negative contrasting.

In this study, acetylcholine receptor-rich postsynaptic membranes from electric tissues of the electric rays Narcine brasiliensis and Torpedo californica are negatively contrasted for thin-section electron microscopy through the use of tannic acid. Both outer (extracellular) and inner (cytoplasmic) membrane surfaces are negatively contrasted, and can be studied together in transverse sections. The hydrophobic portion of the membrane appears as a thin (approximately 2 nm), strongly contrasted band. This band is the only image given by membrane regions which are devoid of acetylcholine receptor. In regions of high receptor density, however, both surfaces of the membrane are seen to bear or be associated with material which extends approximately 6.5 nm beyond the center of the bilayer. The material on the outer surface can be identified with the well-known extracellular portion of the receptor molecule. A major portion of the inner surface image is eliminated by extraction of the membranes at pH 11 to remove peripheral membrane proteins, principally the 43,000 Mr (43K) protein. The images thus suggest a cytoplasmic localization of the 43K protein, with its distribution being coextensive with that of the receptor. They also suggest that the 43K protein extends farther from the cytoplasmic surface than does the receptor.

Animals↗
PubMed1982

Visualization at the mouse neuromuscular junction of a submembrane structure in common with Torpedo postsynaptic membranes.

To test for submembrane structures common to postsynaptic membranes of muscle and electric tissue, mouse sternomastoid muscles were fixed in the presence of tannic acid under conditions such that a heavy layer of densely staining material was built up on plasma membranes. In this section electron micrographs of such muscles, the hydrophobic portion of the bilayer and some membrane-associated structures appear in strong negative contrast. At the endplate, acetylcholine receptor-rich membrane regions appear as a broad (14-15-nm) image approximately bisected by the thin (approximately 2-nm) image of the bilayer. The extracellular half of the image arises from the receptor protein. The cytoplasmic half contains a distinct bar of contrasted material which is precisely co-extensive with the receptor image Quantitative and direct visual comparisons show that the image is almost indistinguishable from that obtained by a similar method applied to isolated postsynaptic membranes from electric tissue of Torpedo californica (Sealock, R. (1982) J. Cell Biol. 92:514-522). In the latter case, the bar probably arises from the 43,000-dalton protein, a major peripheral membrane protein component of the isolated membranes. The results thus suggest that the 43,000-dalton protein in muscle and electric tissue forms similar structures and, conversely, that the structure in the isolated membranes retains the essential features of its in situ state.

Animals↗
PubMed1980

Identification of regions of high acetylcholine receptor density in tannic acid-fixed postsynaptic membranes from electric tissue.

Several distinct membrane specializations in electric tissue of the torpedine ray, Narcine brasiliensis, are rendered visible in thin section electron microscopy by the use of tannic acid during sample preparation. The acetylcholine receptor-containing regions of the postsynaptic membrane have a morphology shared by no other membrane in the tissue: a thick outer lamina, previously ascribed by others to the receptor protein itself, and a thick (approximately 8 nm) inner lamina which is precisely co-extensive with the thick outer lamina. Both of these features are found in biochemical preparations of the post-synaptic membrane upon fixation in the presence of tannic acid. Other membrane specializations having thick outer laminae can be distinguished from that due to the receptor. Thus, receptor-containing regions in isolated membrane fragments can be unambiguously identified by inspection. This provides an important practical tool for the study of these membranes. In addition, the data presented here and elsewhere suggest a model for the receptor-containing membrane in which material, presumably protein, is roughly symmetrically distributed on the two sides of the membrane.

Acetylcholine↗
PubMed1980

Postsynaptic distribution of acetylcholine receptors in electroplax of the torpedine ray, Narcine brasiliensis.

Other investigations have shown that many postsynaptic membranes which contain high densities of the nicotinic acetylcholine receptor appear to be unusually thick in electron micrographs. This peculiarity is believed to arise in part from the receptor protein itself. We show that in electroplax of Narcine brasiliensis, the thick membrane is found in juxtaneural postsynaptic regions, but not in deeper portions of junctional fold-like postsynaptic papillae or in extrasynaptic regions of the innervated face. Autoradiographic localization of receptor-bound [125I]alpha-bungarotoxin confirms that high densities of receptor are confined to juxtaneural regions. Narcine electric tissue thus provides a system for biochemical study of an acetylcholine receptor localization similar to that found at the mammalian or amphibian neuromuscular junction. Limited data suggest that similar postsynaptic distributions of receptor also occur in electroplax of several species of Torpedo.

Acetylcholine↗
PubMed1976

Effects of alpha-toxins from Bungarus multicinctus and Bungarus caeruleus on cholinergic responses in Aplysia neurons.

The effects of alpha-bungarotoxin from Bungarus multicinctus and Bungarus caeruleus were studied on three types of cholinergic response in Aplysia central neurones. These responses are the result of three distinct changes in ionic permeability: selective increases in permeabiliyt to Na, Cl and K, respectively. It was shown that 10(-5) M alpha-bungarotoxin from B. multicinctus completely blocks the response resulting from an increase in Cl permeability, while having no effect on either of the other two responses types (even when the toxin concentration was increased to 5 X 10(-5) M). The block of the Cl-dependent response by alpha-bungarotoxin is reversible. None of the three response types were affected by similar concentrations of alpha-toxin from B. caeruleus. Higher concentrations were not systematically tested. These results contradict reports of other authors on the action of alpha-bungarotoxin on molluscan acetylcholine (ACh) responses. Possible reasons for the discrepancies between our findings and those published by other authors are discussed.

Animals↗