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Biomedical subjects

H B Peng

Publications and source records attributed to H B Peng.

72 records · Page 4Linked to original sources
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

Elimination of preexistent acetylcholine receptor clusters induced by the formation of new clusters in the absence of nerve.

Acetylcholine receptors (AChRs) form clusters spontaneously in cultured Xenopus myotomal muscle cells in the absence of innervation. Innervation causes a dispersal of these clusters, as well as the formation of new clusters along the neuromuscular contact. To examine whether this dispersal process is dependent upon the presence of the nerve or can be effected by the formation of new clusters alone, we examined the stability of the preexistent clusters during the formation of new AChR clusters induced by basic polypeptide-coated latex beads. These beads cause a rapid development of clusters in cultured Xenopus muscle cells, and the size and the number of clusters can be experimentally manipulated by varying the size and number of beads applied to the culture. Accompanying the bead-induced formation of new clusters was an observed suppression of preexistent clusters, as shown by fluorescence microscopy after staining with rhodamine-conjugated alpha-bungarotoxin. This process of extra-bead cluster removal can be speeded up by treating the cultures with more beads or using beads of larger diameter. Furthermore, if the beads are applied before the spontaneous AChR clustering, no extra-bead clusters are formed. Thus, the latex beads can mimic the nerve in both causing the formation of new clusters and the dispersal of preexistent clusters. Although the receptor clusters are dispersed by the beads, the density of diffuse receptors, as shown by autoradiography with 125I-conjugated alpha-bungarotoxin does not decrease in the short-term cultures under study. This indicates that the dispersal of preexistent clusters is not due to a depletion of the surface pool of AChRs.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Membrane-related specializations associated with acetylcholine receptor aggregates induced by electric fields.

The localization of membrane-associated specializations (basal lamina and cytoplasmic density) at sites of acetylcholine receptor (AChR) aggregation is consistent with an involvement of these structures in receptor stabilization. We investigated the occurrence of these specializations in association with AChR aggregates that develop at the cathode-facing edge of Xenopus muscle cells during exposure to a DC electric field. The cultures were labeled with a fluorescent conjugate of alpha-bungarotoxin and the receptor distribution on selected cells was determined before and after exposure to the field. In thin sections taken from the same cells, the cathode-facing edge was characterized by plaques of basal lamina and cytoplasmic density co-extensive with sarcolemma of increased density. In sections cut in a plane similar to the fluorescence image, it was possible to demonstrate that the specializations were concentrated at areas of field-induced AChR aggregation, and at receptor clusters existing on control cells. This finding further indicates that these structures participate in AChR stabilization, and that the mechanisms involved in AChR aggregation that result from field exposure and nerve contact may be similar.

Animals↗
PubMed1985

Association of the postsynaptic 43K protein with newly formed acetylcholine receptor clusters in cultured muscle cells.

The postsynaptic membrane from Torpedo electric organ contains, in addition to the acetylcholine receptor (AChR), a major peripheral membrane protein of approximately 43,000 mol wt (43K protein). Previous studies have shown that this protein is closely associated with AChR and may be involved in anchoring receptors to the postsynaptic membrane. In this study, binding sites for monoclonal antibodies (mabs) to the 43K protein have been compared to the distribution of AChR in Xenopus laevis muscle cells in culture. In double label immunofluorescence experiments, clusters of AChR that occur spontaneously on these cells were stained with anti-43K mabs. Newly formed receptor clusters induced with positive polypeptide-coated latex beads were also stained with anti-43K mabs as early as 12 h after the application of the beads. Exact correspondence in the distribution of the anti-43K protein binding sites and the AChR was found in both types of clusters. These results suggest that the 43K protein becomes associated with AChR clusters during a period of active postsynaptic membrane differentiation. Thus, this protein may participate in the clustering process.

Animals↗
PubMed1984

Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters.

The formation of acetylcholine receptor (AChR) clusters can be experimentally induced in cultured Xenopus myotomal muscle cells by positive polypeptide-coated latex beads (Peng, H.B., P.-C. Cheng, and P.W. Luther, 1981, Nature [Lond.], 292:831-834). This provides a simple procedure for studying the cellular process of AChR clustering. In this study, the involvement of calcium and calmodulin in this process was examined. A deprivation in extracellular calcium by calcium-free medium or by the addition of calcium antagonists such as divalent cations Co2+ and Ni2+ (1-5 mM) or organic compounds verapamil and D-600 (0.1-0.5 mM) suppressed the formation of AChR clusters induced by the latex beads in a largely reversible manner. Antagonists against calmodulin, including trifluoperazine (1-5 microM) and the naphthalene sulfonamide W-7 (20 microM), also suppressed AChR clustering. However, the effect of W-7 was much weaker than that of trifluoperazine (TFP). Although the formation of AChR clusters is inhibited by these drugs, the stability of the existent clusters is relatively insensitive to them. These data suggest that the clustering of AChR involves a Ca2+ and calmodulin-activated process. Immunofluorescence studies using an antibody against calmodulin indicate that calmodulin is diffusely distributed in the cytoplasm in addition to its localization at the I-bands. Thus I propose that a local rise in intracellular calcium caused by a locally applied stimulus, exemplified here by the polypeptide-coated latex beads, may trigger the formation of AChR clusters. Furthermore, the cellular machinery for this process may involve calmodulin and is diffusely distributed in the muscle cell.

Animals↗
PubMed1984

Early cytoplasmic specialization at the presumptive acetylcholine receptor cluster: a meshwork of thin filaments.

Postsynaptic differentiation can be experimentally induced in cultured Xenopus myotomal muscle cells by polyornithine-coated latex beads (Peng, H. B., and P.-C. Cheng, 1982, J. Neurosci., 2:1760-1774). In this study, we examined the time course of this process. Small, punctate acetylcholine receptor (AChR) clusters were detectable as early as 1.5 h after the addition of the beads. Subsequently, both the size and the number of the clusters increased with time until a saturation level was reached between 8-24 h. Because the onset and the site of the AChR clustering could be precisely marked, we were able to examine the early structural specializations associated with presumptive AChR clusters. At 1 h, when less than 20% bead-muscle contacts displayed AChR clusters, 70% of the contacts already exhibited a meshwork of 5-6-nm filaments, which were of the same size as the thin filaments within the myofibrils and thus may contain actin. A system of cisternae similar to the smooth endoplasmic reticulum was suspended within this meshwork, but other organelles were excluded from it. This meshwork, being the earliest cytoplasmic specialization at the presumptive AChR clusters and appearing before the clusters, may be a mechanism for the clustering process.

Animals↗
PubMed1983

Cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor cluster in cell cultures.

Whole-mount stereo electron microscopy has been used to examine the cytoskeletal organization of the presynaptic nerve terminal and the acetylcholine receptor (AChR) clusters in cultures of Xenopus nerve and muscle cells. The cells were grown on Formvar-coated gold electron microscope (EM) finder grids. AChR clusters were identified in live cultures by fluorescence microscopy after labeling with tetramethylrhodamine-conjugated alpha-bungarotoxin. After chemical fixation and critical-point drying, the cytoplasmic specializations of identified cells were examined in whole mount under an electron microscope. In the presynaptic nerve terminal opposite to the AChR cluster, synaptic vesicles were clearly suspended in a lattice of 5-12-nm filaments. Stereo microscopy showed that these filaments directly contacted the vesicles. This lattice was also contiguous with the filament bundle that formed the core of the axon. At the AChR cluster, an increased cytoplasmic density differentiated this area from the rest of the cytoplasm. This density was composed of a meshwork of filaments with a mean diameter of 6 nm and irregularly shaped membrane cisternae 0.1-0.5 micron in width, which resembled the smooth endoplasmic reticulum. These membrane structures were interconnected via the filaments. Organelles that were characteristic of the bulk of the sarcoplasm such as the rough endoplasmic reticulum and the polysomes, were absent from the cytoplasm associated with the AChR cluster. These results indicate that the cytoskeleton may play an important role in the development and/or the maintenance of the neuromuscular synapse, including the release of transmitter in the nerve terminal and the clustering of AChRs in the postsynaptic membrane.

Animals↗
PubMed1982

Formation of postsynaptic specializations induced by latex beads in cultured muscle cells.

An early event in the formation of neuromuscular junctions is an accumulation of acetylcholine receptors (AChRs) in the postsynaptic membrane. This concentration of receptors is essential for the establishment of the synaptic transmission. Previously, we showed that latex beads coated with positively charged polypeptide molecules can induce the formation of AChR clusters at the bead-muscle contacts (Peng, H. B., P.-C. Cheng, and P. W. Luther (1981) Nature 292: 831-834). In this study, we demonstrate that, in addition to AChR clusters, a set of postsynaptic specializations normally present at the adult neuromuscular junction also develops at the bead-muscle contacts in the absence of nerve. These specializations include the membrane-associated cytoplasmic density and its associated thin (6- to 8-nm) filaments, clusters of intramembranous particles, membrane invaginations, the basal lamina, and 60- to 100-nm coated vesicles. Our results indicate that the mechanism for the formation of the postsynaptic specializations is preprogramed in the muscle and can be triggered by an interaction with polypeptide-coated latex beads. Thus, this bead-muscle system can be used as a simple model for understanding the postsynaptic development during synaptogenesis.

Animals↗
PubMed1981

Freeze-fracture study of the crayfish stretch receptor.

The crayfish slow-adapting stretch receptor was fixed under relaxed or stretched conditions (twice the relaxed length) and then processed for freeze-fracture study. The sensory neuron membrane had evenly distributed intramembrane particles mostly on its P face. The density of these particles was higher in the cell body than in the dendritic tips, which are the terminal portions of the dendrites. The dendritic tips were cylindrical under the relaxed condition and showed deformations with stretch stimuli. When they were fixed under the stretched condition with 1.6% glutaraldehyde in 0.12 M phosphate buffer (the total osmolarity of this fixative is isosmotic with the physiological solution), the dendritic tips showed regional swelling and shrinkage. The intramembrane particle density of the swollen parts decreased and there were particle-free patches of membrane, whereas the particle density of the shrunken parts increased. On the other hand when the receptor was fixed with 1.6% glutaraldehyde in 0.2 M phosphate buffer (the total osmolarity is hyperosmotic but buffer osmolarity is isosmotic), the diameter of the dendritic tips became smaller, and their membrane particle densities were almost the same as that under the relaxed condition. The sheath cells covering the sensory neuron were characterized by their sheet-like profiles, gap junctions, and crater-like protrusions. The receptor muscle membrane had longitudinal foldings, occasional invaginations, peripheral couplings, string-shaped particle aggregates, and band-shaped particle aggregates.

Animals↗
PubMed1980

Development of the postsynaptic membrane in Xenopus neuromuscular cultures observed by freeze-fracture and thin-section electron microscopy.

The formation of synapses between cultured neurons and muscle cells from Xenopus embryos has been studied with freeze-fracture and thin-section techniques. Clusters of large P-face intramembranous particles (about 11-12 nm) were observed in both innervated and non-innervated muscle cells. They presumably represented clusters of acetylcholine (ACh) receptors because of their close resemblance to the post-junctional particle clusters at the adult neuromuscular (N-M) junctions. In one-day cocultures, particle aggregates could be observed in more than 50% of the N-M contacts. At this stage, these aggregates were diffusely distributed along the contacts. After two days of coculture, extensive and tight clustering of large particles was seen along the length of persisting N-M contacts. Each particle cluster was composed of many particle aggregates and a particle-free groove demarcated each aggregate from its neighbor, thus producing a convoluted appearance of the membrane, which corresponded well with the thin-section image of the membrane profiles at the N-M contacts. In both freeze-fracture and thin-section images, membrane depressions with a diameter of about 0.1 micron were often observed in the vicinity of N-M contacts in newly innervated muscle cells. Within the pits of these depressions a small aggregate of large particles similar to those in the sarcolemma was often encountered. Such particle-rich membrane depressions were also observed in non-innervated muscle cells. They may represent sites for the incorporation of new ACh receptors in light of current theories. Particle aggregates were also closely associated with certain deep membrane invaginations, suggesting that these structures may be involved in the concentration of ACh receptors. Close membrane contacts were observed between nerve endings and muscle cells in young cocultures thin-sectioned. Gap junction-like particle aggregates were also observed in the muscle membrane along identified young N-M contacts. These data suggest that the formation of transient gap junctions may accompany the initial stages of synaptogenesis in Xenopus N-M cultures.

Acetylcholine↗
PubMed1978

Membrane particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells.

Clusters of membrane particle aggregates were found in the cultures of Xenopus embryonic muscle cells. In innervated cultures, the aggregates were usually found in the vicinity of the nerve. In terms of particle density and morphology, they resembled the postsynaptic particle aggregates of adult skeletal muscle fibers, suggesting that they may be related to acetylcholine receptors. Similar particle aggregates were also found in noninnervated cultures. They may correspond to extrajunctional clusters of acetylcholine receptors or "hot spots."

Animals↗
PubMed1976

A simple, selective method for freeze-fracturing spherical cells.

A simple and selective method for freeze-fracturing spherical cells is described. The cells are loaded into the holes of a thin nickel screen. A metal hat is applied to the cell monolayer and the whole assembly, hat-cells-screen, is frozen and then fractured by ripping the hat off. The fractured face on the screen is replicated. By varying the size of the screen holes and by applying the hat to either side of the screen, this method can selectively expose the E face (or the outer half of plasma membrane), the P face (or the inner half of the plasma membrane), or the cytoplasm of the cells. It also provides a means to produce fractures at a preselected area on the cell, if the cells can be loaded onto the screen in an oriented fashion.

Cell Membrane↗
PubMedDate not supplied

Targeting acetylcholinesterase molecules to the neuromuscular synapse.

The functional integrity of the neuromuscular synapse requires that sufficient numbers of acetylcholinesterase (AChE) molecules be localized on the specialized extracellular matrix between the nerve terminal and the post-synaptic membrane. Multiple interrelated levels of regulation are necessary to accomplish this complex task including the spatial and temporal restriction of AChE mRNA expression within the muscle fiber, local translation and assembly of AChE polypeptides, and focused accumulation of AChE molecules on the extracellular matrix. This is accomplished in part through the organization of other extracellular matrix molecules into a complex which further associates with acetylcholine receptors and their accompanying molecules. Finally, the mature neuromuscular junction contains molecules which can act as receptors for the attachment of AChE which in turn may allow for the turnover of this enzyme at the synapse. This brief review will focus mainly on contributions from our laboratory towards understanding the mechanisms involved in organizing AChE molecules at the neuromuscular synapse.

Acetylcholinesterase↗
PubMedDate not supplied

Changes in cell shape and actin distribution induced by constant electric fields.

The development of motility in cultured cells is usually associated with a polarization of the cell shape. In particular, the leading edge of the cell is extended into a lamella which acts as a locus for the elaboration of cell processes and for the formation of cell-substrate contacts and, at the opposite end, retraction fibres often extend beyond the trailing edge of the cell. The alignment of microfilament bundles (stress fibres) along the direction of migration and the presence of a band of actin at the leading edge of the cell suggest an involvement of this protein in the motile process. The direction of growth and orientation of various cell types in tissue culture can be influenced by externally applied d.c. electric fields but the effect of the field on cellular motile activities is unknown. Here we describe a galvanotropic response of cultured Xenopus epithelial cells. At a field strength of 5 V cm-1 these cells elongate perpendicularly with respect to the field. The anodal side of the cell retracts and both the ends and cathodal edge become active in the extension of ruffling lamellipodia. In parallel with the change in the cell axis, stress fibres are oriented perpendicularly to the field, and a band of actin is associated with the lamellae at the cathodal edge and at the ends of the cell.

Actins↗