Ultrastructure of acetylcholine receptor clusters on cultured muscle fibers.

Clusters of membrane particle aggregates were found in the cultures of Xenopus embryonic muscle cells. In innervated cultures, the agegates were usually found in the vicinity of a 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."Cultures of cells isolated from embryonic muscle and nerve tissue have recently become a major tool in the study of synaptogenesis (1-3). The advantages of this in vitro system include its superb optical resolution and the ease of experimental manipulation. We have studied the ultrastructural aspects of synapse formation in cultures of embryonic Xenopus nerve and muscle cells. This system has recently been studied by several workers (4-8). Its major advantage is the simplicity in preparing and maintaining the early cultures. Furthermore, the in vitro development of cultured muscle cells can be closely compared with their in vivo development-for instance, in the formation of the tail musculature of the tadpole (6, 9).Much insight about the structure of the adult frog neuromuscular junction has come from the use of the freeze-fracture technique (10, 11). Aggregates of membrane particles, which may represent clusters of acetylcholine receptors, have been found in the postjunctional membrane in exact register with the presynaptic release sites of synaptic vesicles. In this paper, we report the existence of similar particle aggregates in innervated and noninnervated cultures of Xenopus embryonic muscle cells.MATERIALS AND METHODS Tissue Culture. Xenopus laevis eggs were obtained and inseminated according to the method of Hamburger (12). Eggs from a female, that had received an injection of 650 international units of human chorionic gonadotropin (Sigma, St. Louis, MO) the night before, were stripped into a dish of 10% Holtfreter solution containing a few drops of concentrated sperm suspension from a homogenized testis (full-strength Holtfreter solution contains 60 mM NaCl, 0.6 mM KCI, 0.9 mM CaC12, and 0.2 mM NaHCO3). After fertilization, single embryos were separated from the egg mass and reared in the same solution at 20 embryos per 60-mm petri dish. Cells for tissue culture were isolated from Nieuwkoop-Faber stage 21 embryos (13). The dissection was done aseptically. Each embryo was dejellied in 10% Holtfreter solution. It was then transferred to Steinberg solution, consisting of 60 mM NaCl, 0.67 mM KCI, 0.34 mM Ca(NO3)2, 0.83 mM MgSO4, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), pH 7.4 (modified from ref. 12). The dorsal skin was opened and the dorsal part of the embryo, including the dorsal mesoderm, the notochord, and the neural tube, was cut away and transferred to Steinberg solution containing collagenase (Sigma, type I), 1 mg/ml. After 15-30 m...

Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

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

Peng¹,

Nakajima²

1978

“…The distribution of AcCho receptors on mouse and rat myotube surfaces was visualized by indirect immunoperoxidase staining of bound a-bungarotoxin (a-btx), as described (10,11), except that: (i) Abbreviations: AcCho, acetylcholine; a-btx, a-bungarotoxin; A/M, acetylcholine receptor aggregates per myotube; Bt2cAMP, N6,02'-dibutyryl-adenosine 3':5'-cyclic monophosphate; DMEM, the Dulbecco-Vogt modification of Eagle's minimal essential medium.…”

Section: Methodsmentioning

confidence: 99%

A factor from neurons increases the number of acetylcholine receptor aggregates on cultured muscle cells.

Christian¹,

Daniels²,

Sugiyama³

et al. 1978

Self Cite

138

There is an increase in the number of acetylcholine (AcCho) receptor aggregates on striated embryonic mouse myotubes when they are cocultured with clonal neuroblastoma-glioma hybrid cells. Medium conditioned by hybrid cells contains a factor which increases the number of AcCho receptor aggregates on myotubes cultured from mouse, rat or chick muscle. AcCho receptor-aggregating activity was present in medium conditioned by the neuroblastoma parent clone but was not detected in medium conditioned by cells of the parent glioma clone, fibroblasts, or HeLa cells. The factor increased the aggregation of AcCho receptors within 24 hr without a significant increase in the total number of AcCho receptors, and its action did not depend on myotube protein synthesis. The factor appears to rearrange the distribution of myotube AcCho receptors either by aggregating mobile AcCho receptors or by stabilizing labile receptor aggregates. Acetylcholine (AcCho) receptors of the innervated skeletal muscle are localized at the neuromuscular junction (1) seemingly by two processes: (i) accumulation of AcCho receptors at the neuromuscular contact region, and (ii) elimination of AcCho receptors from noncontact regions (2). Activation of the muscle fiber by electrical stimulation or synaptic activity can decrease the number of extrajunctional AcCho receptors (3); however, there is only speculation concerning the mechanism of AcCho receptor accumulation at muscle regions aligned with presynaptic acetylcholine release sites (4, 5). In various cells, randomly dispersed surface receptors form patches or caps when the cells are treated with specific immunoglobulins or lectins (6, 7). We here report a similar phenomenon: a factor produced by the cholinergic neuroblastoma-glioma hybrid cell line NG108-15 increases the number of AcCho receptor aggregates on cultured myotubes. We propose that such a factor may be involved in the aggregation of AcCho receptors at the site of nerve contact during synapse formation. MATERIALS AND METHODSCell Culture. Mouse muscle cultures were prepared from the hindlimbs of 18-20-day-old C57B/6N mouse embryos, as described (8), except that cell proliferation was suppressed by the addition of 10-5 M fluorodeoxyuridine on the fifth and sixth days of culture. Cultures were established by adding a suspension of 5 X 105 single cells to 35-mm plastic plates, or 1.5 X 105 cells to the 16-mm wells of plastic multiwell plates (Costar). Conditioned medium produced as described below was added to muscle cultures 10-21 days after plating.Rat muscle cultures were prepared from Fischer strain rat embryos by using the same methods.Chick muscle cultures were prepared from the pectoral muscle of 11-day-old chick embryos (9). A suspension of mechanically dissociated single cells was prepared in a medium of 85% Eagle's minimal essential medium, 10% horse serum, and 5% chick embryo extract, and 2,0 X 104 cells in 40ul 'of medium were added to the 5-mm round wells in Teflon-covered glass slides (Roboz Surgical Instrument Co.). On ...

“…The buffer was removed and the cells were fixed for 60 min in 0.01 M sodium periodate/0.075 M lysine/2% paraformaldehyde/0.037 M phosphate, pH 6.9, with one change of fixative after 30 min. This method of fixation has been shown by others to be compatible with preservation of cellular ultrastructure and surface antigens (11,12). After fixation, the cells were washed with Pi buffer several times and then washed for 30-60 min, with changes every 10 Pi buffer/albumin with changes every 10 min, the cells were treated with either 125I-or fluorescein isothiocyanate-labeled goat anti-rabbit IgG.…”

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

Distribution and activity of endogenous lectin during myogenesis as measured with antilectin antibody.

Podleski¹,

Greenberg²

1980

Antibodies to electrolectin, a lectin endogenous to embryonic skeletal muscle, have been used to study the distribution of electrolectin during myogenesis in L3 cells and rat primary muscle cultures. Antibody binding is highest to mononucleated cells and is low to myotubes in both systems. Binding is much lower to fibroblasts in the primary cultures. Binding appears to be on the surface of these cells, although evidence is presented for there being binding on the inside of cells as well. When observed on myotubes, binding is generally associated with highly stained patches and in some instances is near regions where fusion may be occurring. In L3 cells, binding sites can be exposed by treating mononucleated cells with trypsin. These results are discussed in terms of their possible role in myogenesis and synaptogenesis. Teichberg et al. (1) discovered and partially characterized a lectin from electric organs and other excitable tissues. This lectin, called electrolectin, caused the agglutination of rabbit erythrocytes and this agglutination was inhibited by galactose-containing sugars. Subsequently, a protein with these properties has been isolated and has been found in several different tissues (2-4).Because this lectin was found in high activities in embryonic skeletal muscle, several laboratories investigated the possibility that electrolectin was involved in the differentiation of skeletal muscle. Although all these studies showed that the activity of electrolectin changed as the muscle cells differentiated, no definitive function for electrolectin has been proven (5-8).In our studies, using the L6 line of myogenic cells, we have found that the agglutination due to electrolectin is blocked by another protein, and evidence for the surface location of this protein has been provided (9). We have suggested that this blocking protein may be identical to another agglutinating protein present in these cells, which we have called myonectin. Because the blocking protein is soluble under our homogenization conditions, its presence in the supernatant fraction can prevent the accurate measurement of the soluble form of electrolectin (s-electrolectin). There is another form of the electrolectin which is bound to subcellular organelles but can be solubilized by sugars containing 3-D-galactose; we have called this particulate form of electrolectin p-electrolectin. All three proteins-i.e., s-electrolectin, p-electrolectin, and myonectin-undergo characteristic changes in activity as the cells differentiate. Myonectin and p-electrolectin activities are very low after the cells are plated into fresh medium, but s-electrolectin activity is high. As the cells grow to confluency and begin to fuse, both myonectin and p-electrolectin activities increase, but s-electrolectin activity decreases. These changes in activities can represent changes in specific activities of >10-fold (9).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" ...