Direct observation of the rapid aggregation of acetylcholine receptors on identified cultured myotubes after exposure to embryonic brain extract

Olek, Anthony J.; Pudimat, P. A.; Daniels, Mathew P.

doi:10.1016/0092-8674(83)90156-3

Cited by 36 publications

(24 citation statements)

References 23 publications

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“…In addition, the ultrastructure of myotubes exposed to EBX for 4 hr was compared with that of untreated control cells. The results indicate that the first detectable stage of AChR aggregate assembly, the formation of clouds of microaggregates (Olek et al, 1983) is associated with relatively subtle changes in the cellsurface contour and underlying cytoskeleton. The extensive appearance of a distinct basal lamina and a co-extensive dense cytoplasmic specialization, as well as deeper folds in the myotube surface, appears to occur after microaggregate assembly but is closely correlated with the initial formation of AChR aggregates per se.…”

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confidence: 96%

“…After 4 hr exposure to EBX, approximately 8-10% of the top half of responsive myotubes became occupied by aggregates, with the mean area of aggregates being 12 Mm2 (see Fig. 1; Olek et al, 1983).…”

Section: Video Microscopymentioning

confidence: 99%

“…At various times after the addition of EBX, identified myotubes were photographed using a video imageintensification system, under reduced illumination, to monitor the assembly of AChR aggregates (see Olek et al, 1983). To summarize: Cells were examined with a Zeiss photomicroscope II equipped for epifluorescence with a 50 W mercury lamp and a 63 x n.a.…”

Section: Video Microscopymentioning

confidence: 99%

“…Within 4-6 hr, embryonic brain extract (EBX) induces the formation of sites of AChR density elevated 5-9x above that of surrounding regions, and the appearance of these aggregates is preceded by the formation of clouds of punctate microaggregates (Olek et al, 1983). A video image-intensification system was used to monitor this redistribution of fluorescently labeled AChR, and sites of aggregation were mapped on identified myotubes.…”

mentioning

confidence: 99%

“…To accomplish this, we have examined the ultrastructural changes that occur during the initial induction of AChR-rich endplatelike structures in cultured rat myotubes by a soluble brain extract (Olek et al, 1983;Salpeter et al, 1982;Sanes et al, 1984). In order to study the spatial and temporal relationships precisely, we have directly monitored the induced aggregation of fluorescently labeled AChR, using low incident illumination and a video image-intensification system to eliminate the inhibitory effect of illumination on the aggregation of fluorescently labeled AChR (Olek et al, 1983). The associated ultrastructure at stages within the first 6 hr of AChR aggregate formation was subsequently examined on the same, directly monitored myotubes.…”

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confidence: 99%

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Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes

Olek¹,

Ling²,

Daniels³

1986

J. Neurosci.

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The ultrastructure of cultured rat myotubes was examined at stages in the initial assembly of acetylcholine receptor (AChR) aggregates in order to elucidate the role of cell-surface specializations in aggregate formation. Within 4-6 hr, embryonic brain extract (EBX) induces the formation of sites of AChR density elevated 5-9x above that of surrounding regions, and the appearance of these aggregates is preceded by the formation of clouds of punctate microaggregates (Olek et al., 1983). A video image-intensification system was used to monitor this redistribution of fluorescently labeled AChR, and sites of aggregation were mapped on identified myotubes. After processing the cultures for electron microscopy, thin sections were taken through identified aggregate sites at various stages in assembly. Specializations, including a basal lamina, mound-shaped plasma membrane contours with occasional deep infoldings, and a subjacent dense cytoskeletal specialization, which tended to exclude other cytoplasmic organelles, were associated with newly formed aggregates found 4-6 hr after adding EBX to the cultures. Analysis of random thin sections through EBX-treated and untreated myotubes showed that the extent of specializations of the basal lamina and cytoplasm was approximately threefold greater in cells exposed to EBX for 4 hr, suggesting a concurrent, and possibly interdependent, organization of such specializations with AChR aggregate assembly. Examination of sections through clouds of microaggregates, which formed within 90 min, revealed mound-shaped plasma membrane contours and underlying cytoplasm depleted of organelles but relatively little basal lamina and submembrane cytoskeletal density. These results suggest that the initial stage of AChR aggregate assembly involves relatively subtle changes in the structure of the cell cortex and that the evolution of microaggregates to aggregates may require the formation of additional cytoskeletal and extracellular matrix structures.A critical question in cellular neurobiology is how certain regions within neurons and their synaptic targets become organized to subserve specialized functions. A striking example of such a region is the motor endplate, where a small area (less than 0.0 1% of the cell surface) of the muscle cell becomes highly differentiated in function and structure during synapse formation. While the function of the mature motor endplate in neuromuscular transmission has been well studied, relatively little

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confidence: 96%

Section: Video Microscopymentioning

confidence: 99%

Section: Video Microscopymentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes

Olek¹,

Ling²,

Daniels³

1986

J. Neurosci.

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Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: Refinements and applications

Daniels

Lowe

Shah

et al. 2000

Microsc. Res. Tech.

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Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve‐muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better‐defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon‐induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon‐induced AChR aggregation on muscle cell species, by the use of chick‐rat chimeric co‐cultures. (5) We provide evidence for the role of alternatively‐spliced agrin isoforms in synapse formation by using single cell RT‐PCR with neurons collected from co‐cultures after observation of axon‐induced AChR aggregation. Microsc. Res. Tech. 49:26–37, 2000. Published 2000 Wiley‐Liss, Inc.

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Acetylcholine receptor clustering and triton solubility: Neural effect

Podleski

Salpeter

1988

J. Neurobiol.

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Previous studies by Prives et al. (1980, 1982a and b) have shown that acetylcholine receptors (AchRs) are extracted from muscle cells in vitro by Triton X-100 at different rates, and that clustered receptors extract most slowly. The present study was aimed at comparing the relative extractability of receptors in clusters with those in intercluster regions and the role of neural factors in regulating this extractability. Using primary rat muscle cells in vitro we confirmed that receptor extraction with Triton X-100 does not fit a single exponential but has more than one rate, and that in control cells clustered receptors extract more slowly than do receptors in intercluster regions. The major new observation in this study was that neural extract lowered the overall Triton extraction rate of intercluster receptors to that of clustered receptors. Additional new observations include the findings that (1) both clustered and intercluster receptors show multiphasic extraction rates; (2) stabilization of AchRs against Triton extraction increases with time in the surface membrane; (3) the effect of neural extract on Triton extractability of AChR is dependent on factors that control RNA synthesis, cytoskeletal elements, and collagen; (4) fixation and/or buffer washes accelerate receptor extraction only in cells that are treated with Triton, but not in control cells; (5) in control cells (not exposed to neural factors) Triton X-100 causes new clusters to form. From experiments using Con A we suggest that the Triton-induced new clusters may not be formed by a redistribution of receptors but are, most likely, due to the presence of groups of intercluster receptors with extraction rates lower than those of surrounding receptors.

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Direct observation of the rapid aggregation of acetylcholine receptors on identified cultured myotubes after exposure to embryonic brain extract

Cited by 36 publications

References 23 publications

Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes

Development of ultrastructural specializations during the formation of acetylcholine receptor aggregates on cultured myotubes

Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: Refinements and applications

Acetylcholine receptor clustering and triton solubility: Neural effect

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