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

Abstract

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

Antibody‐induced crosslinking and cholesterol‐sensitive, anomalous diffusion of nicotinic acetylcholine receptors

“…When the mAb‐labeled nAChR clustering events are compared with those of BTX‐labeled receptors (Mosqueira et al ) the lifetimes of the antibody nanoclustering events are found to be 3‐fold longer ( p < 0.01 for control and for cholesterol‐modifying conditions), a finding consistent with the idea that mAb35‐induced crosslinking adds stability to/prolongs the nanocluster formation. These dynamic structures may correspond to the diffraction‐limited ‘microaggregates’ observed along NMJ development (Olek et al ; Olek et al ) reviewed in (Sanes and Lichtman ).…”

Krp1 (Sarcosin) promotes lateral fusion of myofibril assembly intermediates in cultured mouse cardiomyocytes

“…Cells were cultured in 60 mm Permanox dishes (Nunc Nalgene) and processed for transmission electron microscopy as previously described [28], with minor modifications as reported [29]. In brief, cultures were fixed at three or seven days post-transfection with glutaraldehyde and tannic acid (Mallinckrodt), post-fixed with osmium tetroxide, stained en bloc with uranyl acetate, ethanol dehydrated and Epon embedded.…”

Altered Expression of Tropomodulin in Cardiomyocytes Disrupts the Sarcomeric Structure of Myofibrils

“…The cultures were postfixed in OsO 4 , stained en bloc with uranyl acetate, and further processed as described. 21 After embedding, two or three typical areas were selected on the basis of the shape and density of the cells. Thin sections were obtained parallel to the surface of the coverslip.…”