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.