Early stages in the formation and stabilization of acetylcholine receptor aggregates on cultured myotubes: sensitivity to temperature and azide.

We have studied the effects of temperature and sodium azide on the formation and stability of embryonic brain extract (EBX)2-induced acetylcholine receptor (AChR) aggregates on myotubes. Sequential changes in AChR distribution were studied on living myotubes in culture by video-intensified fluorescence microscopy. Aggregate formation was temperature dependent, increasing sharply from 24-36 degrees, maximal at 36-37 degrees, and virtually blocked at 38-40 degrees. Whereas aggregate size increased rapidly with time (up to 4 hr) at 36 degrees, at 18-24 degrees small (less than or equal to 1 micron) "microaggregates" formed and accumulated for up to 10 hr. Aggregates formed within 1.5 hr at the sites of microaggregates (formed after 4 hr at 23 degrees) if the temperature was raised to 36 degrees. However, if EBX was removed, the microaggregates on 50% of myotubes disassembled within 1.5 hr. The formation of microaggregates at 23 degrees and aggregates at 36 degrees was reversibly inhibited by sodium azide. These results show that clusters of microaggregates are the precursors of aggregates, and suggest that microaggregate clouds represent a discrete, labile, ATP-dependent stage in aggregate formation. Aggregates that had formed after 4 hr in the presence of EBX disassembled slowly (within 12-14 hr) following removal of EBX at 36 degrees, and even more slowly at 23-30 degrees. However, a temperature shift to 38 degrees, or the addition of azide, resulted in a rapid but reversible disassembly of aggregates (within 4 hr). Thus, newly formed aggregates appear to be relatively stable structures, while microaggregate clouds are labile, tending to disassemble or evolve into aggregates.