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

Abstract

We have studied the effects of temperature and sodium azide on the formation and stability of embryonic brain extract (EBX) 2 2 Abbreviations used: AChR, acetylcholine receptor, EBX, embryonic brain extract; TMR-BTX, tetramethylrhodamine conjugated α-bungarotoxin -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°, maximal at 36–37°, and virtually blocked at 38–40°. Whereas aggregate size increased rapidly with time (up to 4 hr) at 36°, at 18–24° small (≤1 μm) “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°) if the temperature was raised to 36°. However, if EBX was removed, the microaggregates on 50% of myotubes disassembled within 1.5 hr. The formation of microaggregates at 23° and aggregates at 36° 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°, and even more slowly at 23–30°. However, a temperature shift to 38°, 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.