ACTIN DISASSEMBLES REVERSIBLY DURING ELECTRICALLY-INDUCED RECYCLING OF SYNAPTIC VESICLES IN CULTURED NEURONS

We have studied depolarization-induced regulation of actin assembly in exocytotically active areas of dissociated chick sympathetic neurons. Active areas were identified with the fluorescent dye FM1-43 which la bels synaptic vesicles that recycle in these regions. Exocytosis (elec trically stimulated) was monitored in real time through depletion of F M1-43 fluorescence. To study depolarization-induced disassembly of act in in the FM1-43-stained regions, the cells were fixed after different periods of depolarization and stained with rhodamine phalloidin, whic h binds preferentially to the filamentous form of actin. In active reg ions, actin disassembles and reassembles during continuous 2 min depol arization. Actin disassembly that occurs after the first 25 s of depol arization was detested by a reduction in rhodamine phalloidin staining and confirmed by correlative electron microscopy. Immunogold staining revealed that actin is abundant throughout resting terminals. In some experiments, actin filaments were stabilized by loading cells with un labelled phalloidin before stimulating secretion. Stabilizing the fila ments does not alter the initial release but strongly reduces the rele ase rate at later stages. These data are consistent with a model in wh ich partial disassembly of actin filaments is necessary for facilitati ng the transport of vesicles within the terminal and reassembly is nec essary for Limiting that movement. (C) 1998 Elsevier Science B.V.