Plasmalemmal repair of severed neurites of PC12 cells requires Ca2+ and synaptotagmin

Ca2+ and synaptotagmin (a Ca2+-binding protein that regulates axolemmal fus ion of synaptic vesicles) play essential roles in the repair of axolemmal d amage in invertebrate giant axons. We now report that neurites of a rat phe ochromocytoma (PC12) cell line transected and maintained in a serum medium form a dye barrier (exclude an external hydrophilic fluorescent dye) and su rvive for 24-hr posttransection (based on morphology and retention of anoth er hydrophilic dye internally loaded at 6-hr posttransection), Some (25%) t ransected neurites that form a dye barrier regrow. Most (83%) neurites tran sected in a saline solution containing divalent cations (PBS++) also exclud e entry of an externally placed hydrophilic fluorescent dye at 15-min postt ransection. In contrast, only 14 or 17% of neurites maintained in a divalen t cation-free solution (PBS') or in PBS= + Mg2+, respectively, form a dye b arrier. Neurites that do not form a dye barrier do not survive for 24 hr. W hen PC12 neurites are loaded with an antibody to squid synaptotagmin, most (81 %) antibody-loaded neurites do not form a dye barrier, whereas most (gr eater than or equal to 81%) neurites loaded with heat-inactivated antibody or preimmune IgG do form a barrier. These data show that: I) transected neu rites of PC12 cells have mechanism(s) for plasmalemmal repair (dye barrier formation and survival), 2) Ca2+ is necessary for dye barrier formation, wh ich occurs minutes after transection and is necessary for survival and regr owth; and 3) synaptotagmin is an essential mediator of barrier formation. T he similarity in the requirements for plasmalemmal repair in this mammalian cell preparation with those reported previously for invertebrate axons sug gests that mechanisms necessary for plasmalemmal repair have been conserved phylogenetically. (C) 2000 Wiley-Liss, Inc.