Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)-regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.

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