CALCIUM-REGULATED EXOCYTOSIS IS REQUIRED FOR CELL-MEMBRANE RESEALING

Using confocal microscopy, we visualized exocytosis during membrane re sealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca2+-regulate d exocytosis. The rate of exocytosis was correlated quantitatively wit h successfully resealing, In embryos, whose activated surfaces must fi rst dock vesicles before fusion, exocytosis and membrane resealing wer e inhibited by neurotoxins that selectively cleave the SNARE complex p roteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked wi th externally applied stachyose. If cortical vesicles were undocked bo th 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 compet ent for resealing, although botulinum neurotoxin type A was still inef fective, Cortical vesicles transiently undocked in the presence of tet anus toxin were subsequently fusion incompetent although to a large ex tent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is requ ired and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.