LARGE PLASMA-MEMBRANE DISRUPTIONS ARE RAPIDLY RESEALED BY CA2-DEPENDENT VESICLE-VESICLE FUSION EVENTS()

A microneedle puncture of the fibroblast or sea urchin egg surface rap idly evokes a localized exocytotic reaction that may be required for t he rapid resealing that follow this breach in plasma membrane integrit y (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC ). 263:390-393). How this exocytotic reaction facilitates the resealin g process is unknown. We found that starfish oocytes and sea urchin eg gs rapidly reseal much larger disruptions than those produced with a m icroneedle. When an similar to 40 by 10 mu m surface patch was torn of f, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein de xtran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not d etected by confocal microscopy. Moreover, only a brief (similar to 5-1 0 s) rise in cytosolic Ca2+ was detected at the wound site. Several li nes of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. Whe n we injected FS-containing SW deep into the cells, a vesicle formed i mmediately, entrapping within its confines most of the FS. DiI stainin g and EM confirmed that the barrier delimiting injected SW was a membr ane bilayer. The threshold for vesicle formation was similar to 3 mM C a2+ (SW is similar to 10 mM Ca2+). The capacity of intracellular membr anes for sealing off SW was further demonstrated by extruding egg cyto plasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet-rich domain formed a membrane, suggesting that the yolk platelet is a critical ele ment in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle-vesicle (inc luding endocytic compartments but possibly excluding ER) fusion reacti ons. The function in resealing of this cytoplasmic fusion reaction is to form a replacement bilayer patch. This patch is added to the discon tinuous surface bilayer by exocytotic fusion events.