Calcium-induced fusion of sea urchin egg secretory vesicles with planar phospholipid bilayer membranes

The fusion of sea urchin egg secretory vesicles to planar phospholipid bila yer membranes was studied by differential interference contrast (DIC) and f luorescent microscopy, in combination with electrical recordings of membran e conductance. A strong binding of vesicles to protein-free planar membrane s was observed in the absence of calcium. Calcium-induced fusion of vesicle s was detected using two independent assays: loss of the contents of indivi dual vesicles visible by DIC microscopy; and vesicle content discharge acro ss the planar membrane detected by an increase in the fluorescence of a dye . In both cases, no increase in the membrane conductance was observed unles s vesicles were incubated with either Amphotericin B or digitonin prior to applying them to the planar membrane, an indication that native vesicles ar e devoid of open channels. Pre-incubation of vesicles with n-ethylmaleimide (NEM) abolished calcium-induced fusion, Fusion was also detected when vesi cles were osmotically swollen to the point of lysis. In contrast, no fusion of vesicles to planar bilayers was seen when vesicles on plasma membrane ( native cortices) were applied to a phospholipid membrane, despite good bind ing of vesicles to the planar membrane and fusion of vesicles to plasma mem brane. It is suggested that cortical vesicles (CVs) have sufficient calcium -sensitive proteins for fusion to lipid membranes, but in native cortices g ranular fusion sites are oriented toward the plasma membrane. Removal of ve sicles from the plasma membrane may allow fusion sites on vesicles access t o new membranes.