Polycation inhibition of exocytosis from sea urchin egg cortex

Summary

The Ca²⁺-stimulated release of vesicle contents from cortical fragments prepared from sea urchin eggs is an in vitro model for exocytosis. Cortical fragments have been isolated either in suspension (cell surface complex, CSC preparation), or attached to polycation-coated surfaces (cortical lawn, CL preparation). CL, but not CSC, have been reported to undergo a rapid “aging” process whereby they fail to respond to micromolar free Ca²⁺. Since, in principle, the only difference between the two preparations is the use of polycations in the CL preparation, polycations were suspected of being inhibitory. This hypothesis was tested by evaluating the effects of polycation-containing buffers on the Ca²⁺ threshold, rate, and extent of exocytosis in CL prepared from the eggs ofStrongylocentrotus purpuratus. A sensitive microphotometric assay, based on light scattering by the individual cortical vesicles in the CL, was used to quantitate the exocytotic response. Buffers containing polylysine were found to be potent inhibitors of cortical exocytosis. The Ca²⁺ threshold of CL that had been treated for 15 min at room temperature with 50 μg/ml of polylysine was more than three orders of magnitude greater than that of freshly prepared CL. The other polycations tested (protamine, spermine and neomycin) were also found to be inhibitory, but to a lesser degree than polylysine. Two lines of evidence suggested that the polycations used in the preparation of CL are responsible for the rapid “aging” phenomenon: (i) CSC fragments that had been affixed to polylysine-coated coverslips were shown to aquire “aging” characteristics similar to the CL preparations; control CSC that had been maintained in suspension did not. (ii) Radiolabeled poly-l-lysine was shown to dissociate from coated coverslips and redistribute onto CL.