Polycation inhibition of exocytosis from sea urchin egg cortex

Abstract

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 of Strongylocentrotus 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 micrograms/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: CSC fragments that had been affixed to polylysine-coated coverslips were shown to acquire "aging" characteristics similar to the CL preparations; control CSC that had been maintained in suspension did not. Radiolabeled poly-L-lysine was shown to dissociate from coated coverslips and redistribute onto CL.

Poisson-distributed active fusion complexes underlie the control of the rate and extent of exocytosis by calcium.

“…Using the random model, we can rationalize much of the calcium-dependent exocytotic behavior observed in this preparation. These include (a) the sigmoidal dependence of survival on pCa due to the existence of submaximal responses (4, 60); (b) the increase in fusion rate with increase in [Ca2+]i (10,54); (c) the discrepancy between the calcium concentration required for maximal extents and rates (61); (d) shifting of the calcium activation curve with inhibitors of exocytosis (23,34,58); and (e) aging (45,61), where a progressive decrease in calcium sensitivity is observed with time in the isolated planar cortex. The random model succeeds in describing all of these behaviors in the sea urchin with the simple hypothesis that an increase in [Ca2+]i corresponds to an increase in the average number (<n>) and perhaps the efficacy (p) of active fusion complexes at docking sites.…”

“…By invoking schemes involving a single preassembled fusion complex at a docking site, it may be possible to explain some of the individual calcium-dependent behaviors associated with exocytosis. First, it is known that calcium increases the rate of fusion (10,54) and NEM decreases the fusion rate (45). By invoking an intrinsic, time-dependent inactivation process, one can explain both submaximal extents of fusion and the shift in calcium dose-response observed with NEM treatment.…”

Reassociation of cortical secretory vesicles with sea urchin egg plasma membrane: Assessment of binding specificity

“…The bottom of the slide chamber and the top of the coverslip were carefully cleaned with a cotton tipped applicator that had been dipped in water to remove any spots of crystallized salt (from buffer evaporation) that may have accumulated on these surfaces. Scattered light was quantitated with a photodiode apparatus (Crabb & Jackson, 1986) and displayed on a potentiometric chart recorder. With our apparatus, the spot from which CV binding is recorded has an area of 98,000 p,m 2 (equivalent to approximately 22 egg PM fragments with an average diameter of 75/zm).…”

Interaction of Polylysine with the Cellular Receptor for Herpes Simplex Virus Type 1

“…Stimulation of brain triglyceride lipase (Le Petit et al, 1986), as well as of a protein (casein) kinase (Leiderman et al, 1985) have been reported. Polylysine, like neomycin, inhibits Ca2+-induced exocytosis of the sea urchin egg cortex (Crabb & Jackson, 1986, Whitaker & Aitchison, 1985. Thus, even though neomycin and polylysine may share characteristics and properties, it is important to note that polylysine probably has several additional effects on cell function; indeed it may be of significance that polylysine easily internalizes into cells, while neomycin does not readily do so.…”