Previous investigations have supported an osmotic hypothesis for exocytosis; i.e. water flow into secretory granules drives exocytosis by granule swelling. It is known that exocytosis in sea urchin ...eggs is inhibited by hyperosmotic media, but it is unclear at which stage inhibition occurs. The aim of this project is to determine by electron microscopy (EM) the stage at which osmotic gradients are important. EM examination of eggs activated in hyperosmotic seawater reveals that exocytosis is inhibited due to repositioning of 90% of the granules as far as 1.0 um for the plasma membrane. A small fraction of the granules do remain docked and fuse with the plasma membrane; however, the exocytic pore does not widen. Separation of granules by formation of a granule-free zone (GFZ) is brought about by cytoskeletal reorganization. In platinum replicas of hyperosmotically-treated eggs, beds of filaments can be seen between the granules and the plasma membrane. NBD-phallacidin staining and S1 myosin binding (methods to probe actin polymerization) show an increase in the amount and a change in location of actin filaments, respectively. The GFZ fails to form in the absence of either extracellular Ca$\sp{2+}$ or Na$\sp+$. Indeed, presence of either an intracellular Ca$\sp{2+}$ buffer or a Ca$\sp{2+}$ channel blocker inhibits formation of this zone. Apparently hyperosmolality triggers an influx of exogenous Ca$\sp{2+}$, which initiates polymerization of actin and repositioning of granules, thereby preventing fusion. The addition of hyperosmotic seawater to eggs after insemination arrests the spreading wave of exocytosis. Thin-sections reveal stages of exocytosis and matrix disassembly, which can be arranged in a sequence: (1) membrane fusion, (2) lamellar separation, (3) pore widening, (4) matrix expansion, (5) pocket opening, (6) matrix expulsion, (7) lamellar fragmentation, and (8) particle formation. Both nonionic and ionic hyperosmotic media arrest exocytosis at the third stage: membrane fusion and pore formation occur, but pore widening does not. Dextran polymers containing divalent cations arrest exocytosis at the fourth stage resulting in omega-configured pockets with partially disassembled matrices. These observations suggest that water is required for early stages of matrix disassembly that widen the exocytic pore and open the pocket, but not for fusion. In contrast, later stages of disassembly are not inhibited by hyperosmolality, but can be blocked by high ionic strength media.
