Beginning of exocytosis captured by rapid-freezing of Limulus amebocytes.

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Abstract
Structural changes underlying exocytosis evoked by the application of endotoxin to Limulus amebocytes were studied at the level of detail afforded by freeze-fracture and freeze-substitution techniques combined with the time resolution of direct rapid-freezing. The results with amoebocytes prepared in this manner differed from those with other secretory cells prepared by conventional means. Exocytosis begins within seconds of endotoxin treatment when the plasmalemma invaginates to form pedestal-like appositions with peripheral secretory granules. The juxtaposed membranes at these pedestal appositions form several punctate pentalaminar contacts, but examination of freeze-fractured pedestals revealed no corresponding changes in the intramembrane particle distribution. Small secretory granule openings or pores, very infrequent, appear within the first 5 s after endotoxin treatment. These pores rapidly widen and this widening is immediately followed by the sequential dissolution of the granule contents, which then move into the surrounding extracellular space. Cytoplasmic filaments connecting the plasmalemma with the granule membrane are suitably deployed to be responsible for the plasmalemmal invaginations. How pores begin is not certain, but the appearance of clear spaces between the granule core and the granule membrane at this point in exocytosis supports the possibility of a role for osmotic forces.