Freeze-fracture studies of chemotactic peptide-induced exocytosis in neutrophils: evidence for two patterns of secretory granule fusion

Rabbit neutrophils were fixed in 2% glutaraldehyde and then either quick-frozen and freeze-fractured or embedded and thin-sectioned. Cells exposed to 10^-8 M N-formylmethionyl-leucylphenylalanine (FMLP) and 5 μg/ml cytochalasin B at 22°C underwent a rapid, compound exocytosis. Within 10 sec after stimulation, narrow pores were seen joining individual peripheral granules with the plasma membrane. Sequential fusion of interior granules occurred between 20 and 60 sec and took on two different patterns. The first consisted of a linearly directed series of fusion events resulting in a radial array of tapering invaginations directed toward the cell center. The second pattern consisted of an undirected fusion of larger granules to form highly branched structures. These granules were often connected by narrow tubules and in some cases a series of fused granules would end in a small, vesiclelike tip. This suggested that sequential fusion may involved a set of smaller vesicles as well as the granule membranes themselves.