A freeze-fracture study of early membrane events during mast cell secretion

The early membrane events taking place during mast cell secretion were followed in transmission and freeze-fracture electron microscopy. In order to slow down exocytosis and capture intermediate stages of membrane fusion, special conditions of incubation and stimulation were used. These were as follows: (a) the use of incubation media with altered ionic composition, and (b) stimulation with a low dosage of polymyxin B sulfate (4 microgram/ml) at low temperature (18 degrees C) for very short incubation times (30-60 s), with or without the presence of formaldehyde (0.8%). Under these conditions, unetchable circular impressions are found on the E face of the plasma membrane, 80-100 nm in diameter, with particles associated with their perimeters. In granule-to-granule fusion, the zone involved is demarcated by one or two rows of particles on the E face. In addition, raised circular areas of varying diameters (43-87 nm) surrounded by similar particles, also found on the E face, may represent potential sites before completion of fusion. Neither the circular impressions on the plasma membrane nor the sites on the granule membrane are permanent, but their appearance coincides with initiation of membrane fusion.