Ultrastructural characterization of tannic acid-arrested degranulation of permeabilized guinea pig eosinophils stimulated with GTP-gamma-S.

We have used ultrastructural techniques to investigate secretion in permeabilized eosinophils. As each exocytotic event is rapid we have used tannic acid incubation to trap the maximum number of fusion figures; tannic acid has been used previously in other secretory systems to arrest exocytosis at the cell surface whilst still allowing the preceding events to occur. Using this approach, in conjunction with ultrathin sectioning and cryoreplication, it is possible to demonstrate clear evidence of exocytosis in permeabilized eosinophils after stimulation by GTP-gamma-S. Large numbers of arrested fusion sites are found, including early fusion pedestals, visible in freeze-fracture replicas, having single narrow necked pores as small as 12 x 43 nm. Both individual and compound exocytoses are found, with retention of the secretory product, in particular the crystalline granule core, occurring at many sites. Large numbers of coated pits are also found in cells following extended tannic acid incubation, membrane coats even occurring on arrested granule membranes, suggesting a role in post-fusion membrane recovery. The accessibility of the cell interior and the large number of arrested fusion sites, particularly the presence of very early stages of exocytosis (evident as pedestals in freeze-fracture replicas), makes this a suitable preparation for the localization of key regulators of exocytosis at their sites of action. Although this approach, utilizing permeabilization coupled with tannic acid incubation is not without inherent problems-as with any electron microscopic technique care must be taken to understand the potential for artefacts-there are a number of advantages, particularly with regard to labeling studies, over techniques utilizing ultra rapid freezing.