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 pe rmeabilized eosinophils. As each exocytotic event is rapid we have use d tannic acid incubation to trap the maximum number of fusion figures; tannic acid has been used previously in other secretory systems to ar rest exocytosis at the cell surface whilst still allowing the precedin g events to occur, Using this approach, in conjunction with ultrathin sectioning and cryoreplication, it is possible to demonstrate clear ev idence of exocytosis in permeabilized eosinophils after stimulation by GTP-gamma-S, Large numbers of arrested fusion sites are found, includ ing early fusion pedestals, visible in freeze-fracture replicas, havin g single narrow necked pores as small as 12 x 43 nm, Both individual a nd compound exocytoses are found, with retention of the secretory prod uct, in particular the crystalline granule core, occurring at many sit es, Large numbers of coated pits are also found in cells following ext ended tannic acid incubation, membrane coats even occurring on arreste d granule membranes, suggesting a role in post-fusion membrane recover y The accessibility of the cell interior and the large number of arres ted fusion sites, particularly the presence of very early stages of ex ocytosis (evident as pedestals in freeze-fracture replicas), makes thi s a suitable preparation for the localization of key regulators of exo cytosis at their sites of action. Although this approach, utilizing pe rmeabilization coupled with tannic acid incubation is not without inhe rent problems - as with any electron microscopic technique care must b e taken to understand the potential for artefacts - there are a number of advantages, particularly with regard to labeling studies, over tec hniques utilizing ultrarapid freezing.