Paramecium trichocysts isolated with their membranes are stable in the presence of millimolar Ca2+

We have developed a simple and rapid procedure for the isolation of a pure fraction of Paramecium trichocysts (mature secretory vesicles) with their membranes. Since in wild-type Paramecium cells essentially all trichocysts are docked at pre-formed cortical sites, trichocysts were isolated from cells in which functional trichocysts remain free in the cytoplasm owing to a mutation, tam6, that affects the docking site. Examination of the preparations by freeze-fracture electron microscopy confirms the presence of the membranes. The distribution of particles in the membranes of the isolated trichocysts and in the membranes of wild-type trichocysts in situ are nearly identical and this argues against any rearrangement of the membranes during the isolation procedure. Although the trichocyst matrix undergoes a dramatic structural transition in the presence of Ca2+ and water (matrix expansion), the isolated vesicles with intact membranes are perfectly stable in the presence of millimolar free Ca2+. This result supports a chronology in which the first step in exocytosis is membrane fusion, the swelling of vesicle contents occurring only afterwards, once the contents come into contact with the water and Ca2+ of the external medium. The role of swelling would then be to help disperse, propel or otherwise empty the contents of the vesicle outside the cell.