Quenched flow analysis of exocytosis in Paramecium cells: time course, changes in membrane structure, and calcium requirements revealed after rapid mixing and rapid freezing of intact cells

Synchronous exocytosis in Paramecium cells was analyzed on a subsecond time scale. For this purpose we developed a quenched flow device for rapid mixing and rapid freezing of cells without impairment (time resolution in the millisecond range, dead time approximately 30 ms). Cells frozen at defined times after stimulation with the noncytotoxic secretagogue aminoethyldextran were processed by freeze substitution for electron microscopic analysis. With ultrathin sections the time required for complete extrusion of secretory contents was determined to be less than 80 ms. Using freeze-fracture replicas the time required for resealing of the fused membranes was found to be less than 350 ms. During membrane fusion (visible 30 ms after stimulation) specific intramembranous particles in the cell membrane at the attachment sites of secretory organelles ("fusion rosette") disappear, possibly by dissociation of formerly oligomeric proteins. This hitherto unknown type of rapid change in membrane architecture may reflect molecular changes in protein-protein or protein-lipid interactions, presumably crucial for membrane fusion. By a modification of the quenched flow procedure extracellular [Ca++] during stimulation was adjusted to less than or equal to 3 x 10(-8) M, i.e., below intracellular [Ca++]. Only extrusion of the secretory contents, but not membrane fusion, was inhibited. Thus it was possible to separate both secretory events (membrane fusion from contents extrusion) and to discriminate their Ca++ requirements. We conclude that no Ca++ influx is necessary for induction of membrane fusion.