FACILITATION OF MEMBRANE-FUSION DURING EXOCYTOSIS AND EXOCYTOSIS-COUPLED ENDOCYTOSIS AND ACCELERATION OF GHOST DETACHMENT IN PARAMECIUM BY EXTRACELLULAR CALCIUM - A QUENCHED-FLOW FREEZE-FRACTURE ANALYSIS/

We had previously shown that an influx of extracellular Ca2+ (Ca-e(2+) ), though it occurs, is not strictly required for aminoethyldextran (A ED)-triggered exocytotic membrane fusion in Paramecium. We now analyze , by quenched-flow/freeze-fracture, to what extent Ca-e(2+) contribute s to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ''ghosts'' - a process difficult to analyze by any other method or in any other system. Maximal exocytotic membran e fusion (analyzed within 80 msec) occurs readily in the presence of [ Ca2+](e) greater than or equal to 5 x 10(-6) M, while normally a [Ca2](e) = 0.5 mM is in the medium. A new finding is that exocytosis and e ndocytosis is significantly stimulated by increasing [Ca2+](e) even be yond levels usually available to cells. Quenching of [Ca2+](e) by EGTA application to levels of resting [Ca2+](i) or slightly below does red uce (by similar to 50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden ei ther by increasing stimulation time or by readdition of an excess of C a-e(2+). Our data are compatible with the assumption that normally exo cytotic membrane fusion will include a step of rapid Ca2+-mobilization from subplasmalemmal pools (''alveolar sacs'') and, as a superimposed step, a Ca2+-influx, since exocytotic membrane fusion can occur at [C a2+](e) even slightly below resting [Ca2+](i). The other important con clusion is that increasing [Ca2+](e) facilitates exocytotic and endocy totic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca2+](e) also drives detachment of ''ghosts'' - a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca2+, orders of magnit ude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle.