A CALCIUM INFLUX IS NEITHER STRICTLY ASSOCIATED WITH NOR NECESSARY FOR EXOCYTOTIC MEMBRANE-FUSION IN PARAMECIUM-CELLS

Exocytosis of trichocysts in Paramecium cells was generally believed to depend on extracellular Ca, since it is accompanied by a Ca influx and not seen in the absence of Ca. However, by short term removal of Ca we showed recently that only extrusion of secretory contents, but not membrane fusion after stimulation with aminoethyldextran (AED), depends on extracellular Ca. We have now extended these studies to longer times and shown that membrane fusion is stimulated by AED even after 1 min at low Ca (less-than-or-equal-to 30 nM). At prolonged times membrane fusion was induced by sole removal of Ca. In the presence of AED, trichocyst contents were slowly extruded followed by resealing of the fused membranes, indicating independency of endocytotic membrane fusion from extracellular Ca (though we observed aberrant resealing). Later on, Ca removal is followed by cell death. By using videomicroscopy, we further provide the first evidence that exocytosis is not necessarily accompanied by an influx of Ca in the presence of the usual high concentrations (1 mM), since local exocytosis at the rear end of the cells is not followed by ciliary reversal which is triggered by Ca influx. We conclude that a Ca influx is neither regularly associated with, nor necessary for, induction of exocytotic membrane fusion in Paramecium cells. As a source for a possible alternative intracellular liberation of calcium during exocytosis, we analyzed the subplasmalemmal alveolar sac system by electron spectroscopic imaging and found indications for Ca redistributions shortly after stimulation.