Stimulation and inhibition of secretion in Paramecium: Role of divalent cations

The effects of Ca²⁺ and Mg²⁺ on exocytosis in Paramecium tetraurelia cells were examined with light microscopy, freeze fracture (FEM) and transmission electron microscopy (TEM) of thin-sectioned embedded cells. Picric acid-Ca²⁺-induced secretion in wild type (wt) cells was captured by 'quick' fixation with OsO₄, and TEM demonstrated membrane fusion occurring before trichocyst matrix (tmx) expansion. Cells stimulated with picric acid in the presence of high extracellular Mg²⁺ showed very few sites of membrane fusion and no tmx expansion, suggesting that Ca²⁺ is required for both membrane fusion and tmx expansion. Further information was obtained by comparing secretory responses of wt cells with a temperature-sensitive secretory mutant, nd 9. These cells when grown at the permissive temperature (18°C) possess normal rosettes at the secretory site and secrete in response to picric acid-Ca²⁺, but when grown at 27°C they lack rosettes and do not secrete. Quantitation of picric acid-Ca²⁺-induced secretion revealed that: (a) the number of tmx secreted by wt and nd 9 cells was independent of their cultural growth phase, (b) wt cells secreted the same number of tmx when grown either at 18 or 27°C, and (c) nd 9 18°C cells secreted the same number of tmx when grown either at 18 or 27°C. Wild type and nd 9 cells had the same frequencies of occupied and unoccupied secretory sites as determined by quantitative analysis of freeze-fracture replicas. After stimulation with divalent cation ionophore A23187 and Ca²⁺, wt cells showed a significant reduction in the frequency of occupied sites. FEM and TEM studies revealed that A23187-Ca²⁺ induced tmx expansion and normal fusion of the plasma and trichocyst membranes in wt and nd 9 18°C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27°C cells. The lack of membrane fusion in nd 9 27° C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.