Gilligan D M, Satir B H
J Cell Biol. 1983 Jul;97(1):224-34. doi: 10.1083/jcb.97.1.224.
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 degrees C) possess normal rosettes at the secretory site and secrete in response to picric acid-Ca2+, but when grown at 27 degrees C they lack rosettes and do not secrete (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). 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 degrees C, and (c) nd 9 18 degrees C cells secreted the same number of tmx as wt 18 or 27 degrees C cells. 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 degrees C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27 degrees C cells. The lack of membrane fusion in nd 9 27 degrees C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.
利用光学显微镜、冷冻断裂（FEM）和超薄切片包埋细胞的透射电子显微镜（TEM），研究了Ca2+和Mg2+对四膜虫细胞胞吐作用的影响。苦味酸-Ca2+诱导的野生型（wt）细胞分泌通过OsO4“快速”固定捕获，TEM显示在刺丝泡基质（tmx）膨胀之前发生了膜融合。在高细胞外Mg2+存在下用苦味酸刺激的细胞显示很少有膜融合位点，并且没有tmx膨胀，这表明Ca2+对于膜融合和tmx膨胀都是必需的。通过比较wt细胞与温度敏感分泌突变体nd 9的分泌反应获得了进一步的信息。这些细胞在允许温度（18摄氏度）下生长时，在分泌位点具有正常的玫瑰花结，并且对苦味酸-Ca2+有分泌反应，但在27摄氏度下生长时，它们缺乏玫瑰花结并且不分泌（Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143）。对苦味酸-Ca2+诱导的分泌进行定量分析表明：（a）wt和nd 9细胞分泌的tmx数量与其培养生长阶段无关，（b）wt细胞在18或27摄氏度下生长时分泌的tmx数量相同，并且（c）nd 9 18摄氏度细胞分泌的tmx数量与wt 18或27摄氏度细胞相同。通过对冷冻断裂复制品的定量分析确定，野生型和nd 9细胞具有相同频率的被占据和未被占据的分泌位点。在用二价阳离子载体A23187和Ca2+刺激后，wt细胞显示被占据位点的频率显著降低。FEM和TEM研究表明，A23187-Ca2+在wt和nd 9 18摄氏度细胞中诱导tmx膨胀以及质膜和刺丝泡膜的正常融合，但在nd 9 27摄氏度细胞中诱导tmx膨胀而没有伴随的膜融合。nd 9 27摄氏度细胞中缺乏膜融合表明玫瑰花结颗粒所代表的分子是膜融合所特需的。
