Knochel M, Kissmehl R, Wissmann J D, Momayezi M, Hentschel J, Plattner H, Burgoyne R D
Faculty of Biology, University of Konstanz, Konstanz, Germany.
Histochem Cell Biol. 1996 Apr;105(4):269-81. doi: 10.1007/BF01463930.
Annexins were isolated from Paramecium cell homogenates by standard ethylene glycol tetraacetic acid (EGTA) extraction and 100 000-g centrifugation. Two different antibodies (Abs) against synthetic peptides were used, Call-15 and B15, which in mammalian cells recognize a sequence of annexin II or a common sequence occurring in several annexins (except for annexin II), respectively. With anti-Call-15 Abs, western blots from EGTA extracts showed strongly reactive bands of 44.5 and 46 kDa and of higher values. Some of these bands bound to the 100 000-g pellet fraction when Ca(2+) was added. Immuno- and affinity labelling revealed selective, Ca(2+)-dependent labelling of the cell cortex, with enrichment around trichocyst docking sites (facing subplasmalemmal Ca(2+) stores). Cortical fluorescence labelling decreased in wild-type (7S) cells when trichocyst ghosts were detached after synchronous exocytosis. Similarly, cortical labelling was reduced when intact trichocysts were detached from the cell surface of non-discharge mutant cells (nd9-28 degrees C, showing identical bands on blots), which then contained numerous heavily labelled phagolysosomes. This strongly suggests annexin downregulation. All together, the dynamic labelling of cortical structures we observed strongly supports involvement of calpactin-like annexins in trichocyst docking. Anti-B15 Abs recognized a band of 51 kDa and some of higher values. These Abs selectively labelled the outlines of the cytoproct, the site of spent phagolysosome exocytosis. In conclusion, our data indicate involvement of specific sets of annexins in site-specific positioning and attachment of widely different secretory organelles at the cell surface in Paramecium cells.
通过标准的乙二醇四乙酸（EGTA）提取和100000g离心从草履虫细胞匀浆中分离出膜联蛋白。使用了两种针对合成肽的不同抗体（Abs），Call-15和B15，它们在哺乳动物细胞中分别识别膜联蛋白II的一个序列或几种膜联蛋白（膜联蛋白II除外）中出现的一个共同序列。用抗Call-15抗体，EGTA提取物的蛋白质免疫印迹显示出44.5 kDa和46 kDa以及更高值的强反应带。当添加Ca(2+)时，其中一些条带与100000g沉淀部分结合。免疫和亲和标记显示细胞皮层有选择性的、Ca(2+)依赖性标记，在刺丝泡对接位点（面向质膜下Ca(2+)储存处）周围富集。当同步胞吐后刺丝泡空壳分离时，野生型（7S）细胞中的皮层荧光标记减少。同样，当完整的刺丝泡从非放电突变细胞（nd9 - 28℃，在印迹上显示相同条带）的细胞表面分离时，皮层标记减少，此时这些细胞含有许多高度标记的吞噬溶酶体。这强烈表明膜联蛋白下调。总之，我们观察到的皮层结构的动态标记有力地支持了类钙蛋白酶膜联蛋白参与刺丝泡对接。抗B15抗体识别出一条51 kDa和一些更高值的条带。这些抗体选择性地标记了胞肛的轮廓，即用过的吞噬溶酶体胞吐的部位。总之，我们的数据表明特定的膜联蛋白组参与了草履虫细胞中广泛不同的分泌细胞器在细胞表面的位点特异性定位和附着。
