van Deurs B, Hansen S H, Petersen O W, Melby E L, Sandvig K
Department of Anatomy, University of Copenhagen, Denmark.
Eur J Cell Biol. 1990 Feb;51(1):96-109.
The toxic plant protein ricin binds to both the apical and basolateral surface domains of MDCK (strain I) cells grown on polycarbonate filters. Endocytosis of 125I-labeled ricin was not only higher from the basolateral than from the apical surface--an observation which can be explained by the higher surface area of the basolateral surface--but it also appeared to be more efficient when measured as a percentage of total cell-associated ricin. Monovalent ricin-horseradish peroxidase (Ri-HRP), which is known to behave like native ricin with respect to intracellular transport, also binds to, and is taken up from, both the apical and the basolateral surfaces. Initially, after 10 to 15 min, molecules taken up from the two surface domains at 37 degrees C are present in two separate (basolateral and apical) early endosomal populations. This can also be obtained by incubating for 60 min at 18 degrees C. However, after 30 to 60 min at 37 degrees C, most internalized ligand is found in apical lysosomes, regardless from which surface endocytosis took place. Experiments with endocytosis of cationized ferritin from the apical pole and HRP or Ri-HRP from the basolateral pole showed that intermixing in apical lysosomes (or prelysosomes) of molecules taken up from the two poles occurs. Bidirectional transcytosis involving coated pits of both 125I-labeled ricin and Ri-HRP was demonstrated and was found to be most efficient (as measured in per cent of endocytosed toxin) from the apical pole. Transcytosis was strongly reduced at 18 degrees C, and no transepithelial transport of ricin could be measured at 4 degrees C. Transcytosed ricin was intact and could intoxicate new cells. Finally, delivery of ricin internalized from both the apical and the basolateral surface to the apically localized trans-Golgi network occurred at 37 degrees C but not at 18 degrees C, and ricin inhibited protein synthesis largely with the same kinetics following uptake from the two poles. Incubation at 18 degrees C strongly inhibited the toxic effect of ricin. These data show that ricin can intoxicate epithelia from both sides and also penetrate tight epithelial barriers in intact form.
有毒植物蛋白蓖麻毒素可结合在聚碳酸酯滤膜上生长的MDCK（I株）细胞的顶端和基底外侧表面结构域。125I标记的蓖麻毒素从基底外侧表面的内吞作用不仅高于从顶端表面——这一现象可以用基底外侧表面更大的表面积来解释——而且当以与细胞结合的总蓖麻毒素的百分比来衡量时，其似乎也更有效。单价蓖麻毒素-辣根过氧化物酶（Ri-HRP），已知其在细胞内运输方面与天然蓖麻毒素表现相似，也能结合并从顶端和基底外侧表面被摄取。最初，在10到15分钟后，在37℃从两个表面结构域摄取的分子存在于两个分开的（基底外侧和顶端）早期内体群体中。这也可以通过在18℃孵育60分钟来实现。然而，在37℃孵育30到60分钟后，无论内吞作用发生在哪个表面，大多数内化的配体都存在于顶端溶酶体中。从顶端摄取阳离子化铁蛋白以及从基底外侧摄取HRP或Ri-HRP的内吞作用实验表明，从两极摄取的分子在顶端溶酶体（或前溶酶体）中会发生混合。证明了125I标记的蓖麻毒素和Ri-HRP通过包被小窝的双向转胞吞作用，并且发现从顶端极转胞吞作用最有效（以摄取的毒素百分比衡量）。在18℃时转胞吞作用显著降低，在4℃时未检测到蓖麻毒素的跨上皮运输。转胞吞的蓖麻毒素是完整的，并且能够使新细胞中毒。最后，在37℃时，从顶端和基底外侧表面内化的蓖麻毒素都能运输到顶端定位的反式高尔基体网络，但在18℃时则不能，并且蓖麻毒素从两极摄取后抑制蛋白质合成的动力学基本相同。在18℃孵育强烈抑制了蓖麻毒素的毒性作用。这些数据表明，蓖麻毒素可以从两侧使上皮细胞中毒，并且还能以完整形式穿透紧密的上皮屏障。
