Webster P, Grab D J
International Laboratory for Research on Animal Diseases, Nairobi, Kenya.
J Cell Biol. 1988 Feb;106(2):279-88. doi: 10.1083/jcb.106.2.279.
Endocytosis and intracellular transport has been studied in the bloodstream forms of Trypanosoma brucei by light and electron microscopy, using colloidal gold coupled to bovine transferrin (transferrin-gold). The endocytosed transferrin-gold, visualized by silver intensification for light microscopy, was present in vesicular structures between the cell nucleus and flagellar pocket of the organism. At the ultrastructural level, transferrin-gold was present after a 10-min incubation in the flagellar pocket, coated vesicles, cisternal networks, and lysosomelike structures. Endocytosis and intracellular processing of T. brucei variable surface glycoprotein (VSG) was studied using two preparations of affinity-purified rabbit IgG directed against different parts of the VSG. One preparation of IgG was directed against the cross-reacting determinant (CRD): a complex glycolipid side chain covalently linked to the COOH-terminus of the VSG molecule. The other was directed against determinants on the rest of the VSG molecule. When the two IgG preparations were used on thawed, thin cryosections of trypanosomes that had been incubated in transferrin-gold before fixation, the organelles involved with transferrin-gold endocytosis labeled with both antibodies, as well as many vesicular, tubular, and vacuolar structures that did not contain endocytosed transferrin-gold. Both antibodies also labeled the cell surface. In double-labeling experiments both antibodies were closely associated except that IgG directed against the VSG molecule labeled all the cisternae of the Golgi apparatus, whereas anti-CRD IgG was shown to label only half of the Golgi apparatus. Evidence for sorting of VSG molecules from endocytosed transferrin-gold was found. Double-labeling experiments also showed some tubular profiles which labeled on one side with anti-CRD IgG and on the other side with anti-VSG IgG, suggesting a possible segregation of parts of the VSG molecule.
通过光学显微镜和电子显微镜，利用与牛转铁蛋白偶联的胶体金（转铁蛋白 - 金），对布氏锥虫血流形式的内吞作用和细胞内运输进行了研究。通过银增强用于光学显微镜观察的内吞转铁蛋白 - 金，存在于该生物体细胞核和鞭毛袋之间的囊泡结构中。在超微结构水平上，孵育10分钟后，转铁蛋白 - 金出现在鞭毛袋、被膜小泡、扁平囊网络和溶酶体样结构中。使用两种针对可变表面糖蛋白（VSG）不同部位的亲和纯化兔IgG制剂，研究了布氏锥虫可变表面糖蛋白（VSG）的内吞作用和细胞内加工过程。一种IgG制剂针对交叉反应决定簇（CRD）：一种与VSG分子COOH末端共价连接的复杂糖脂侧链。另一种针对VSG分子其余部分的决定簇。当将这两种IgG制剂用于解冻的、在固定前已在转铁蛋白 - 金中孵育的锥虫薄冰冻切片时，参与转铁蛋白 - 金内吞作用的细胞器被两种抗体标记，以及许多不包含内吞转铁蛋白 - 金的囊泡、管状和液泡结构。两种抗体也标记了细胞表面。在双重标记实验中，两种抗体紧密相关，只是针对VSG分子的IgG标记了高尔基体的所有扁平囊，而抗CRD IgG仅标记了高尔基体的一半。发现了从内吞转铁蛋白 - 金中分拣VSG分子的证据。双重标记实验还显示了一些管状结构，一侧用抗CRD IgG标记，另一侧用抗VSG IgG标记，这表明VSG分子的部分可能存在分离。
