Frölich Sonja, Wallach Michael
The iThree Institute, School of Medical and Molecular Biosciences,University of Technology Sydney,PO Box 123,Broadway,Sydney,New South Wales 2007,Australia.
Parasitology. 2015 Jun;142(7):855-64. doi: 10.1017/S0031182015000207. Epub 2015 Mar 24.
To determine the involvement of the actin cytoskeleton in macrogametocyte growth and oocyst wall formation, freshly purified macrogametocytes and oocysts were stained with Oregon Green 514 conjugated phalloidin to visualize F-actin microfilaments, while Evans blue staining was used to detect type 1 wall forming bodies (WFB1s) and the outer oocyst wall. The double-labelled parasites were then analysed at various stages of sexual development using three-dimensional confocal microscopy. The results showed F-actin filaments were distributed throughout the entire cytoplasm of mature Eimeria maxima macrogametocytes forming a web-like meshwork of actin filaments linking the type 1 WFBs together into structures resembling 'beads on a string'. At the early stages of oocyst wall formation, F-actin localization changed in alignment with the egg-shaped morphology of the forming oocysts with F-actin microfilaments making direct contact with the WFB1s. In tissue oocysts, the labelled actin cytoskeleton was situated underneath the forming outer layer of the oocyst wall. Treatment of macrogametocytes in vitro with the actin depolymerizing agents, Cytochalasin D and Latrunculin, led to a reduction in the numbers of mature WFB1s in the cytoplasm of the developing macrogametocytes, indicating that the actin plays an important role in WFB1 transport and oocyst wall formation in E. maxima.
为了确定肌动蛋白细胞骨架在大配子体生长和卵囊壁形成中的作用，将新鲜纯化的大配子体和卵囊用 Oregon Green 514 偶联鬼笔环肽染色，以观察 F-肌动蛋白微丝，同时用伊文思蓝染色检测 1 型壁形成体（WFB1s）和卵囊外壁。然后使用三维共聚焦显微镜在有性发育的各个阶段对双标记的寄生虫进行分析。结果显示，F-肌动蛋白丝分布在成熟的巨型艾美耳球虫大配子体的整个细胞质中，形成了一个肌动蛋白丝的网状结构，将 1 型 WFBs 连接在一起，形成类似“串珠”的结构。在卵囊壁形成的早期阶段，F-肌动蛋白的定位随着形成的卵囊的卵形形态而变化，F-肌动蛋白微丝与 WFB1s 直接接触。在组织卵囊中，标记的肌动蛋白细胞骨架位于卵囊壁形成的外层之下。用肌动蛋白解聚剂细胞松弛素 D 和拉春库林在体外处理大配子体，导致发育中的大配子体细胞质中成熟 WFB1s 的数量减少，表明肌动蛋白在巨型艾美耳球虫的 WFB1 运输和卵囊壁形成中起重要作用。
