Gormley J A, Howard R J, Taraschi T F
Department of Pathology and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
J Cell Biol. 1992 Dec;119(6):1481-95. doi: 10.1083/jcb.119.6.1481.
During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.
在疟疾感染的无性阶段，细胞内寄生虫将膜转运至红细胞胞质，并将脂质和蛋白质转运至宿主细胞膜，实质上是在“改造”红细胞。为了研究恶性疟原虫感染的红细胞内脂质和蛋白质的转运途径，通过共聚焦荧光成像显微镜对同步培养物进行时间分析，以观察输出膜（囊泡）和寄生虫蛋白的产生、位置和形态。早在侵入后4小时，在体外与C6-NBD标记的磷脂一起孵育的感染红细胞的红细胞胞质中就观察到了高度移动的囊泡。这些囊泡在滋养体阶段最为普遍。开发了一种免疫荧光技术，以同时确定单个被寄生红细胞内荧光膜和多种寄生虫蛋白的形态和分布。用异硫氰酸荧光素（FITC）或德克萨斯红标记的单克隆抗体使寄生虫蛋白可视化。双标记免疫荧光显示，在所检测的五种寄生虫抗原中，只有一种主要与红细胞胞质中的膜相关。另外两种寄生虫抗原仅部分定位于这些囊泡，大多数输出抗原存在于宿主细胞质中的无脂聚集体中。另一种转运至红细胞胞质的寄生虫抗原仅定位于无脂聚集体中。未输出的寄生虫质膜（PPM）和/或寄生泡膜（PVM）抗原总是与PPM/PVM中的荧光脂质共定位。使用FITC和德克萨斯红标记的二抗同时可视化两种寄生虫蛋白，结果显示一些寄生虫蛋白在相同的囊泡中组成性转运，而其他蛋白在输出前被分隔开。在四种输出抗原中，只有一种似乎通过膜介导的事件穿过PPM和PVM的屏障，而其他抗原则通过脂质（囊泡）非依赖途径穿过PPM/PVM到达宿主细胞质和表面膜。
