Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A.
Redistribution and shedding of flagellar membrane glycoproteins visualized using an anti-carbohydrate monoclonal antibody and concanavalin A.
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DOI：
10.1083/jcb.102.5.1797
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发表时间：
1986-05
期刊：
影响因子：
--
通讯作者：
Salomonsky NL
中科院分区：
文献类型：
--
作者：
Bloodgood RA;Woodward MP;Salomonsky NL
两种碳水化合物结合探针，即凝集素伴刀豆球蛋白 A 和名为 FMG-1 的抗碳水化合物单克隆抗体，已用于研究它们各自表位在莱茵衣藻 pf-18 菌株表面上的分布。这两种配体均一地结合到鞭毛膜的外表面和一般细胞体质膜上，尽管标记在鞭毛膜上更强烈。此外，两种配体与细胞壁糖蛋白发生交叉反应。对于鞭毛膜，伴刀豆球蛋白 A 和 FMG-1 单克隆抗体均优先与表观分子量为 350,000 的主要高分子量糖蛋白结合，尽管此外还与许多次要糖蛋白存在交叉反应。高分子量鞭毛糖蛋白的 V-8 蛋白酶消化物的蛋白质印迹表明，虽然凝集素和抗体不竞争相同的结合位点，但凝集素和抗体识别的表位在糖蛋白内重复多次并一起出现。将活细胞与单克隆抗体或凝集素在 4°C 下孵育，会导致鞭毛表面均匀标记；当细胞变暖时，这些配体以特有的方式沿着鞭毛表面重新分布。所有鞭毛表面结合的抗体或凝集素在每个鞭毛的尖端聚集成单个聚集体；该聚集物随后迁移到鞭毛的基部，并在那里脱落到培养基中。重新分布的速率取决于温度，并且这些配体识别的糖蛋白与凝集素或单克隆抗体共同重新分布。这种动态的鞭毛表面现象与在许多哺乳动物细胞类型中描述的加帽现象非常相似。然而，它发生在缺乏大多数通常与其他系统中的加帽相关的细胞骨架成分的结构（鞭毛）上。 FMG-1 单克隆抗体抑制鞭毛表面运动，表现为聚苯乙烯微球的快速双向易位。
Two carbohydrate-binding probes, the lectin concanavalin A and an anti- carbohydrate monoclonal antibody designated FMG-1, have been used to study the distribution of their respective epitopes on the surface of Chlamydomonas reinhardtii, strain pf-18. Both of these ligands bind uniformly to the external surface of the flagellar membrane and the general cell body plasma membrane, although the labeling is more intense on the flagellar membrane. In addition, both ligands cross- react with cell wall glycoproteins. With respect to the flagellar membrane, both concanavalin A and the FMG-1 monoclonal antibody bind preferentially to the principal high molecular weight glycoproteins migrating with an apparent molecular weight of 350,000 although there is, in addition, cross-reactivity with a number of minor glycoproteins. Western blots of V-8 protease digests of the high molecular weight flagellar glycoproteins indicate that the epitopes recognized by the lectin and the antibody are both repeated multiple times within the glycoproteins and occur together, although the lectin and the antibody do not compete for the same binding sites. Incubation of live cells with the monoclonal antibody or lectin at 4 degrees C results in a uniform labeling of the flagellar surface; upon warming of the cells, these ligands are redistributed along the flagellar surface in a characteristic manner. All of the flagellar surface-bound antibody or lectin collects into a single aggregate at the tip of each flagellum; this aggregate subsequently migrates to the base of the flagellum, where it is shed into the medium. The rate of redistribution is temperature dependent and the glycoproteins recognized by these ligands co-redistribute with the lectin or monoclonal antibody. This dynamic flagellar surface phenomenon bears a striking resemblance to the capping phenomenon that has been described in numerous mammalian cell types. However, it occurs on a structure (the flagellum) that lacks most of the cytoskeletal components generally associated with capping in other systems. The FMG-1 monoclonal antibody inhibits flagellar surface motility visualized as the rapid, bidirectional translocation of polystyrene microspheres.