Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Kyoto, Japan.
Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan.
Nat Chem Biol. 2016 Jun;12(6):402-10. doi: 10.1038/nchembio.2059. Epub 2016 Apr 4.
Gangliosides, glycosphingolipids containing one or more sialic acid(s) in the glyco-chain, are involved in various important physiological and pathological processes in the plasma membrane. However, their exact functions are poorly understood, primarily because of the scarcity of suitable fluorescent ganglioside analogs. Here, we developed methods for systematically synthesizing analogs that behave like their native counterparts in regard to partitioning into raft-related membrane domains or preparations. Single-fluorescent-molecule imaging in the live-cell plasma membrane revealed the clear but transient colocalization and codiffusion of fluorescent ganglioside analogs with a fluorescently labeled glycosylphosphatidylinisotol (GPI)-anchored protein, human CD59, with lifetimes of 12 ms for CD59 monomers, 40 ms for CD59's transient homodimer rafts in quiescent cells, and 48 ms for engaged-CD59-cluster rafts, in cholesterol- and GPI-anchoring-dependent manners. The ganglioside molecules were always mobile in quiescent cells. These results show that gangliosides continually and dynamically exchange between raft domains and the bulk domain, indicating that raft domains are dynamic entities.
神经节苷脂是糖脂的一种，在糖链中含有一个或多个唾液酸，参与质膜中的多种重要生理和病理过程。然而，由于缺乏合适的荧光神经节苷脂类似物，其确切功能仍不清楚。在这里，我们开发了一种系统合成类似物的方法，这些类似物在分配到与筏相关的膜域或制剂方面与天然类似物的行为相似。在活细胞质膜中单分子荧光成像显示，荧光神经节苷脂类似物与荧光标记的糖基磷脂酰肌醇（GPI）锚定蛋白人 CD59 之间存在明显但短暂的共定位和共扩散，CD59 单体的寿命为 12 毫秒，静止细胞中 CD59 的瞬时同源二聚体筏的寿命为 40 毫秒，而结合 CD59 簇筏的寿命为 48 毫秒，这些方式均依赖于胆固醇和 GPI 锚定。在静止细胞中，神经节苷脂分子始终处于可移动状态。这些结果表明，神经节苷脂不断地在筏域和主体域之间动态交换，表明筏域是动态实体。
