Schindler M, Koppel D E, Sheetz M P
Proc Natl Acad Sci U S A. 1980 Mar;77(3):1457-61. doi: 10.1073/pnas.77.3.1457.
The lateral mobility of fluorescein-labeled membrane glycoproteins was measured in whole unlysed erythrocytes and erythrocyte ghosts by the technique of "fluorescence redistribution after fusion." Measurements were made on polyethylene glycol-fused cell pairs in which only one member of the couplet was initially fluorescently labeled. Diffusion coefficients were estimated from the rate of fluorescence redistribution determined from successive scans with a focused laser beam across individual fused pairs. This technique allows for the analysis of diffusion within cell membranes without the possible damaging photochemical events caused by photobleaching. It was found that lateral mobility of erythrocyte proteins can be increased by the addition of polyphosphates (i.e., ATP and 2,3-diphosphoglycerate) and decreased by the addition of organic polyamines (i.e., neomycin and spermine). This control is exerted by these molecules only when they contact the cytoplasmic side of the membrane and is not dependent upon high-energy phosphates. Microviscosity experiments employing diphenylhexatriene demonstrated no changes in membrane lipid state as a function of these reagents. Our results, in conjunction with data on the physical interactions of cytoskeletal proteins, suggest that the diffusion effector molecules alter the lateral mobility of erythrocyte membrane proteins through modifications of interactions in the shell, which is composed of spectrin, actin, and component 4.1.
通过“融合后荧光再分布”技术，在完整未裂解的红细胞和红细胞影中测量了荧光素标记的膜糖蛋白的侧向流动性。对聚乙二醇融合的细胞对进行测量，其中在融合对中只有一个成员最初被荧光标记。扩散系数是根据用聚焦激光束对单个融合对进行连续扫描所确定的荧光再分布速率来估计的。该技术允许分析细胞膜内的扩散，而不会因光漂白导致可能的破坏性光化学事件。发现添加多磷酸盐（即ATP和2,3-二磷酸甘油酸）可增加红细胞蛋白的侧向流动性，而添加有机多胺（即新霉素和精胺）则会降低其侧向流动性。这些分子仅在它们接触膜的细胞质侧时才发挥这种控制作用，并且不依赖于高能磷酸盐。使用二苯基己三烯的微粘度实验表明，膜脂质状态不会因这些试剂而发生变化。我们的结果与关于细胞骨架蛋白物理相互作用的数据相结合，表明扩散效应分子通过改变由血影蛋白、肌动蛋白和4.1组分组成的外壳中的相互作用，来改变红细胞膜蛋白的侧向流动性。
