LATERAL MOBILITY OF LIPID ANALOGS AND GPI-ANCHORED PROTEINS IN SUPPORTED BILAYERS DETERMINED BY FLUORESCENT BEAD TRACKING
LATERAL MOBILITY OF LIPID ANALOGS AND GPI-ANCHORED PROTEINS IN SUPPORTED BILAYERS DETERMINED BY FLUORESCENT BEAD TRACKING
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DOI：
10.1007/bf00234654
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发表时间：
1993-07-01
影响因子：
2.4
通讯作者：
EISINGER, J
中科院分区：
文献类型：
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作者：
FEIN, M;UNKELESS, J;EISINGER, J
脂质类似物和糖基磷脂酰肌醇（GPI）锚定的蛋白质纳入玻璃支持的磷脂双层（SBL）耦合到小（30 nm直径）的荧光珠，其在液相中的运动被跟踪通过增强荧光视频显微镜。链霉亲和素（St）共价连接到聚苯乙烯珠的羧基修饰表面，结合生物素化膜组分或针对特定膜组分的生物素化单克隆抗体（mAb）。以0.2秒的间隔记录随机扩散膜分子的珠子的位置，持续约1分钟。发现珠子的均方位移（rho）是扩散时间t的线性函数，扩散系数D来自关系式rho（t）= 4Dt。通过该方法获得的分散在卵磷脂：胆固醇（80：20%）双层中的生物素化磷脂酰乙醇胺（Bi-PE）的D值在0.05至0.6 mum 2/sec的范围内，平均值（D）= 0.26 mum 2/sec，类似于通过抗荧光素mAb 4-4-20或其Fab片段连接到St偶联珠的荧光素缀合的磷脂酰乙醇胺（Fl-PE）的[D] = 0.24 /mum 2/sec的值。D的这些值与连接到30 nm金颗粒的Fl-PE报道的值相当，但比在相同平面双层中的Fl-PE的值低几倍，如通过荧光光漂白恢复测量的，D = 1.3 mum 2/sec。还通过使用适当的生物素化mAb测定了相似SBL中两种GPI锚定蛋白的迁移率，发现衰变加速因子（CD 55）和人Fc γ RIIIB（CD 16）受体的[D]分别为0.25和0.56 mum 2/sec。这里描述的方法适用于跟踪任何可接近的膜组件。
Lipid analogues and glycosylphosphatidylinositol (GPI)-anchored proteins incorporated in glass-supported phospholipid bilayers (SBL) were coupled to small (30 nm diameter) fluorescent beads whose motion in the liquid phase was tracked by intensified fluorescence video microscopy. Streptavidin (St), covalently attached to the carboxyl modified surface of the polystyrene bead, bound either the biotinylated membrane component, or a biotinylated monoclonal antibody (mAb) directed against a specific membrane constituent. The positions of the beads tethered to randomly diffusing membrane molecules were recorded at 0.2 sec intervals for about 1 min. The mean square displacement (rho) of the beads was found to be a linear function of diffusion time t, and the diffusion coefficient, D, was derived from the relation, rho(t) = 4Dt. The values of D for biotinylated phosphatidylethanolamine (Bi-PE) dispersed in an egg lecithin: cholesterol (80:20%) bilayer obtained by this methodology range from 0.05 to 0.6 mum2/sec with an average of (D) = 0.26 mum2/sec, similar to the value of [D] = 0.24 /mum2/sec for fluorescein-conjugated phosphatidylethanolamine (Fl-PE) linked to St-coupled beads by the anti-fluorescein mAb 4-4-20 or its Fab fragment. These values of D are comparable to those reported for Fl-PE linked to 30 nm gold particles but are several times lower than that of Fl-PE in the same planar bilayer as measured by fluorescence photobleaching recovery, D = 1.3 mum2/sec. The mobilities of two GPI-anchored proteins in similar SBL were also determined by use of the appropriate biotinylated mAb and were found to be [D] = 0.25 and 0.56 mum2/sec for the decay accelerating factor (DAF, CD55) and the human FcgammaRIIIB (CD16) receptors, respectively. The methodology described here is suitable for tracking any accessible membrane component.