DIRECT OBSERVATION OF BROWNIAN-MOTION OF LIPIDS IN A MEMBRANE
DIRECT OBSERVATION OF BROWNIAN-MOTION OF LIPIDS IN A MEMBRANE
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DOI：
10.1073/pnas.88.14.6274
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发表时间：
1991-07-01
影响因子：
11.1
通讯作者：
JACOBSON, KA
中科院分区：
文献类型：
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作者：
LEE, GM;ISHIHARA, A;JACOBSON, KA
Nanovid 显微镜使用 30 至 40 nm 胶体金探针与视频增强对比度相结合，可用于检查活细胞质膜中单个分子的随机和定向运动。为了在模型系统中验证该技术，在含有荧光素共轭磷脂酰乙醇胺 (Fl-PtdEtn) 的支撑平面双层中跟踪脂质分子的运动，该双层用 30 nm 金抗荧光素 (anti-Fl) 标记。通过仅将抗-F1缀合至金来制备多价金探针。通过在缀合之前将不相关的抗体与抗-F1混合来制备寡价探针。膜结合的金颗粒以随机模式移动，这与二维随机运动的计算机模拟产生的模式没有区别。多价金探针的平均横向扩散系数(D)为0.26×10(-8)cm2/秒，少价探针的平均D为0.73×10(-8)cm2/秒。 16% 的多价探针和 50% 的少价探针的 D 值超过 0.6 x 10(-8) cm2/秒，在考虑随机变化后，该值与平面双层中 Fl-PtdEtn 光漂白后荧光恢复测量的 1.3 x 10(-8) cm2/秒 D 一致。价态对扩散的影响表明，多价金结合多种脂质，形成直径达 30-40 nm 的圆盘，导致相对于结合一种或极少数脂质的少价金而言，扩散减少。如果在解释结果时考虑金探针的化合价，Nanovid 显微镜是分析膜内单个或小分子团运动的有效方法。
Nanovid microscopy, which uses 30- to 40-nm colloidal gold probes combined with video-enhanced contrast, can be used to examine random and directed movements of individual molecules in the plasma membrane of living cells. To validate the technique in a model system, the movements of lipid molecules were followed in a supported, planar bilayer containing fluorescein-conjugated phosphatidylethanolamine (Fl-PtdEtn) labeled with 30-nm gold anti-fluorescein (anti-Fl). Multivalent gold probes were prepared by conjugating only anti-Fl to the gold. Paucivalent probes were prepared by mixing an irrelevant antibody with the anti-Fl prior to conjugation. The membrane-bound gold particles moved in random patterns that were indistinguishable from those produced by computer simulations of two-dimensional random motion. The multivalent gold probes had an average lateral diffusion coefficient (D) of 0.26 x 10(-8) cm2/sec, and paucivalent probes had an average D of 0.73 x 10(-8) cm2/sec. Sixteen percent of the multivalent and 50% of the paucivalent probes had values for D in excess of 0.6 x 10(-8) cm2/sec, which, after allowance for stochastic variation, are consistent with the D of 1.3 x 10(-8) cm2/sec measured by fluorescence recovery after photobleaching of Fl-PtdEtn in the planar bilayer. The effect of valency on diffusion suggests that the multivalent gold binds several lipids forming a disk up to 30-40 nm in diameter, resulting in reduced diffusion with respect to the paucivalent gold, which binds one or a very few lipids. Provided the valency of the gold probe is considered in the interpretation of the results, Nanovid microscopy is a valid method for analyzing the movements of single or small groups of molecules within membranes.