Single-molecule imaging analysis of Ras activation in living cells
Single-molecule imaging analysis of Ras activation in living cells
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DOI：
10.1073/pnas.0401354101
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发表时间：
2004-05-11
影响因子：
11.1
通讯作者：
Kusumi, A
中科院分区：
文献类型：
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作者：
Murakoshi, H;Iino, R;Kusumi, A
单分子荧光共振能量转移（FRET）方法已经发展到观察激活的小G蛋白Ras在单个分子的水平。用荧光GTP类似物BodipyTR-GTP（受体）显微注射表达与YFP（供体）融合的H-或K-Ras的KB细胞，并通过单分子FRET监测表皮生长因子诱导的BodipyTR-GTP与YFP-（H或K）-Ras的结合。在活化时，Ras扩散被大大抑制/固定，表明形成大的活化Ras信号传导复合物。这些复合物可以作为平台用于将Ras信号转导至效应分子，进一步表明Ras信号转导需要比与效应分子的简单碰撞更多的东西。募集到膜上的GAP 334-GFP也是静止的，表明其与信号复合物结合。这里开发的单分子FRET方法提供了一个强大的技术来研究各种G蛋白的信号转导机制。
A single-molecule fluorescence resonance energy transfer (FRET) method has been developed to observe the activation of the small G protein Ras at the level of individual molecules. KB cells expressing H- or K-Ras fused with YFP (donor) were microinjected with the fluorescent GTP analogue BodipyTR-GTP (acceptor), and the epidermal growth factor-induced binding of BodipyTR-GTP to YFP-(H or K)-Ras was monitored by single-molecule FRET. On activation, Ras diffusion was greatly suppressed/immobilized, suggesting the formation of large, activated Ras-signaling complexes. These complexes may work as platforms for transducing the Ras signal to effector molecules, further suggesting that Ras signal transduction requires more than simple collisions with effector molecules. GAP334-GFP recruited to the membrane was also stationary, suggesting its binding to the signaling complex. The single-molecules FRET method developed here provides a powerful technique to study the signal-transduction mechanisms of various G proteins.