Differential ras activation between caveolae/raft and non-raft Microdomains
Differential ras activation between caveolae/raft and non-raft Microdomains
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DOI：
10.1247/csf.06019
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发表时间：
2007-01-01
影响因子：
1.5
通讯作者：
Miyawaki, Atsushi
中科院分区：
文献类型：
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作者：
Fukano, Takashi;Sawano, Asako;Miyawaki, Atsushi
虽然Ras激活的后果已经在肿瘤发生的背景下进行了广泛的研究，但其在信号转导的生理模式中的调节还没有得到很好的理解。将荧光指示剂Raichu-Ras与H-Ras和K-Ras的C-末端高变区融合，以分别产生质膜的小窝/筏（Raichu-tH）和非筏结构域（Raichu-tK）内Ras活化的指示剂. Raichu-tH也大量存在于内膜中。为了以高空间分辨率监测Ras激活，必须观察信号的切片图像。我们已经开发了一种宽视场荧光显微镜配备了数字微镜装置（DMD），以获取光学切片图像，使用条纹投影。该系统提供了可靠的信号，从荧光共振能量转移（FRET）之间的青色和黄色突变体的绿色荧光蛋白。我们已经使用这个系统来证明，在刺激与生长因子，这两个指标被激活在空间和时间上独特的模式。
Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu- Ras, was fused to the C- terminal hypervariable regions of H- Ras and K- Ras to create indicators for Ras activation within caveolae/ rafts ( Raichu- tH) and non- raft domains ( Raichu- tK) of the plasma membrane, respectively. Raichu- tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device ( DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer ( FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.