H-ras膜锚的单分子成像揭示了细胞膜胞质小叶中的结构域
Single-molecule imaging of the H-ras membrane-anchor reveals domains in the cytoplasmic leaflet of the cell membrane
摘要：
在过去的十年中，有证据表明，直径为50-700 nm的小区域位于质膜的外质小叶中。这些结构域中的大多数被认为由特定的脂质和蛋白质组成，并且被认为协调信号转导级联。类似的结构域是否也存在于质膜的细胞质小叶中，目前尚不清楚。为了研究胞质小叶结构域的存在，将H-Ras膜靶向序列融合到增强型黄色荧光蛋白的C末端。使用单分子荧光显微镜，记录单个分子在质膜的细胞质小叶中扩散的轨迹。从这些轨迹，个别膜锚定的增强黄色荧光蛋白分子的扩散进行了研究，在活细胞的时间尺度从5到200毫秒。结果表明，30-40%的分子的扩散被限制在域与一个典型的大小为200 nm。肌动蛋白的分解和胆固醇的提取都没有显著改变结构域的特征，这表明所观察到的结构域可能与迄今为止确定的膜结构域无关。
英文摘要：
In the last decade evidence has accumulated that small domains of 50-700 nm in diameter are located in the exoplasmic leaflet of the plasma membrane. Most of these domains supposedly consist of specific sets of lipids and proteins, and are believed to coordinate signal transduction cascades. Whether similar domains are also present in the cytoplasmic leaflet of the plasma membrane is unclear so far. To investigate the presence of cytoplasmic leaflet domains, the H-Ras membrane-targeting sequence was fused to the C-terminus of the enhanced yellow fluorescent protein. Using single-molecule fluorescence microscopy, trajectories of individual molecules diffusing in the cytoplasmic leaflet of the plasma membrane were recorded. From these trajectories, the diffusion of individual membrane-anchored enhanced yellow fluorescent protein molecules was studied in live cells on timescales from 5 to 200 ms. The results show that the diffusion of 30-40% of the molecules is constrained in domains with a typical size of 200 nm. Neither breakdown of actin nor cholesterol extraction changed the domain characteristics significantly, indicating that the observed domains may not be related to the membrane domains identified so far.
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