Nanoscale analysis of signal trans l-ctionbyelecfron microscopy

横向异质性在经典的流体镶嵌模型的细胞膜，现在设想为微域或“筏”，富含神经节苷脂，胆固醇和特定的膜蛋白。单粒子跟踪和荧光共振能量转移技术的分析表明，正常未刺激细胞中的筏非常小，可能持续不到1 ms。如果筏在纳米尺寸范围内，预计电子显微镜（EM）将具有足够的分辨率来可视化筏，但是需要用于常规样品制备的化学固定剂不太可能保持膜分子（特别是脂质）的原位定位，甚至可能导致人为结果。因此，我们使用了快速冷冻和冷冻断裂的复制品，它们可以在原位物理地固定分子，从而最大限度地减少了人为扰动的可能性。使用该方法，神经节苷脂GM 1和GM 3（细胞膜中假定的筏分子）的分布可以在纳米尺寸范围内可视化，并且它们在正常小鼠成纤维细胞中显示簇，其对胆固醇消耗和冷却敏感（Mol. Cell，18，2112-2122，2007）

Lateral heterogeneities in the classical fluid-mosaic model of cell membranes are now envisaged as microdomains or 'rafts' that are enriched in gangliosides, cholesterol and specific membrane proteins. The analyses with single-particle tracking and fluorescent resonance energy transfer techniques have suggested that rafts in normal unstimulated cells are extremely small and may last for less than 1 ms. Electron microscopy (EM) would be expected to have sufficient resolving power to visualize rafts if they are in the nanometer size range, but chemical fixatives that need to be used for conventional sample preparations are unlikely to preserve the in situ localization of membrane molecules, particularly lipids, and may even cause artifactual results. Therefore, we used quick-frozen and freeze-fracture replicas which can physically immobilize molecules in situ and thus minimized the possibility of artifactual perturbation. Using this method, the distribution of gangliosides GM1 and GM3, putative raft molecules in the cell membrane, can be visnalized in the nanometer size range, and they show clusters in normal mouse fibroblasts, which are susceptible to cholesterol depletion and chilling (Mol. Biol. Cell, 18, 2112-2122, 2007)