Nanoscale analysis of signal trans l-ctionbyelecfron microscopy

在经典的细胞膜流体镶嵌模型中，横向异质性现在被认为是富含神经节苷脂、胆固醇和特定膜蛋白的微域或‘筏’。单粒子跟踪和荧光共振能量转移技术的分析表明，正常未受刺激的细胞中的木筏非常小，可能持续不到1ms。如果木筏在纳米尺寸范围内，电子显微镜(EM)有望具有足够的分辨率来可视化木筏，但传统样品制备需要使用的化学固定剂不太可能保持膜分子的原位定位，特别是脂质，甚至可能导致人造结果。因此，我们使用了快速冷冻和冷冻断裂的复制品，这种复制品可以物理地将分子固定在原位，从而将人工干扰的可能性降至最低。利用这种方法，神经节苷脂GM1和GM3可能是RAFT分子在细胞膜上的分布，可以在纳米级范围内观察到，它们在正常小鼠成纤维细胞中呈现簇状，而正常小鼠成纤维细胞对胆固醇耗竭和寒冷(Mol.比奥尔。手机，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)