Direct visualization of Ras proteins in spatially distinct cell surface microdomains.
Direct visualization of Ras proteins in spatially distinct cell surface microdomains.
复制标题
DOI：
10.1083/jcb.200209091
复制
发表时间：
2003-01-20
影响因子：
7.8
通讯作者：
Hancock, John F
中科院分区：
文献类型：
--
作者：
Prior, Ian A;Muncke, Cornelia;Parton, Robert G;Hancock, John F
信号复合物定位于特定的微区协调质膜上的信号转导。使用免疫金电子显微镜的质膜片加上空间点模式分析，我们已经可视化形态特征的微域，包括脂筏，在原位和高分辨率。我们发现，一个内质膜脂筏标记显示胆固醇依赖的聚集在微区的平均直径为44 nm，占35%的细胞表面。交联的外部小叶筏蛋白的结果在重新分配的内部小叶筏，但他们保留其模块化结构。Ras微定位分析表明，非活性H-ras分布在脂筏和胆固醇独立的微域之间。相反，激活的H-ras和K-ras主要存在于非重叠的、胆固醇独立的微区中。半乳糖凝集素-1稳定的协会激活的H-ras与这些nonraft微结构域，而K-ras聚类法尼基化，但不支持geranylgeranylation。这些结果表明，内质膜包括一个复杂的马赛克离散微区。在这个框架内的差异空间定位可能会占不同的信号输出的高度同源的Ras蛋白。
Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.