High-throughput, single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking
High-throughput, single-particle tracking reveals nested membrane domains that dictate KRasG12D diffusion and trafficking
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DOI：
10.7554/elife.46393
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发表时间：
2019-11-01
期刊：
影响因子：
7.7
通讯作者：
Nan, Xiaolin
中科院分区：
文献类型：
--
作者：
Lee, Yerim;Phelps, Carey;Nan, Xiaolin
膜纳米结构域与Ras信号转导有关，但这些结构域是什么以及它们如何与Ras相互作用仍然不清楚。在这里，使用单粒子跟踪与光活化定位显微镜（spt-PALM）和详细的轨迹分析，我们表明，不同的膜结构域决定KRas（G12 D）（一个活跃的KRas突变体）扩散和贩运U2 OS细胞。KRas（G12 D）在类似于70 nm域中表现出不动状态，每个域嵌入在赋予中等流动性的较大域（类似于200 nm）中，而膜的其余部分支持快速扩散。此外，KRas（G12 D）通过不动状态从膜连续移除并补充到快速状态，这让人想起Ras内化和再循环。重要的是，KRas（G12 D）的扩散和运输性质在广泛的蛋白质表达水平范围内保持不变。我们的研究结果揭示了膜组织如何决定Ras的膜扩散和运输，并为Ras信号的空间调控提供了新的见解。
Membrane nanodomains have been implicated in Ras signaling, but what these domains are and how they interact with Ras remain obscure. Here, using single particle tracking with photoactivated localization microscopy (spt-PALM) and detailed trajectory analysis, we show that distinct membrane domains dictate KRas(G12D) (an active KRas mutant) diffusion and trafficking in U2OS cells. KRas(G12D) exhibits an immobile state in similar to 70 nm domains, each embedded in a larger domain (similar to 200 nm) that confers intermediate mobility, while the rest of the membrane supports fast diffusion. Moreover, KRas(G12D) is continuously removed from the membrane via the immobile state and replenished to the fast state, reminiscent of Ras internalization and recycling. Importantly, both the diffusion and trafficking properties of KRas(G12D) remain invariant over a broad range of protein expression levels. Our results reveal how membrane organization dictates membrane diffusion and trafficking of Ras and offer new insight into the spatial regulation of Ras signaling.