Using single-particle tracking to study nuclear trafficking of viral genes
Using single-particle tracking to study nuclear trafficking of viral genes
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DOI：
10.1529/biophysj.104.042234
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发表时间：
2004-10-01
影响因子：
3.4
通讯作者：
Zhuang, XW
中科院分区：
文献类型：
--
作者：
Babcock, HP;Chen, C;Zhuang, XW
遗传物质如何进出细胞核的问题不仅对于了解病毒感染而且对于推进基因递送技术都是一个非常重要的问题。在这里，我们展示了一种物理技术，使基因贩运研究在单基因水平相结合的灵敏的荧光显微镜显微注射。作为一个模型系统，我们研究了流感基因的核进口，在核糖核蛋白（vRNP）的形式，通过成像单个vRNP在活细胞中的真实的时间。我们的单粒子轨迹表明，vRNP通过扩散运输到核膜。我们已经观察到vRNP和核孔复合物之间的异质相互作用，解离速率常数跨越两个数量级。我们的单粒子跟踪实验也提供了新的见解vRNP的核输入的调节机制：流感M1蛋白，进口过程的调节蛋白，下调vRNP的核输入通过抑制vRNP和核孔复合物之间的相互作用，但对vRNP的运输性能没有显着的影响。我们希望这种单粒子跟踪方法在基因贩运调查中得到广泛应用。
The question of how genetic materials are trafficked in and out of the cell nucleus is a problem of great importance not only for understanding viral infections but also for advancing gene-delivery technology. Here we demonstrate a physical technique that allows gene trafficking to be studied at the single-gene level by combining sensitive fluorescence microscopy with microinjection. As a model system, we investigate the nuclear import of influenza genes, in the form of ribonucleoproteins (vRNPs), by imaging single vRNPs in living cells in real time. Our single-particle trajectories show that vRNPs are transported to the nuclear envelope by diffusion. We have observed heterogeneous interactions between the vRNPs and nuclear pore complexes with dissociation rate constants spanning two orders of magnitude. Our single-particle tracking experiments also provided new insights into the regulation mechanisms for the nuclear import of vRNPs: the influenza M1 protein, a regulatory protein for the import process, downregulates the nuclear import of vRNPs by inhibiting the interactions between vRNPs and nuclear pore complexes but has no significant effect on the transport properties of vRNPs. We expect this single-particle tracking approach to find broad application in investigations of genetic trafficking.