Development of visualization technique which enables us to monitor the molecular dynamics between the nucleus and cytoplasm on real time in living cells

我们已经开发了一种体内分析系统，使我们能够在活细胞中实时三维监测功能分子的行为。此外，我们已经能够在一个活细胞中同时可视化四种分子，如染色体蛋白质和微管。利用该系统，我们分析了核蛋白和核转运因子输入蛋白α和输入蛋白β的行为。因此，我们得到了以下数据：研究发现，在有丝分裂后，尽管核膜的完整性似乎得到了恢复，但核蛋白的输入活性仍有一小段时间未恢复。将绿色荧光蛋白（GFP）标记的输入蛋白α微注射到活细胞的细胞质中，在没有固定的活状态下观察到GFP输入蛋白α的胞内定位，该蛋白主要定位在细胞质中。相比之下，注射gfp输入蛋白α后将核蛋白注射到相同的细胞中，它在1分钟内被转运到细胞核中，然后在1分钟内返回到细胞质中。此外，在有丝分裂前约2分钟，gfp输入蛋白α的总体分布发生了改变。我们发现gfp输入蛋白β定位于整个细胞质和细胞核，并且在间期总体定位没有改变。此外，核蛋白的注射对gfp输入蛋白β的细胞内分布没有影响。

We have developed an in vivo assay system which enables us to monitor the behavior of functional molecules three-dimensionally on real time in living cells. Moreover, we have been able to visualize four kinds of molecules such as chromosomeaml proteins and microtubules simultaneously in one living cell. By using this system, we analyzed the behavior of nuclear proteins and nuclear transport factors such as importin α and importin β. As a result, we have obtained the following data.2. It was found that after mitosis, there exists a short period when the nuclear protein import activity is not yet recovered, even though the integrity of nuclear envelope appears to be restored.3. When green fluorescent protein (GFP)-tagged importin α was microinjected into the cytoplasm of living cells and the intracellular localization of the GFP-importin α was observed in a live state without fixation, the protein was primarily localized in the cytoplasm. In contrast, when nuclear proteins were injected to the same cells after injection of the GFP-importin α, it was transported into the nucleus within one minute and then returned to the cytoplasm within the next one minute. Moreover, it was found that the overall distribution of the GFP-importin α was altered to the nucleus about two minutes just before mitosis.4. We found that GFP-importin β was localized throughout the cytoplasm and nucleus and that the overall localization did not change during interphase. Moreover, the injection of nuclear proteins did not affect the intracellular distribution of the GFP-importin β.