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

我们开发了一种活体检测系统，使我们能够实时三维地监测活细胞中功能分子的行为。此外，我们已经能够在一个活细胞中同时观察到染色体蛋白和微管等四种分子。利用这个系统，我们分析了核蛋白和核转运因子如Importinα和Importinβ的行为。因此，我们获得了以下数据。研究发现，有丝分裂后，尽管核膜的完整性似乎得到了恢复，但核蛋白输入活性仍存在一段较短的时间。将绿色荧光蛋白标记的Importinα显微注射到活细胞的胞浆中，观察到绿色荧光蛋白Importinα在无固定的活体状态下的细胞内定位，蛋白主要定位在细胞质中。相反，当在注射绿色荧光蛋白进口素α后将核蛋白注射到相同的细胞中时，它在一分钟内被输送到细胞核，然后在接下来的一分钟内返回细胞质。此外，研究还发现，绿色荧光蛋白输入素α的总体分布在有丝分裂前两分钟左右发生了变化。我们发现绿色荧光蛋白导入蛋白β定位于细胞质和细胞核，并且整个定位在间期没有改变。此外，注射核蛋白并不影响绿色荧光蛋白导入蛋白β在细胞内的分布。

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.