| Research Abstract |
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.
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