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

开发可视化技术，使我们能够实时监测活细胞中细胞核和细胞质之间的分子动力学

• 批准号: 08558079
• 负责人: YONEDA Yoshihiro
• 金额: $ 11.65万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08558079/
• 关键词: Molecular visualization technique; Nuclear protein transport; Green fluorescent protein; Importin; Fluorescent dye; Nuclear localization signal; Cell cycle; Microinjection; 核-細胞質間物質輸送; importin; 三次元可視化技術; 細胞核; green fluorescent protein; 画像解析

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.

我们开发了一种体内检测系统，使我们能够实时监测活细胞中功能分子的三维行为。此外，我们已经能够在一个活细胞中同时观察四种分子，例如染色体蛋白和微管。通过使用该系统，我们分析了核蛋白和核转运因子（例如 importin α 和 importin β）的行为。结果，我们得到了以下数据： 2．研究发现，有丝分裂后，尽管核膜的完整性似乎已恢复，但仍存在一小段时间核蛋白输入活性尚未恢复。3．当将绿色荧光蛋白（GFP）标记的导入蛋白α显微注射到活细胞的细胞质中并且在未固定的活细胞状态下观察到GFP-导入蛋白α的细胞内定位时，该蛋白质主要定位于细胞质中。相比之下，当注射GFP-输入蛋白α后将核蛋白注射到同一细胞中时，它在一分钟内被转运到细胞核中，然后在接下来的一分钟内返回到细胞质中。此外，发现GFP-importin α的整体分布在有丝分裂前约两分钟改变到细胞核。4．我们发现 GFP-importin β 定位于整个细胞质和细胞核，并且整体定位在间期期间没有改变。此外，核蛋白的注射并不影响GFP-importin β的细胞内分布。

项目成果

Togo Ikuta: "Nuclear Localization and Export Signals of the Human Aryl Hydrocarbon Receptor" THE JOURNAL OF BIOLOGICAL CHEMISTRY. 273 (5). 2895-2904 (1998)

Togo Ikuta：“人类芳基烃受体的核定位和输出信号”生物化学杂志。

Hidetaka Eguchi: "A Nuclear Localization Signal of Human Aryl Hydrocarbon Receptor Nuclear Translocator/Hypoxia-inducible Factor 1β is a Novel Bipartite Type Recognized by the Two Components of Nuclear Pore-targeting Complex" THE JOURNAL OF BIOLOGICAL CHE

Hidetaka Eguchi：“人芳基烃受体核转位子/缺氧诱导因子 1β 的核定位信号是一种由核孔靶向复合物的两个成分识别的新型二分类型”《生物化学杂志》

Shingo Kose: "β-Subunit of Nuclear Pore-targeting Complex (Importin-β) Can Be Exported from the Nucleus in a Ran-independent Manner"THE JOURNAL OF BIOLOGICAL CHEMISTRY. 274.7. 3946-3952 (1999)

Shingo Kose：“核孔靶向复合物的β-亚基（导入蛋白-β）可以以独立于Ran的方式从细胞核中输出”生物化学杂志274.7 3946-3952（1999）。

Yosuke Matsuoka: "Identification and Characterization of Nuclear Pore Subcomplexes in Mitotic Extract of Human Somatic Cells" Biochemical and Biophysical Research Communications. 254. 417-423 (1999)

Yosuke Matsuoka：“人体细胞有丝分裂提取物中核孔亚复合物的鉴定和表征”生物化学和生物物理研究通讯。

Fumihiko Yokoya: "β-Catenin Can Be Transported into the Nucleus in a Ran-unassisted Manner" Molecular biology of the Cell. (in press). (1999)

Fumihiko Yokoya：“β-连环蛋白可以以无助的方式转运到细胞核中”细胞的分子生物学（1999 年出版）。