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.

我们已经开发了一个体内测定系统，使我们能够在活细胞中实时监测功能分子的行为。通过使用该系统，我们分析了核蛋白和诸如Emportinα和Emptractβ之类的转运因子。虽然将某些EEN荧光蛋白（GFP）标记的Importinα的完整性显微注射到活细胞的细胞质中，并且在活细胞的细胞细胞中，在具有固定性的活体状态下观察到GFP -Irmirtiralα的细胞内定位，但蛋白质是在固定的，但在蛋白细胞质。在注射GFP-impertinα之后，将S注射到同一细胞中，在一分钟内将其转移到核中，并在下一个核中返回到细胞质。 4。我们发现，GFP - 要素β被定位为细胞质，并且整体蛋白质的总体定位变化不影响GFP-rimint蛋白β的细胞内分布。

项目成果

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》

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。

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）。

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）。

Yoichi Miyamoto: "Differential Modes of Nuclear Localization Signal (NLS) Recognition by Three Distinct Classes of NLS Receptors" THE JOURNAL OF BIOLOGICAL CHEMISTRY. 272・42. 26375-26381 (1997)

宫本洋一：“三种不同类型的 NLS 受体的核定位信号（NLS）识别的不同模式”生物化学杂志 272・42（1997）。