Multidimensional analysis of small GTPases as key molecules in the neural network formation by using activity-imaging

使用活动成像对小 GTP 酶作为神经网络形成中的关键分子进行多维分析

• 批准号: 16500242
• 负责人: NAKAMURA Takeshi
• 金额: $ 2.37万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16500242/
• 关键词: G protein; neural network formation; FRET; imaging; 海馬神経細胞; Rho; 神経細胞; FRETプローブ; PI3キナーゼ; 神経回路; Rhoファミリー; Rasファミリー; 成長円錐; RNAi

In this study, we performed the activity-imaging of Ras and Rho GTPases using GFP-based FRET probes in PC12 Cells, DRG neurons, and hippocampal neurons.1.During NGF-induced neurite outgrowth in PC12 cells, the activity-imaging and RNA interference study showed that local PIP3 accumulation after NGF stimulation recruited Vav2 and Vav3 to the plasma membrane and thereby activated Rac1 and Cdc42 ; this Vav2/3-dependent activation of Rac1 and Cdc42 promoted the formation of short processes leading to neurite outgrowth. We also provide evidence that Vav2 and Vav3 are involved in the local activation of PI3-kinase at the protrusions after NGF stimulation. Therefore, this study demonstrates that Vav proteins are constituents for the first time that Vav proteins are constituents of the PIP3-dependent positive feedback loop that cycles locally with morphological changes.2.We visualized RhoA/Rac1/Cdc42 activities during laminin-induced growth cone advance of DRG neurons and N1E-115 cells. The Rac1 and Cdc42 activities were high in the P-domain of growth cones. Against a model involving RhoA down-regulation at the periphery of protruding growth cones, we found that the RhoA activity was higher in the P-domain than in the C-domain and axon shaft, and that a high level of RhoA activity was maintained in the extending part of growth cones. In LPA-treated N1E-115 cells, well-developed neurites with growth cones showed RhoA activation, but sustained their extended morphology until they were drawn toward the contracting somata. Thus, RhoA activation in the shaft results in neurite retraction, whereas high RhoA activity in the P-domain is necessary to retain the spread morphology of nerve growth cone.3.We visualized the activities of Ras and Rho GTPases within the postsynaptic sites using high-density cultures of hippocampal neurons. This study showed that each member of these GTPases had a peculiar pattern of activation in the postsynaptic sites.

在本研究中，我们使用基于gfp的FRET探针在PC12细胞、DRG神经元和海马神经元中对Ras和Rho gtpase进行了活性成像。在NGF诱导的PC12细胞神经突生长过程中，活性成像和RNA干扰研究表明，NGF刺激后局部PIP3的积累将Vav2和Vav3招募到质膜上，从而激活了Rac1和Cdc42；这种依赖于vav2 /3的Rac1和Cdc42的激活促进了导致神经突生长的短过程的形成。我们还提供证据表明，在NGF刺激后，Vav2和Vav3参与了突起处pi3激酶的局部激活。因此，本研究首次证明了Vav蛋白是pip3依赖的正反馈回路的组成部分，该回路随形态变化局部循环。我们观察了在层粘胶蛋白诱导的DRG神经元和N1E-115细胞生长锥推进过程中RhoA/Rac1/Cdc42的活性。在生长锥的p域，Rac1和Cdc42活性较高。通过对生长锥外周RhoA下调的模型，我们发现p区RhoA活性高于c区和轴突轴，并且生长锥延伸部分RhoA活性维持在较高水平。在lpa处理的N1E-115细胞中，具有生长锥的发育良好的神经突显示RhoA激活，但保持其延伸的形态，直到它们被吸引到收缩的体细胞。因此，轴上RhoA的激活导致神经突缩回，而p结构域的高RhoA活性对于保持神经生长锥的扩散形态是必要的。我们利用高密度海马神经元培养可视化突触后位点Ras和Rho gtpase的活性。这项研究表明，这些gtpase的每个成员在突触后位点都有一种特殊的激活模式。

项目成果

RalA activation at nascent lamellipodia of EGF-stimulated Cos7 cells and migrating MDCK cells.

RalA 在 EGF 刺激的 Cos7 细胞和迁移 MDCK 细胞的新生片状伪足中激活。

• 发表时间: 2004
• 期刊: Mol. Biol. Cell 15
• 作者: Takaya;A.;Ohba;Y.;Kurokawa;K.;Matsuda,M.
• 通讯作者: Matsuda,M.

Monitoring spatio-temporal regulation of Ras and Rho GTPase with GFT-based FRET probes.

使用基于 GFT 的 FRET 探针监测 Ras 和 Rho GTPase 的时空调节。

• 发表时间: 2005
• 期刊: Methods 37
• 作者: Nakamura;T.;Aoki;K.;Matsuda;M.
• 通讯作者: M.

生細胞内情報分子の蛍光可視化プローブ:タンパク質プローブ、G protein先端の分析法-理工学からナノ・バイオまで

活细胞中信息分子的荧光可视化探针：蛋白质探针、G 蛋白的先进分析方法 - 从科学和工程到纳米生物

• 发表时间: 2004
• 作者: 中村岳史;松田道行
• 通讯作者: 松田道行

Local PIP3 accumulation recruits Vav2 and Vav3 to activate Rac1/Cdc42 and initiate neurite outgrowth in nerve growth factor-stimulated PC12 cells.

局部 PIP3 积累会招募 Vav2 和 Vav3 来激活 Rac1/Cdc42 并启动神经生长因子刺激的 PC12 细胞中的神经突生长。

• 发表时间: 2004
• 期刊: Mol. Biol. Cell (In press)
• 作者: Aoki;K.;Nakamura;T.;Fujikawa;K.;Matsuda;M.
• 通讯作者: M.

Involvement of the c-Src-Crk-C3G-Rap1 signaling in the nectin-induced activation of Cdc42 and formation of adherens junctions

• DOI: 10.1074/jbc.m411099200
• 发表时间: 2005-01-07
• 期刊: JOURNAL OF BIOLOGICAL CHEMISTRY
• 影响因子: 4.8
• 作者: Fukuyama, T;Ogita, H;Takai, Y
• 通讯作者: Takai, Y