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Analyses of Rho small GTPase-dependent regulation of septin structure and functions

Septin 结构和功能的 Rho 小 GTP 酶依赖性调节分析

基本信息

批准号：
17590259
负责人：
NAGATA Koh-ichi
金额：
$ 2.3万
依托单位：
Institute for Developmental Research, Aichi Human Service Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590259/
关键词：
signal transduction biomolecules septin cytoskeleton Rho

项目摘要

We showed that active Rho disrupts septin filament structures in REF52 cells. Among Rho effector molecules tested, Rhotekin induced morphological changes similar to those by activated Rho. In neuroblastoma cells, Rhotekin and Sept9b were enriched in the tip of neurites, a location where cortical actin reorganization is induced. We proposed that Rhotekin is a novel regulator organizing septin structures and provide a new link between the septin and Rho-signaling. We then identified PIST as a binding partner for Rhotekin. Rhotekin associated with PIST in vitro and in MDCK cells. The C-terminal SPV motif of Rhotekine xhibited binding to the PDZ domain of PIST. The binding was markedly inhibited by an activated Rho in COS7 cells. PIST and Rhotekin was co-localized at the Golgi-apparatus in non-polarized fibroblast-like MDCK cells and adherens junctions (AJs) in the fully polarized cells. PIST and Rhotekin were recruited from the cytosol to AJs as the cell becomes polarized. Expression of active Rho or prevention of Rhotekin-PIST interaction induced diffuse cytoplasmic distribution of Rhotekin in MDCK cells. We also identified a PDZ protein, Lin-7B, as a binding partner for Rhotekin. We found that Rhotekin interacts with Lin-7B in in vitro pull-down assays, and forms an immunocomplex in the rat brain. Their binding affinity became increased in the presence of active Rho in the COS7 cells. In addition, immunohistochemical analyses demonstrated that Lin-7 as well as Rhotekin is enriched in neurons. These results suggest that Lin-7 plays some role in neuronal functions in concert with Rho/Rhotekin signals.

我们发现活性 Rho 会破坏 REF52 细胞中的隔膜丝结构。在测试的 Rho 效应分子中，Rhotekin 诱导的形态变化与激活的 Rho 相似。在神经母细胞瘤细胞中，Rhotekin 和 Sept9b 在神经突尖端富集，这是诱导皮质肌动蛋白重组的位置。我们提出 Rhotekin 是一种组织 septin 结构的新型调节因子，并在 septin 和 Rho 信号传导之间提供了新的联系。然后我们确定 PIST 是 Rhotekin 的结合伙伴。 Rhotekin 在体外和 MDCK 细胞中与 PIST 相关。 Rhotekin 的 C 端 SPV 基序抑制与 PIST 的 PDZ 结构域的结合。 COS7 细胞中激活的 Rho 显着抑制了这种结合。 PIST 和 Rhotekin 在非极化成纤维样 MDCK 细胞中共定位于高尔基体，在完全极化细胞中共定位于粘附连接 (AJ)。当细胞极化时，PIST 和 Rhotekin 从细胞质被招募到 AJ。活性 Rho 的表达或 Rhotekin-PIST 相互作用的阻止诱导了 MDCK 细胞中 Rhotekin 的弥漫性细胞质分布。我们还鉴定了 PDZ 蛋白 Lin-7B，作为 Rhotekin 的结合伴侣。我们发现 Rhotekin 在体外 Pull-down 实验中与 Lin-7B 相互作用，并在大鼠大脑中形成免疫复合物。当 COS7 细胞中存在活性 Rho 时，它们的结合亲和力会增加。此外，免疫组织化学分析表明 Lin-7 和 Rhotekin 在神经元中富集。这些结果表明 Lin-7 与 Rho/Rhotekin 信号协同在神经元功能中发挥一定作用。