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

我们发现，活性Rho破坏了REF 52细胞中的隔蛋白丝结构。在测试的Rho效应分子中，Rhotekin诱导的形态学变化类似于活化的Rho。在神经母细胞瘤细胞中，Rhotekin和Sept 9 b富集在神经突的尖端，这是诱导皮质肌动蛋白重组的位置。我们提出Rhotekin是一种新的调节剂，组织septin结构，并提供了一个新的septin和Rho信号之间的联系。然后，我们将PIST鉴定为Rhotekin的结合伴侣。Rhotekin在体外和MDCK细胞中与PIST相关。Rhotekine的C-末端SPV基序抑制与PIST的PDZ结构域的结合。在COS 7细胞中，这种结合被激活的Rho显著抑制。PIST和Rhotekin在非极化成纤维细胞样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（AJs）。随着细胞极化，PIST和Rhotekin从细胞质中被募集到AJs中。表达活性Rho或阻止Rhotekin-PIST相互作用诱导Rhotekin在MDCK细胞中弥散性胞质分布。我们还确定了一个PDZ蛋白，林-7B，作为一个结合伴侣的罗泰金。我们发现Rhotekin与Lin-7 B在体外下拉试验中相互作用，并在大鼠脑中形成免疫复合物。在COS 7细胞中存在活性Rho时，它们的结合亲和力增加。此外，免疫组织化学分析表明，Lin-7以及Rhotekin在神经元中富集。这些结果表明，Lin-7与Rho/Rhotekin信号一起在神经元功能中起一定作用。

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.