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

我们发现，活性Rho破坏了REF52细胞中的隔膜细丝结构。在被测试的Rho效应分子中，Rhotekin引起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在神经突起的顶端富含，这是一个诱导皮质肌动蛋白重组的位置。我们认为Rhotekin是一种新的组织Septin结构的调节子，并在Septin和Rho信号之间提供了一种新的联系。然后，我们确定PIST是Rhotekin的结合伙伴。在体外和MDCK细胞中，Rhotekin与PIST相关。Rhotekine的C-末端SPV基序抑制了与Pist的PDZ结构域的结合。在COS7细胞中，活化的Rho能显著抑制这种结合。Pist和Rhotekin共同定位于未极化的成纤维细胞样MDCK细胞的高尔基体和完全极化的细胞中的黏附连接(AJ)。随着细胞的极化，皮斯特和罗特金被从胞浆中招募到AJ。表达活性Rho或阻止Rhotekin-Pist相互作用诱导Rhotekin在MDCK细胞中弥漫性胞浆分布。我们还确定了PDZ蛋白LIN-7B作为Rhotekin的结合伙伴。我们发现Rhotekin在体外下拉试验中与Lin-7B相互作用，并在大鼠大脑中形成免疫复合体。在COS7细胞中，活性Rho的存在使它们的结合亲和力增强。此外，免疫组织化学分析表明，LIN-7和Rhotekin在神经元中都有丰富的表达。这些结果表明，LIN-7通过Rho/Rhotekin信号在神经元功能中发挥一定的作用。

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.