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

我们发现活跃的Rho破坏了REF52细胞中的septin丝结构。在Rho效应分子中，Rhotekin诱导的形态学变化与活化的Rho相似。在神经母细胞瘤细胞中，Rhotekin和Sept9b富集于神经突尖端，这是诱导皮层肌动蛋白重组的位置。我们提出Rhotekin是一种组织septin结构的新型调节剂，并在septin和rho信号传导之间提供了新的联系。然后，我们确定了ist作为Rhotekin的结合伙伴。Rhotekin与体外和MDCK细胞中的PIST相关。Rhotekine的c端SPV基序与PIST的PDZ结构域结合。在COS7细胞中，活化的Rho明显抑制了这种结合。在非极化的成纤维细胞样MDCK细胞和完全极化细胞的粘附连接（AJs）中，PIST和Rhotekin共定位于高尔基体。当细胞极化时，PIST和Rhotekin从细胞质中被招募到AJs中。活性Rho的表达或阻止Rhotekin- ist相互作用诱导了Rhotekin在MDCK细胞中的弥漫性细胞质分布。我们还发现了PDZ蛋白Lin-7B作为Rhotekin的结合伙伴。我们在体外拉下实验中发现Rhotekin与Lin-7B相互作用，并在大鼠大脑中形成免疫复合物。在COS7细胞中，活性Rho的存在增加了它们的结合亲和力。此外，免疫组化分析表明，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.