Roles of cofilin in regulation of actin cytoskeleton dynamics

肌动蛋白细胞骨架的动力学和重组在细胞迁移、胞质分裂、内吞和极性形成等多种细胞内事件中起着重要的作用。肌动蛋白的动态和重组受大量肌动蛋白结合蛋白和上游信号分子的时空调控。Cofilin是肌动蛋白细丝动力学的关键调节因子，它刺激肌动蛋白细丝的解聚和断裂。Cofilin通过LIM-Kinase(LIMK)对Ser-3的磷酸化而失活。Ser-3-磷酸化的Cofilin(P-Cofilin)被Slingshot磷酸酶去磷酸化并重新激活。本项目的目的是揭示cofilin在肌动蛋白细胞骨架重组中的作用。在本研究中，我们开发了一种新的探针，使用双分子荧光互补(BIFC)方法检测Cofilin的肌动蛋白结合活性。在本研究中，探头的强度和灵敏度都有所提高。我们利用杆状病毒表达系统在昆虫细胞中表达和纯化了这些探针蛋白，并建立了利用互补GFP的荧光检测Cofilin的肌动蛋白结合活性、LIMK的激酶活性和Slingshot的磷酸酶活性的方法。我们用500多种化合物对Cofilin、LIMK和Slingshot的抑制剂进行了筛选。因此，LIMK被两种被称为Src酪氨酸激酶抑制剂的化合物抑制。这些化合物抑制了LIMK过表达细胞中肌动蛋白的超聚合。据报道，其中一种化合物在LIMK抑制浓度下抑制细胞迁移和轴突延伸。我们正试图利用活细胞中的化合物来分析cofilin磷酸化在肌动蛋白细胞骨架重组中的作用。我们将在化合物的衍生形式中筛选更具体的LIMK抑制剂。这些抑制剂将成为抑制肿瘤转移和炎症的药物的种子。

Actin cytoskeletal dynamics and reorganization play essential roles in various intracellular events, including cell migration, cytokinesis, endocytosis, and polarity formation. Actin dynamics and reorganization are spatiotemporally regulated by a large number of actin-binding proteins and upstream signaling molecules. Cofilin, a key regulator of actin filament dynamics, stimulates depolymerization and severing of actin filaments. Cofilin is inactivated by phosphorylation of Ser-3 by LIM-kinase (LIMK). The Ser-3-phosphorylated cofilin (P-cofilin) is dephosphorylated and reactivated by the Slingshot phosphatase. The purposes of this project were to reveal the function of cofilin in actin cytoskeleton reorganization. In the present study, we have developed a new probe that detects the actin-binding activity of cofilin using a bimolecular fluorescence complementation (BiFC) method. The probe was improved higher intensity and sensitivity in this study. We expressed and purified these probe proteins in insect cells using a baculovirus expressing system, and developed methods to measure the actin-binding activity of cofilin, the kinase activity of LIMK, and the phosphatase activity of Slingshot with the fluorescence of complemented-GFP. We screened the inhibitors of cofilin, LIMK and Slingshot with over five hundred chemical compounds. As a consequence, LIMK was inhibited two compounds which was known as Src tyrosine kinase inhibitor. The compounds inhibited the hyper-polymerization of actin in LIMK overexpressing cells. It was reported that one of the compounds inhibited cell migration and neurite extension at the concentration of LIMK inhibition. We are tring to analyze the role of cofilin phosphorylation in the actin cytoskeleton reorganization using the compounds in living cells. We are going to screen more specific inhibitors for LIMK in derivative forms of the compounds. These inhibitors will be seeds of the drugs for inhibition of metastasis and inflammation.