The Role of Ezrin in Membrane and Actin Interactions

 描述(申请人提供)：Ezrin在广泛的转移性癌症类型中过度表达，但对Ezrin在促进转移表型中所起的作用知之甚少。目前尚不清楚为什么过度表达会促进转移，部分原因是我们缺乏对Ezrin是如何控制和调节的了解。已经描述了将RhoA信号通路与Ezrin激活联系起来的联系，RhoA信号通路也与转移表型有关。当Ezrin被激活时，它被转移到质膜和肌动蛋白细胞骨架之间的界面，在那里Ezrin起到交联剂的作用。RhoA信号还会启动蛋白质激活级联反应，使细胞产生力量。RhoA力的产生和Ezrin作为质膜和肌动蛋白细胞骨架交联剂的作用相结合，表明Ezrin作用于增强质膜上RhoA产生的细胞内力。此外，RhoA还与RhoGDI和PKA相互作用，后者可被质膜上的Ezrin激活。这项建议的目的是确定Ezrin在调节细胞在质膜和肌动蛋白细胞骨架上产生的力量中的作用。在具体目标1中，我们将使用一种新开发的光学生物传感器来测量活细胞中跨越Ezrin的张力，以研究Ezrin负荷是如何控制的。我们使用一系列药物治疗来干扰Ezrin与质膜和细胞骨架的连接，以确定Ezrin何时处于紧张状态。为了确定Ezrin负载如何受细胞形态和运动的控制，我们还将使用微图案化技术控制单个细胞的形状、大小和迁移行为，同时跟踪Ezrin负载。在特定的目标2中，我们将通过RhoA信号通路来研究Ezrin和运动性之间的联系。我们将使用Ezrin基因敲除细胞系来测试Ezrin的去除如何通过RhoGDI和PKA影响RhoA信号级联。为了进一步了解这一系统，我们还将测量Ezrin载量与RhoA和PKA活性的比较，为了解这一复杂系统提供一个新的视角。这些目标的结果将有助于我们理解转移性癌细胞是如何迁移的，可能导致 针对转移运动的治疗方法的发展。这项奖学金将为我提供新的技能，使我的职业生涯发展成为一名独立的科学家，同时也提高了我们对肿瘤转移一个未被研究的方面的理解。

 DESCRIPTION (provided by applicant): Ezrin is overexpressed in a wide range of metastatic cancer types, but relatively little is known about what role Ezrin plays in promoting the metastatic phenotype. It is unclear why overexpression promotes metastasis due in part to our lack of understanding of how Ezrin is controlled and regulated. Connections have already been described that link the RhoA signaling pathway, which is also associated with metastatic phenotypes, to Ezrin activation. When Ezrin is activated it translocates to the interface between the plasma membrane and the actin cytoskeleton, where Ezrin acts as a cross linker. RhoA signaling also initiates protein activation cascades which cause the cell to produce force. This combination of RhoA force production and Ezrin's role as a plasma membrane and actin cytoskeleton cross linker suggests that Ezrin acts to reinforce RhoA generated intra- cellular forces at the plasma membrane. In addition, RhoA also interacts with RhoGDI and PKA, which can be activated by Ezrin at the plasma membrane. The goal of this proposal is to determine the role of Ezrin in mediating cell generated force at the plasma membrane and actin cytoskeleton. In Specific Aim 1, we will use a newly developed optically-based biosensor that measures tension across Ezrin in live cells to investigate how Ezrin loading is controlled. We use a series of drug treatments designed to perturb how Ezrin connects to the plasma membrane and cytoskeleton to determine when Ezrin is under tension. To determine how Ezrin loading is controlled by cell morphology and movement, we will also use micropatterning techniques control the shape, sizes and migration behavior of individual cells, while tracking Ezrin loading. In Specific Aim 2, we will investigate the connection between Ezrin and motility, through the RhoA signaling pathway. We will use an Ezrin knockdown cell line to test how the RhoA signaling cascade is affected through RhoGDI and PKA by the removal of Ezrin. To further our understanding of this system, we will also measure Ezrin loading as it compares to the activity of RhoA and PKA, providing a novel view into this complex system. The results of these aims will contribute to our understanding of how metastatic cancer cells migrate, potentially leading to the development of therapeutic treatments targeted at metastatic motility. This fellowship will provide me with new skills to allow my career to develop as an independent scientist, while also improving our understanding of an understudied aspect of metastasis.