The Role of Ezrin in Membrane and Actin Interactions

 描述（由申请人提供）：Ezrin在广泛的转移性癌症类型中过表达，但关于Ezrin在促进转移性表型中起什么作用知之甚少。目前还不清楚为什么过度表达会促进转移，部分原因是我们对Ezrin是如何控制和调节的缺乏了解。已经描述了将RhoA信号传导途径（其也与转移表型相关）与Ezrin活化联系起来的连接。当Ezrin被激活时，它移位到质膜和肌动蛋白细胞骨架之间的界面，在那里Ezrin充当交联剂。RhoA信号还启动蛋白质激活级联反应，导致细胞产生力。RhoA力产生和Ezrin作为质膜和肌动蛋白细胞骨架交联剂的作用的这种组合表明Ezrin用于增强RhoA在质膜处产生的细胞内力。此外，RhoA还与RhoGDI和PKA相互作用，其可以在质膜上被Ezrin激活。该提议的目标是确定Ezrin在介导细胞在质膜和肌动蛋白细胞骨架上产生的力中的作用。在具体目标1中，我们将使用一种新开发的基于光学的生物传感器来测量活细胞中Ezrin的张力，以研究如何控制Ezrin负载。我们使用了一系列药物治疗，旨在扰乱Ezrin如何连接到质膜和细胞骨架，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。在具体目标2中，我们将通过RhoA信号通路研究Ezrin和运动之间的联系。我们将使用Ezrin敲低细胞系来测试RhoA信号级联如何通过RhoGDI和PKA被Ezrin的去除而受到影响。为了进一步了解这个系统，我们还将测量Ezrin负载，因为它与RhoA和PKA的活性相比，为这个复杂的系统提供了一个新的视角。这些目标的结果将有助于我们理解转移性癌细胞如何迁移，可能导致 针对转移性运动的治疗性治疗的发展。这个奖学金将为我提供新的技能，让我的职业生涯发展为一个独立的科学家，同时也提高了我们对转移的一个未充分研究的方面的理解。

 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.