Actions of the Sodium-Hydrogen Exchanger Subtype, NHE1

描述（由申请人提供）：质膜Na-H交换器NHE1在细胞内pH稳态中具有确定的作用。在过去的资助期内的工作表明，NHE1还通过直接结合肌动蛋白结合蛋白的ERM家族作为肌动蛋白丝的锚点。在成纤维细胞中，ERM结合保留了NHE1在板足前缘的定位，这对于肌动蛋白丝在膜突起中的组装是必要的。NHE1在离子易位和肌动蛋白锚定中的协调功能是细胞定向迁移所必需的，也是细胞极性、肌动蛋白聚合和局灶粘连重塑所必需的。在果蝇中发现了一种结合ERM同源物的新型DNHE1，在盘状盘基骨柱中发现了一种细胞极化和趋化所必需的新型DNHE1，这表明NHE1锚定肌动蛋白和细胞定向运动对NHE1的要求在进化上是保守的。当前提案的目的是通过研究NHE1的两种功能协同作用以产生维持极性所必需的不对称局部H+外排的假设来确定NHE1如何调节定向细胞运动。Aim 1的研究将通过探究NHE1的局部H+通量如何促进细胞突出、无肌动蛋白的刺端形成以及迁移细胞前沿Rac和Cdc42的空间受限激活，来确定NHE1如何调节哺乳动物成纤维细胞的膜突出和肌动蛋白聚合。NHE1是否在迁移细胞前沿的自生正反馈回路中起作用也将被确定。Aim 2探讨了NHE1在离子易位和肌动蛋白锚定中的两种功能是如何协调地重塑哺乳动物成纤维细胞中的细胞-底物黏附的，方法是询问NHE1的肌动蛋白锚定是否对整合素激活或局灶黏附相关蛋白组装的后续步骤是必要的，以及NHE1的离子易位是否通过pH依赖性的整合素-细胞骨架连接的破坏促进局灶黏附的分解。新形成的局灶粘连如何促进nhe1依赖的迁移表型也将被确定。Aim 3的重点是为什么DdNHE1是Dictyostelium趋化性所必需的，通过询问它是否产生局部H+外排，它是否通过作用于细胞前缘或后缘的信号来维持极性，以及遗传筛选是否识别介导极性和趋化性的DdNHE1效应物。总的来说，这项研究计划解决了与极性如何发展和维持以及空间限制信号传导过程如何驱动细胞迁移相关的重要问题。

DESCRIPTION (provided by applicant): The plasma membrane Na-H exchanger NHE1 has an established role in intracellular pH homeostasis. Work during the past funding period revealed that NHE1 also acts as an anchor for actin filaments by binding directly the ERM family of actin-binding proteins. In fibroblasts, ERM binding retains the localization of NHE1 at the leading edge of lamellipodia and is necessary for the assembly of actin filaments in membrane protrusions. The coordinate functions of NHE1 in ion translocation and actin anchoring are necessary for directed cell migration and are required for cell polarity, actin polymerization, and remodeling of focal adhesions. A novel DNHE1 in Drosophila that binds an ERM ortholog, and a novel DdNHE1 in Dictyostelium discoideum that is necessary for cell polarity and chemotaxis were identified, suggesting that actin anchoring by NHE1 and a requirement for NHE1 in directional cell movement are evolutionarily conserved. The objective of the current proposal is to determine how NHE1 regulates directional cell movement by investigating the hypothesis that its two functions act coordinately to generate an asymmetrically localized H+ efflux that is necessary for maintaining polarity. Studies in Aim 1 will determine how NHE1 regulates membrane protrusions and actin polymerization in mammalian fibroblasts by asking how localized H+ fluxes by NHE1 promote cell protrusion, the formation of actin free barbed ends, and the spatially restricted activation of Rac and Cdc42 at the leading edge of migrating cells. Whether NHE1 acts in a self-generating positive feedback loop at the leading edge of migrating cells will also be determined. Aim 2 addresses how the two functions of NHE1 in ion translocation and actin anchoring coordinately remodel cell-substrate adhesions in mammalian fibroblasts by asking whether actin anchoring by NHE1 is necessary for integrin activation or for subsequent steps in the assembly of focal adhesion-associated proteins, and whether ion translocation by NHE1 promotes focal adhesion disassembly through a pH dependent disruption of the integrin-cytoskeleton linkage. How newly formed focal adhesions contribute to the NHE1-dependent migratory phenotype will also be determined. Aim 3 focuses on why DdNHE1 is necessary for Dictyostelium chemotaxis by asking whether it generates localized H+ efflux, whether it maintains polarity by acting on signals at leading or trailing edges of the cell, and whether genetic screens identify DdNHE1 effectors mediating polarity and chemotaxis. Overall, this research plan addresses significant questions related to how polarity is developed and maintained and how spatially restricted signaling processes drive cell migration.