Anti-integrin nanowires as a platform to determine mechanisms regulating transepithelial permeability

摘要 上皮细胞形成选择性屏障，将器官隔开并保护它们免受外部环境的影响 从而实现了特殊的生理功能。上皮屏障通透性有两个调节成分， 跨细胞传递的跨细胞途径和细胞与细胞接触部位之间的细胞旁途径 受紧密连接(TJ)调节。我们之前确定了通过接触刺激上皮细胞 带有纳米结构膜(NSFs)的根尖质膜可显著增加跨上皮细胞的通透性 大分子，如Alexa-594标记的Fab，通过跨细胞和细胞旁途径。 一些证据表明，顶端定位的1整合素在神经干细胞的能力中具有潜在的作用 增加基材的渗透性。然而，使用NSF的一个缺点是，它们的行动方式是与大型、 顶端质膜的异质性斑块使精确定义分子变得困难 将特定表面蛋白与上皮屏障功能变化联系起来的机制。因此，具体到 研究1整合素是否直接参与屏障通透性增加，我们开发了一种抗 由抗-1整合素抗体与功能化聚己内酯偶联的整合素纳米线系统 纳米线。这些反整合素纳米线作为特异性聚集顶部定位的1整合素的平台 确定整合素刺激是否具有调节上皮屏障功能的能力。治疗 含抗整合素纳米线的上皮单层显著降低人脐静脉内皮细胞的跨上皮阻力 并增加Alexa-594Fab跨极化单层的跨上皮通量速率。这些 功能效应与纳米线诱导的TJ支架蛋白定位的变化有关 封闭带-1(ZO-1)和整合素相关肌动蛋白结合蛋白talin，以及重排 肌动蛋白的细胞骨架由应力纤维转变为更具皮质化的组织模式。为了定义 整合素调节上皮通透性的作用机制我们将检验这一假说 整合素通过抗整合素纳米线聚集，通过整合素的改变增加通透性。 相关的肌动蛋白结合蛋白通过以下目的导致细胞骨架重塑。在目标1中， 我们将测量整合素后跨细胞和细胞旁对溶质渗透性的贡献 测量抗整合素纳米线治疗如何引起TJs结构和功能变化的刺激 和跨细胞渗透性。在目标2中，我们将确定整合素介导的通透性变化是否受到驱动 通过改变整合素相关肌动蛋白结合蛋白的募集并测量其对肌动蛋白的影响 组织。我们还将评估整合素相关肌动蛋白结合蛋白和肌动蛋白细胞骨架的变化 组织是对整合素介导的通透性变化的要求。这项提议的目标是 识别顶部定位的整合素调节上皮屏障功能的新机制。

ABSTRACT Epithelia form selective barriers that compartmentalize organs and protect them from external environments which allows for specialized physiologic function. Epithelial barrier permeability has two regulated components, the transcellular path mediated by transcytosis, and the paracellular path between cell-cell contact sites regulated by tight junctions (TJs). We previously determined that stimulation of epithelial cells by contact between the apical plasma membrane with nanostructured films (NSFs) increases transepithelial permeability to large macromolecules, such as Alexa-594 labeled Fab, through both the transcellular and paracellular pathways. Several lines of evidence identified a potential role for apically localized 1 integrin in the ability of NSFs to increase substrate permeability. However, a drawback to using NSFs is that they act by engaging large, heterogeneous patches of the apical plasma membrane making it difficult to precisely define molecular mechanisms linking specific surface proteins to changes in epithelial barrier function. Thus, to specifically investigate whether 1 integrin is directly involved in increased barrier permeability, we developed an anti- integrin nanowire system consisting of anti-1 integrin antibodies conjugated to functionalized polycaprolactone nanowires. These anti-integrin nanowires served as a platform to specifically cluster apically localized 1 integrin to determine whether integrin stimulation has the capacity to regulate epithelial barrier function. Treatment of epithelial monolayers with anti-integrin nanowires significantly decreased the transepithelial resistance of the monolayer and increased the rate of transepithelial flux of Alexa-594 Fab across polarized monolayers. These functional effects were associated with nanowire-induced changes in the localization of the TJ scaffolding protein zonula occludens-1 (ZO-1) and the integrin-associated actin binding protein talin, as well as rearrangement of the actin cytoskeleton from stress fibers into a more cortical pattern of organization. In order to define the mechanisms of action for integrin-mediated regulation of epithelial permeability we will test the hypothesis that integrin clustering by anti-integrin nanowires increases permeability through changes in integrin- associated actin binding proteins leading to cytoskeletal remodeling through the following Aims. In Aim 1, we will measure the transcellular and paracellular contributions to solute permeability following integrin stimulation by measuring how anti-integrin nanowire treatment causes structural and functional changes in TJs and transcellular permeability. In Aim 2, we will determine if integrin mediated changes in permeability are driven by changes in the recruitment of integrin-associated actin binding proteins and measure their impact on actin organization. We will also assess if changes in integrin-associated actin binding proteins and actin cytoskeleton organization are requirements for integrin mediated changes in permeability. The goal of this proposal is to identify novel mechanisms whereby apically localized integrins regulate epithelial barrier function.