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

抗整合素纳米线作为确定跨上皮通透性调节机制的平台

• 批准号: 9760829
• 负责人: Raven Peterson
• 金额: $ 4.5万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9760829
• 关键词: Actin-Binding Protein; Actinin; Actins; Address; Alexa594; Apical; Binding Proteins; Biological Assay; CD47 gene; Cell membrane; Cells; Cultured Cells; Cytoskeleton; Data; Dyes; Electrophysiology (science); Environment; Epithelial; Epithelial Cells; Epithelium; Filipin; Film; Genistein; Goals; Immunofluorescence Immunologic; Immunofluorescence Microscopy; Integrin Binding; Integrins; Ion Transport; Ions; Label; Ligation; Link; Measurement; Measures; Mediating; Membrane Proteins; Methods; Microfilaments; Microscopy; Modeling; Molecular; Morphology; Nanostructures; Organ; Pathway interactions; Pattern; Permeability; Physiological; Positioning Attribute; Regulation; Resistance; Resolution; Role; Route; Scaffolding Protein; Series; Site; Stress Fibers; Structure; System; Talin; Techniques; Testing; Tight Junctions; Work; antibody conjugate; base; blebbistatin; electric impedance; experience; experimental study; inhibitor/antagonist; jasplakinolide; macromolecule; monolayer; nanowire; novel; polycaprolactone; recruit; screening; small molecule; solute; tensin; transcytosis; zonula occludens-1 protein

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.

抽象的 上皮形成选择性障碍，使器官隔离并保护它们免受外部环境的侵害 上皮屏障渗透性具有两个调节的组件， 由转胞胞病介导的跨细胞路径和细胞 - 细胞接触部位之间的细胞细胞路径 由紧密连接（TJ）调节。我们先前确定了通过接触上皮细胞的刺激 带有纳米结构膜（NSF）的顶质膜膜增加了对大型的透化性 大分子（例如Alexa-594）通过跨细胞和细胞细胞途径标记为FAB。 几条证据确定了顶尖局部1整联蛋白在NSF能力中的潜在作用 增加底物渗透性。但是，使用NSF的缺点是，它们通过大量参与而行动 顶端质膜的异质斑块使得难以精确定义分子 将特定表面蛋白与上皮屏障功能变化联系起来的机制。特别是 研究1整联蛋白是否直接参与屏障渗透性的增加，我们开发了一种抗 整联蛋白纳米线系统由抗-1整合素抗体组成，该抗体与功能化的多碳酸酯结合 纳米线。这些抗整合蛋白纳米线是一个平台，可以特异性地局部局部1整合素 确定整联蛋白刺激是否具有调节上皮屏障功能的能力。处理 具有抗整合蛋白纳米线的上皮单层显着提高 单层并提高了跨偏光单层中Alexa-594 Fab的透射速率。这些 功能效应与纳米线诱导的TJ脚手架蛋白定位变化有关 Zonula occludens-1（ZO-1）和结构蛋白相关的肌动蛋白结合蛋白塔林，以及重新排列 肌动蛋白细胞骨架从应力纤维变成更皮质的组织模式。为了定义 整联蛋白介导的上皮渗透性调节的作用机制我们将检验以下假设。 抗整合蛋白纳米线的整合素聚类通过整合素的变化增加了渗透性 相关的肌动蛋白结合蛋白通过以下目标导致细胞骨架重塑。在AIM 1中， 我们将测量整联蛋白后对可溶性渗透性的跨细胞和细胞细胞的贡献 通过测量抗整合蛋白纳米线处理如何引起TJ的结构和功能变化来刺激 和跨细胞渗透性。在AIM 2中，我们将确定整联蛋白介导的渗透性变化是否驱动 通过改变与整联蛋白相关肌动蛋白结合蛋白的募集的变化并衡量其对肌动蛋白的影响 组织。我们还将评估整联蛋白相关肌动蛋白结合蛋白和肌动蛋白细胞骨架的变化是否变化 组织是整联蛋白介导的渗透性变化的要求。该提议的目的是 确定新的机制，顶尖局部整联蛋白调节上皮屏障功能。