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 上皮形成选择性屏障，分隔器官并保护它们免受外部环境的影响 其允许特殊的生理功能。上皮屏障通透性有两个受调节的组成部分， 由转胞吞作用介导的跨细胞路径，以及细胞与细胞接触位点之间的旁细胞路径 我们之前确定，上皮细胞的刺激是通过紧密连接（TJ）来调节的。 具有纳米结构膜（NSF）的顶端质膜可增加跨上皮通透性 大分子，例如 Alexa-594 标记的 Fab，通过跨细胞和细胞旁途径。 多项证据表明，顶部定位的 1 整合素在 NSF 的能力中具有潜在作用。 然而，使用 NSF 的一个缺点是它们通过接合大的、 顶端质膜的异质斑块使得很难精确定义分子 将特定表面蛋白与上皮屏障功能的变化联系起来的机制。 为了研究 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 整合素纳米线系统由与功能化聚己内酯缀合的抗1整合素抗体组成 这些抗整合素纳米线充当了特异性聚集顶端局部 1 整合素的平台。 以确定整合素刺激是否具有调节上皮屏障功能的能力。 具有抗整合素纳米线的上皮单层显着降低了跨上皮阻力 单层并增加了 Alexa-594 Fab 穿过极化单层的跨上皮通量速率。 功能效应与纳米线诱导的 TJ 支架蛋白定位变化相关 zonula occlusionns-1 (ZO-1) 和整合素相关肌动蛋白结合蛋白 talin，以及重排 肌动蛋白细胞骨架从应力纤维转变为更皮质的组织模式。 整合素介导的上皮通透性调节的作用机制，我们将检验以下假设： 抗整合素纳米线的整合素簇通过整合素的变化增加了渗透性 在目标 1 中，相关肌动蛋白结合蛋白导致细胞骨架重塑。 我们将测量整合素后跨细胞和旁细胞对溶质渗透性的贡献 通过测量抗整合素纳米线治疗如何引起 TJ 的结构和功能变化来刺激 在目标 2 中，我们将确定整合素介导的渗透性变化是否受到驱动。 通过整合素相关肌动蛋白结合蛋白募集的变化并测量其对肌动蛋白的影响 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是否发生变化。 组织是整合素介导的渗透性变化的要求。该提案的目标是 确定顶端局部整合素调节上皮屏障功能的新机制。