Unveiling Functional Roles of Apical Surface Interactions Between Opposing Cell Layers

项目摘要 在相对的三维结构中细胞之间产生的顶端表面相互作用（ASI） 在组织结构中相对常见，包括各种不同器官和阶段中的血管和管道， 但对这些相互作用的功能或机制知之甚少。这项提案的目的是阐明 ASIs在组织结构和反应中的作用，作为细胞-细胞相互作用的一个未充分探索的维度。 根据我们的数据和文献，我们假设近距离ASI（< 1 µm）由以下因素控制： 膜糖蛋白之间的静电荷相互作用，而长程ASI（1-20 µm）起作用 通过从细胞表面向上延伸的初级纤毛来组织信号通路。 制定准确的方法来衡量和描述整体之间产生的力量 我们设计了一种新的方法，称为双层分子间 力显微镜（BIFM）用于诱导和测量两个相对表面之间的ASI。BIFM将应用于 测量当两个细胞层从相对侧彼此接近时在两个细胞层之间产生的力。 对于近距离ASI，我们预测影响静电荷相互作用的化学物质将调节 强制反应在PODXL中具有敲除突变的细胞片层，编码顶端唾液粘蛋白（足糖萼蛋白） 具有所提出的抗粘附性能的导电材料将表现出与减少的静电成比例的较低的阻力， 电荷排斥还将评估治疗开发中靶向足糖萼蛋白的抗体。 对于远距离ASI，我们将确定初级纤毛、触角样细胞器与感觉和神经元的作用。 信号功能。使用我们的BIFM设备，我们将诱导睫状体ASI并评估它们对信号传导的影响。作为 作为阴性对照，我们将使用我们已经基因工程化的细胞系来消融初级纤毛（KIF 3A-/- 或KIF 3B-/-）。我们将进一步修改我们的装置，以使微流体流能够在两个薄片之间灌注。 以可调节的速度在BIFM内的细胞，以评估生理环境中的流动开始/停止，并监测 信号活动的变化。这些研究将揭示纤毛如何作为ASI传感器。 为了在体内验证研究结果，我们将分析表现出一系列根尖细胞的生理组织结构。 表面相互作用，重点是树枝状网络，如导管树和血管丛。表达 足糖萼蛋白或纤毛将与几何性质和差异基因表达模式相关。 总之，我们的项目将为理解ASIs提供新的概念和技术进步， 组织结构和生理学的新维度。跨领域影响包括：（1）揭示职能 近距离和远距离ASI的作用;（2）建立一种新的生物物理设备来测量 细胞片层之间的相互作用;和（3）测试细胞粘附和信号传导的机制。我们以往的经验 在建模和开发生物物理工具中的作用使我们能够取得成功。总的来说，这些活动将 为今后研究建立一个具有根本重要性的创新新领域。

PROJECT SUMMARY Apical surface interactions (ASIs) arising between cells in opposing three dimensional architectures are relatively common in tissue structures, including vessels and tubes in a variety of different organs and stages, but little is known about the function or mechanisms of these interactions. The goal of this proposal is to illuminate the role of ASIs in tissue architecture and responses as an under-explored dimension of cell-cell interactions. Based on our data and the literature, we hypothesize that close-range ASIs (< 1 µm) are governed by electrostatic charge interactions between membrane glycoproteins, while long-range ASIs (1-20 µm) function through primary cilia which extend up from the cell surface to organize signaling pathways. To develop accurate methodologies to measure and characterize the forces arising between whole sheets of cells with geometrical separation, we have designed a novel method called Bilayer Intermolecular Force Microscopy (BIFM) to induce and measure ASIs between two opposite surfaces. BIFM will be applied to measure the force generated between two cell layers as they approach each other from opposite sides. For close-range ASIs, we predict that chemicals affecting electrostatic charge interactions will modulate force-response. Cell sheets with knockout mutations in PODXL, encoding an apical sialomucin (podocalyxin) with proposed anti-adhesive properties, will exhibit lower resistance force in proportion to reduced electrostatic charge repulsion. Antibodies targeting podocalyxin, in therapeutic development, will also be assessed. For long-range ASIs, we will determine the role of primary cilia, antenna-like organelles with sensory and signaling functions. Using our BIFM device, we will induce ciliary ASIs and assess their effects on signaling. As a negative control, we will employ cell lines that we have genetically engineered to ablate primary cilia (KIF3A-/- or KIF3B-/-). We will furthermore modify our device to enable microfluidic flow to perfuse between two sheets of cells within the BIFM at adjustable speed, to assess flow start/stop in a physiological context, and monitored for changes in signaling activity. These studies will reveal how cilia serve as ASI sensors. To validate findings in vivo, we will analyze physiological tissue structures exhibiting a range of apical surface interactions, focusing on arborized networks such as ductal trees and blood vessel plexi. Expression of podocalyxin or cilia will be correlated with geometric properties and differential gene expression patterns. In summary, our project will provide novel conceptual and technical advances for understanding ASIs as a novel dimension in tissue architecture and physiology. Cross-cutting impact includes (1) revealing functional roles for both close-range and long-range ASIs; (2) establishing a novel biophysical device to measure interactions between cell sheets; and (3) testing mechanisms of cell adhesion and signaling. Our prior experience in modeling and developing biophysical tools positions us well to succeed. Collectively these activities will establish an innovative new area for future investigation, with fundamental importance.