Unveiling Functional Roles of Apical Surface Interactions Between Opposing Cell Layers

揭示相对细胞层之间顶端表面相互作用的功能作用

• 批准号: 10629101
• 负责人: Hongxia Fu
• 金额: $ 44.13万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10629101
• 关键词: 3-Dimensional; Ablation; Adhesives; Affect; Antibodies; Apical; Architecture; Area; Atomic Force Microscopy; Biological Phenomena; Biology; Biophysics; Blood Vessels; Cell Adhesion; Cell Communication; Cell Line; Cell surface; Cells; Charge; Chemicals; Cilia; Data; Devices; Dimensions; Duct (organ) structure; Electrostatics; Exhibits; Future; Gene Expression Profile; Genetic Engineering; Geometry; Goals; Investigation; Knock-out; Literature; Measures; Membrane Glycoproteins; Methodology; Methods; Microfluidics; Microscope; Modeling; Monitor; Mutation; Organ; Organelles; Perfusion; Physiological; Physiology; Positioning Attribute; Property; Resistance; Role; Sensory; Side; Signal Pathway; Signal Transduction; Speed; Static Electricity; Structure; Surface; Testing; Tissue-Specific Gene Expression; Tissues; Trees; Tube; biophysical tools; cell dimension; design; experience; in vivo; innovation; meter; new technology; novel; podocalyxin; response; sensor; sialomucins; therapeutic development; 3-Dimensional; Ablation; Adhesives; Affect; Antibodies; Apical; Architecture; Area; Atomic Force Microscopy; Biological Phenomena; Biology; Biophysics; Blood Vessels; Cell Adhesion; Cell Communication; Cell Line; Cell surface; Cells; Charge; Chemicals; Cilia; Data; Devices; Dimensions; Duct (organ) structure; Electrostatics; Exhibits; Future; Gene Expression Profile; Genetic Engineering; Geometry; Goals; Investigation; Knock-out; Literature; Measures; Membrane Glycoproteins; Methodology; Methods; Microfluidics; Microscope; Modeling; Monitor; Mutation; Organ; Organelles; Perfusion; Physiological; Physiology; Positioning Attribute; Property; Resistance; Role; Sensory; Side; Signal Pathway; Signal Transduction; Speed; Static Electricity; Structure; Surface; Testing; Tissue-Specific Gene Expression; Tissues; Trees; Tube; biophysical tools; cell dimension; design; experience; in vivo; innovation; meter; new technology; novel; podocalyxin; response; sensor; sialomucins; therapeutic development

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.

项目摘要 在相反的三维体系结构中细胞之间产生的顶部表面相互作用（ASI） 在组织结构中相对常见，包括各种不同的器官和阶段中的容器和管， 但是，这些相互作用的功能或机制知之甚少。该提议的目的是阐明 ASIS在组织结构和反应中的作用是细胞细胞相互作用的探索尺寸不足。 根据我们的数据和文献，我们假设近距离ASI（<1 µm）受 膜糖蛋白之间的静电电荷相互作用，而远距离ASIS（1-20 µM）功能 通过原发性纤毛，从细胞表面延伸到组织信号通路。 开发准确的方法来衡量和表征整体之间产生的力 几何分离的细胞板，我们设计了一种新型方法，称为双层分子 力显微镜（BIFM）诱导并测量两个相对表面之间的ASIS。 BIFM将应用于 测量两个细胞层之间产生的力，它们从相对侧相互接近。 对于近距离ASIS，我们预测影响静电电荷相互作用的化学物质会调节 力反应。 PODXL中具有基因敲除突变的细胞表，编码根尖的唾液素蛋白（Podocalyxin） 具有拟议的抗粘附特性，将与静电降低成比例地存在较低的耐药力 充电排斥。在热发育中靶向足肠蛋白的抗体也将进行评估。 对于远程ASIS，我们将确定原发性纤毛，具有感觉和的天线样细胞器的作用 信号功能。使用我们的BIFM设备，我们将诱导睫状ASIS并评估它们对信号的影响。作为 负面对照，我们将采用一般设计为消融原发性纤毛的细胞系（KIF3A - / - 或KIF3B - / - ）。我们将修改我们的设备以使微流体流动以在两张纸之间进行灌注 BIFM内的单元格在可调速度下，以评估在物理环境中的流量开始/停止，并监视 信号活动的变化。这些研究将揭示纤毛如何充当ASI传感器。 为了验证体内的发现，我们将分析表现出一系列顶端的物理组织结构 表面相互作用，重点是养育网络，例如导管树和血管丛。表达 足小蛋白或纤毛将与几何特性和差异基因表达模式相关。 总而言之，我们的项目将为理解ASIS提供新颖的概念和技术进步 组织结构和生理学的新颖维度。横切影响包括（1）揭示功能 近距离和远程ASI的角色； （2）建立一种新型的生物物理设备来测量 细胞表之间的相互作用； （3）细胞粘合剂和信号传导的测试机制。我们先前的经验 在建模和开发生物物理工具时，我们可以很好地定位我们的成功。这些活动将 建立一个具有基本重要性的未来投资的创新新领域。