Visualizing and Elucidating the Role of Force on Type IV Collagen in Development

可视化和阐明力对 IV 型胶原蛋白发育的作用

• 批准号: 9324296
• 负责人: David R Sherwood
• 金额: $ 18.61万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324296
• 关键词: Affect; Area; Basement membrane; Biochemical; Biophysical Process; Biosensor; Brain; Caenorhabditis elegans; Cells; Collagen; Collagen Type IV; Complex; Defect; Deposition; Development; Disease; Embryo; Embryonic Development; Energy Transfer; Experimental Models; Extracellular Matrix; Family; Fluorescence; Food; Genes; Genetic; Genetic study; Goals; Health; Human; Image; Imagery; In Situ; Lung; Mechanics; Membrane Proteins; Mission; Modeling; Morphology; Mus; Muscle; Mutation; National Institute of Child Health and Human Development; Optics; Organ; Organogenesis; Pathogenesis; Pathology; Pharyngeal structure; Process; Protein Family; Pump; RNA Interference; Recruitment Activity; Regulation; Research; Role; Site; Thinness; Tissues; United States National Institutes of Health; Ursidae Family; Venus; Work; base; crosslink; developmental disease; extracellular; genetic analysis; human disease; in vivo; in vivo Model; insight; knock-down; live cell imaging; mechanical force; mechanical load; mechanical properties; novel therapeutic intervention; novel therapeutics; rapid growth; receptor; response; scaffold; sensor; tool; vertebrate embryos

Visualizing and elucidating the role of force on type IV collagen in development PROJECT SUMMARY Basement membranes are highly conserved, dense, sheet-like extracellular matrices that surround most tissues and organs. Basement membranes provide mechanical strength to developing tissues, and loss or mutations in basement membrane components results in embryonic lethality, developmental defects, and numerous human diseases. Type IV collagen has been proposed to be the key structural component in basement membranes that provides mechanical stability, and mutations in collagen result in devastating developmental disorders that affect multiple dynamically growing and mechanically active tissues, including the vasculature, muscles, and brain. Owing to the difficulty of visualizing and experimentally examining type IV collagen dynamics and basement membrane components in complex vertebrate tissues in vivo, however, it is unknown how type IV collagen is assembled in basement membranes and whether it directly bears load. We have made C. elegans strains expressing functional GFP, Venus, and mCherry tagged versions of collagen and most other basement membrane proteins and receptors. We have also developed a photoconvertible Dendra-tagged collagen strain to optical highlight and track collagen deposited in basement membrane. C. elegans encodes all major basement membrane components with only a single gene representing each family, making it a powerful experimental model to dissect type IV collagen function and basement membrane regulation in vivo. The C. elegans pharynx is encased in a BM and is a rapidly growing contractile organ that initiates pumping in the embryo. The posterior terminal pharyngeal bulb is the site of food grinding, a region of high mechanical activity. The C. elegans pharynx is first covered with basement membrane during early embryogenesis, prior to pharyngeal pumping. We have found that type IV collagen is initially localized uniformly around the developing pharynx in the embryo, but after the pharynx initiates pumping, becomes enriched specifically around the terminal bulb. Loss of type IV collagen leads to pharyngeal pumping and morphological defects, indicating a critical role for collagen in pharyngeal development and function. The goal of this proposal is to combine live-cell imaging of type IV collagen with genetic analysis, RNAi knockdown, and force manipulations and development of a new collagen Fluorescence Energy Transfer (FRET)-based force sensor to: (1) Elucidate the biochemical and biophysical mechanisms of collagen addition to the basement membrane of the growing pharynx and the role of mechanical force in collagen recruitment; and (2) Visualize the load on type IV collagen in situ and determine if collagen is preferentially recruited to BM in areas of high mechanical activity. These studies are relevant to NIH's mission as they will lead to new mechanistic insights into the function, regulation, and assembly of type IV collagen in BMs, thus allowing a better understanding of the basis of human developmental disorders that result from defects in type IV collagen.

可视化并阐明力对 IV 型胶原蛋白发育过程的作用 项目概要 基底膜是高度保守、致密、片状细胞外基质，包围着大多数细胞。 组织和器官。基底膜为发育中的组织提供机械强度，并且损失或 基底膜成分的突变会导致胚胎死亡、发育缺陷和 多种人类疾病。 IV 型胶原蛋白已被认为是关键的结构成分 提供机械稳定性的基底膜和胶原蛋白的突变会导致毁灭性的后果 影响多个动态生长和机械活性组织的发育障碍，包括 脉管系统、肌肉和大脑。由于难以观察和实验检查 IV 型 然而，体内复杂脊椎动物组织中的胶原动力学和基底膜成分 目前尚不清楚IV型胶原蛋白如何在基底膜中组装以及是否直接承受负荷。我们 已制作出表达功能性 GFP、Venus 和 mCherry 标记版本胶原蛋白的线虫菌株 以及大多数其他基底膜蛋白和受体。我们还开发了光电转换器 Dendra 标记的胶原蛋白菌株可光学突出显示并跟踪沉积在基底膜中的胶原蛋白。 C. 线虫编码所有主要的基底膜成分，每个家族只有一个基因， 使其成为剖析 IV 型胶原蛋白功能和基底膜的强大实验模型 体内调节。线虫的咽部被包裹在 BM 中，是一个快速生长的收缩器官， 开始泵入胚胎。后终末咽球是研磨食物的部位，是 高机械活性。线虫的咽部在早期阶段首先被基底膜覆盖。 胚胎发生，在咽部泵送之前。我们发现 IV 型胶原蛋白最初是局部化的 均匀地围绕胚胎中正在发育的咽部，但在咽部开始泵送后，变成 特别在终端灯泡周围丰富。 IV 型胶原蛋白的损失导致咽部泵血和 形态缺陷，表明胶原蛋白在咽部发育和功能中发挥着关键作用。目标 该提案的目的是将 IV 型胶原蛋白的活细胞成像与遗传分析、RNAi 敲除和 力操纵和新型胶原荧光能量转移（FRET）力的开发 传感器：(1) 阐明胶原蛋白添加到基底的生化和生物物理机制 生长中的咽膜和机械力在胶原蛋白募集中的作用； (2) 可视化 原位 IV 型胶原蛋白的负载，并确定胶原蛋白是否优先招募到高浓度区域的 BM 机械活动。这些研究与 NIH 的使命相关，因为它们将带来新的机制见解 深入了解 BM 中 IV 型胶原蛋白的功能、调节和组装，从而更好地了解 IV 型胶原蛋白缺陷导致人类发育障碍的基础。