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.

可视化和阐明力对IV型胶原在发育中的作用 项目摘要 基底膜是高度保守的、致密的、片状的细胞外基质， 组织和器官。基底膜为发育中的组织提供机械强度，并使组织丧失或 基底膜成分的突变导致胚胎死亡，发育缺陷， 许多人类疾病。IV型胶原蛋白已被认为是胶原蛋白的关键结构组分。 提供机械稳定性的基底膜，胶原蛋白的突变导致破坏性的 影响多种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 胶原动力学和基底膜成分在体内复杂的脊椎动物组织，然而， 目前还不清楚IV型胶原是如何在基底膜中组装的，以及它是否直接承受载荷。我们 我做了C。表达功能性GFP、Venus和mCherry标记形式的胶原蛋白的线虫菌株 以及大多数其他基底膜蛋白和受体。我们还开发了一种可光转换的 Dendra标记的胶原蛋白应变，以光学突出显示和跟踪沉积在基底膜中的胶原蛋白。C. 秀丽线虫编码所有主要的基底膜成分，每个家族只有一个基因， 使其成为解剖IV型胶原功能和基底膜的强有力的实验模型 体内调节。梭秀丽线虫咽被包裹在BM中，并且是快速生长的收缩器官， 启动胚胎泵送。咽球后端是食物研磨的部位， 高机械活性。梭线虫咽部首先覆盖有基底膜， 胚胎发生，在咽泵之前。我们发现IV型胶原蛋白最初定位于 在胚胎中，这些细胞均匀地围绕在发育中的咽周围，但是在咽开始抽吸之后， 特别是在终端灯泡周围。IV型胶原蛋白的损失导致咽部泵血， 形态缺陷，表明胶原蛋白在咽部发育和功能中的关键作用。目标 该建议的一个重要方面是联合收割机IV型胶原蛋白的活细胞成像与遗传分析、RNAi敲除和 力操纵和开发一种新的胶原荧光能量转移（FRET）为基础的力 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 生长的咽膜和机械力在胶原蛋白募集中的作用;和（2）可视化 原位IV型胶原的负载，并确定胶原是否优先募集到高密度区域的BM中。 机械活动。这些研究与NIH的使命相关，因为它们将导致新的机制见解 研究BM中IV型胶原蛋白的功能、调节和组装，从而更好地理解 IV型胶原蛋白缺陷导致的人类发育障碍的基础。