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

可视化并阐明力对IV型胶原在发育中的作用 项目总结 基底膜是高度保守、致密、片状的细胞外基质，包围着大多数 组织和器官。基底膜为发育中的组织提供机械强度， 基底膜成分的突变会导致胚胎死亡、发育缺陷和 无数的人类疾病。IV型胶原被认为是 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 影响多种动态生长和机械活动的组织的发育障碍，包括 血管、肌肉和大脑。由于很难对类型IV进行可视化和实验检查 然而，在活体复杂的脊椎动物组织中，胶原动力学和基底膜成分 尚不清楚IV型胶原是如何在基底膜上组装的，以及它是否直接承受负荷。我们 已经制造出表达功能性GFP、Venus和mCherry标记版本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。我们还开发了一款照相转换车 Dendra标记的胶原菌株，以光学突出和跟踪沉积在基底膜中的胶原。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， 使其成为解剖IV型胶原功能和基底膜的有力实验模型 体内调节。线虫咽部被包裹在BM中，是一种快速生长的收缩器官， 提升者将胚胎注入体内。后末端咽球是食物研磨的地方，一个区域 机械活动性高。线虫咽部在早期首先被基底膜覆盖 胚胎发生，在咽部抽血之前。我们发现IV型胶原最初定位于 在胚胎发育的咽部周围均匀分布，但在咽部启动泵送后，变成 特别是在终端球茎周围富含。IV型胶原丢失会导致咽部泵血和 形态缺陷，表明胶原蛋白在咽部发育和功能中起着关键作用。目标是 这一建议的核心是将IV型胶原的活细胞成像与遗传分析、RNAi敲除以及 力的操纵和基于胶原荧光能量转移(FRET)的新型力的发展 传感器：(1)阐明基底胶原蛋白加成的生化和生物物理机制 生长咽膜和机械力在胶原蛋白募集中的作用；和(2)可视化 在原位检测IV型胶原的负载，并确定在高密度区胶原是否优先被吸收到骨髓。 机械活动。这些研究与美国国立卫生研究院的任务相关，因为它们将带来新的机械论见解 了解骨髓间质干细胞中IV型胶原的功能、调节和组装，从而更好地了解 由IV型胶原缺陷引起的人类发育障碍的基础。

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.