Illuminating apical extracellular matrix structure and biogenesis

顶端细胞外基质（aECMs）与所有上皮细胞相关，并且是动物生命所必需的。 它们形成了一个外部保护层，可以抵御来自生物和非生物（物理损害）的挑战。 环境与aECM功能障碍相关的人类疾病表现出一系列病理学 包括但不限于纤维化、骨关节炎和癌症。aECM也发挥重要的结构作用 并且在机械传导中，例如在脊椎动物内耳中是关键的。但总的来说，我们缺乏 在分子水平上理解aECM的3D组织，包括其在 发展和疾病状态。这项工作的前提是建立一套系统的荧光 标记的内源性顶端细胞外基质（aECM）组分，并进一步通过表征 使用超分辨率显微镜，揭示这些分子是如何组织成纳米级图案的， vivo. aECM与所有上皮细胞相关，并且是动物生命所必需的。aECM的一个特点是它们 形成亚微米级复杂3D结构图案的能力。这种纳米级架构具有 已经在各种系统中通过电子显微镜进行了描述，但大多数情况下缺乏分子 相互关联尽管它们的重要性得到了公认，但aECM的组成和生物起源仍然很差 这是因为aECM生物学的许多方面只能在体内进行充分研究。而且 aECM生物学中的关键知识缺口是分泌的分子如何在细胞外组装 环境来形成和维持具有纳米级图案化的复杂3D结构。基本特征的 aECM成分空间和时间定位仍然知之甚少，因为这些 挑战基因编辑技术日益增加的易处理性使得能够标记内源性基因。 蛋白质（knockins）;加上先进的成像，敲入标记的蛋白质允许严格的分析 aECM纳米级结构和生物发生。我们将利用这些技术进步， 在C. elegans，aECM 工具箱这些试剂不仅将揭示aECM生物学的新方面， 用于在其他系统中标记和实时成像aECM。我们的工具包将打开纳米尺度的领域 aECM模式化到分子遗传学解剖，并导致对aECM的深入了解 疾病我们将使用CRISPR/Cas9基因组编辑产生一组100个敲入菌株，并使我们的 广泛提供工具包和协议，为社区提供关键资源。我们将确定 aECM组分的定位、生物发生和纳米级结构。我们将使用实时成像， 确定aECM成分如何分泌并组装成复杂的3D结构， 发展我们的aECM工具包将阐明aECM组装和动力学，并将填补关键知识 aECM结构和发展与疾病动态的差距。

Apical extracellular matrices (aECMs) are associated with all epithelia and are essential for animal life. They form an outer protective layer against biotic and abiotic (physical damage) challenges from the environment. Human diseases associated with aECM dysfunction manifest a range of pathologies including, but not limited to, fibrosis, osteoarthritis, and cancer. aECMs also play important structural roles and are critical in mechanotransduction, such as in the vertebrate inner ear. However, in general we lack an understanding of the 3D organization of aECM at a molecular level, including its dynamics in development and disease states. The premise for this work is to create a systematic set of fluorescently tagged endogenous apical extracellular matrix (aECM) components, and further, through characterization using super-resolution microscopy, to reveal how these molecules are organized into nanoscale patterns in vivo. aECMs are associated with all epithelia and are essential for animal life. A hallmark of aECMs is their ability to form complex 3D structures patterned at the sub-micron scale. Such nanoscale architectures have been described by electron microscopy in a variety of systems but have for the most part lacked molecular correlates. Despite their recognized importance, the composition and biogenesis of aECMs remain poorly understood, partly because many aspects of aECM biology can only be fully studied in vivo. Moreover, a critical knowledge gap in aECM biology is how secreted molecules assemble in the extracellular environment to form and maintain complex 3D structures with nanoscale patterning. Basic features of the spatial and temporal localization of aECM components remain poorly understood because of these challenges. The increasing tractability of gene editing technology has enabled tagging of endogenous proteins (knockins); coupled with advanced imaging, knockin tagged proteins allow rigorous analysis of aECM nanoscale structure and biogenesis. We will exploit these technical advances to develop a standardized set of strains expressing fluorescently tagged aECM proteins in C. elegans, the ‘aECM toolkit’. These reagents will not only reveal novel aspects of aECM biology but will also establish principles for tagging and live imaging of the aECM in other systems. Our toolkit will open up the field of nanoscale aECM patterning to molecular genetic dissection, as well as leading to enhanced understanding of aECM in disease. We will generate a set of 100 knockin strains using CRISPR/Cas9 genome editing and make our toolkit and protocols widely available, generating a critical resource for the community. We will determine localization, biogenesis, and nanoscale architecture of aECM components. We will use live imaging to determine how aECM components are secreted and assemble into complex 3D structures during development. Our aECM toolkit will illuminate aECM assembly and dynamics and will fill critical knowledge gaps in aECM structure and dynamics in development and diseases.