Illuminating apical extracellular matrix structure and biogenesis

顶端细胞外基质(AECM)与所有的上皮细胞相关，是动物生命所必需的。 它们形成了一层外部保护层，抵御来自 环境。与AECM功能障碍相关的人类疾病表现出一系列的病理 包括但不限于纤维化、骨关节炎和癌症。AECM也发挥着重要的结构性作用 在机械转导中至关重要，例如在脊椎动物的内耳中。然而，总的来说，我们缺乏 在分子水平上理解AECM的三维组织，包括其在 发展和疾病状况。这项工作的前提是创建一套系统的荧光 标记的内源性心尖细胞外基质(AECM)成分，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 活着。AECM与所有的上皮细胞相关，是动物生命所必需的。AECM的一个标志是他们的 能够形成亚微米级图案的复杂3D结构。这种纳米级的架构具有 被电子显微镜描述在各种系统中，但在大多数情况下缺乏分子 相互关联。尽管认识到AECM的重要性，但AECM的组成和生物发生仍然很差 了解，部分原因是AECM生物学的许多方面只能在体内进行全面研究。此外，一个 AECM生物学中的关键知识缺口是分泌分子如何在细胞外组装 形成和维护具有纳米级图案的复杂3D结构的环境。的基本功能 由于这些原因，AECM组件的空间和时间定位仍然知之甚少 挑战。基因编辑技术日益增长的可操作性使内源基因的标记成为可能 蛋白质(敲打蛋白)；与先进的成像技术相结合，敲打标记的蛋白质可以对 AECM纳米结构与生物发生。我们将利用这些技术进步来开发一种 在线虫中表达荧光标记AECM蛋白的标准化菌株集 工具包‘。这些试剂不仅将揭示AECM生物学的新方面，而且还将建立原理 用于在其他系统中对AECM进行标记和实时成像。我们的工具包将开辟纳米尺度的领域 AECM模式到分子遗传解剖，以及导致对AECM在 疾病。我们将使用CRISPR/Cas9基因组编辑技术生成一组100个敲门菌，并使我们的 广泛可用的工具包和协议，为社区生成关键资源。我们将决定 AECM组件的本地化、生物发生和纳米级结构。我们将使用实时成像来 确定AECM组件是如何分泌并组装成复杂的3D结构的 发展。我们的AECM工具包将说明AECM的装配和动力学，并将填充关键知识 发展和疾病方面的AECM结构和动态方面的差距。

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.