Illuminating apical extracellular matrix structure and biogenesis

阐明顶端细胞外基质结构和生物发生

• 批准号: 10654029
• 负责人: Andrew D Chisholm
• 金额: $ 19.5万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA CRUZ
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10654029
• 关键词: 3-Dimensional; Animals; Apical; Architecture; Area; Biogenesis; Bioinformatics; Biology; C. elegans genome; CRISPR/Cas technology; Caenorhabditis elegans; Cells; Communities; Complex; Coupled; Data; Degenerative polyarthritis; Development; Disease; Dissection; Electron Microscopy; Environment; Epithelium; Extracellular Matrix; Extracellular Matrix Proteins; Fibrosis; Functional disorder; Genes; Goals; Human Development; Image; Individual; Investigation; Knock-in; Knowledge; Labyrinth; Libraries; Life; Lighting; Location; Malignant Neoplasms; Methods; Microscopy; Modification; Molecular; Molecular Genetics; Nanostructures; Nematoda; Pathology; Pattern; Phenotype; Play; Protein Family; Proteins; Proteomics; Protocols documentation; Reagent; Research; Resolution; Resources; Role; Standardization; Structure; Surface; System; Technology; Testing; Validation; Work; design; extracellular; flexibility; fluorophore; gene function; genetic resource; genome editing; human disease; improved; in vivo; mechanotransduction; nanoscale; novel; submicron; superresolution imaging; superresolution microscopy; three dimensional structure; transcriptomics

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.

顶端细胞外矩阵（AECM）与所有上皮相关，对于动物的生命至关重要。 它们形成了针对生物和非生物（身体损害）挑战的外部保护层 环境。与AECM功能障碍相关的人类疾病表现出一系列病理 包括但不限于纤维化，骨关节炎和癌症。 AECM还扮演重要的结构角色 并且在机械转导至关重要的情况下，例如脊椎动物内耳。但是，总的来说我们缺乏 对分子层面AECM的3D组织的理解，包括其动态 发展和疾病状态。这项工作的前提是创建一组系统的荧光 标记的内源性顶尖细胞外基质（AECM）组件，然后通过表征进一步 使用超分辨率显微镜，以揭示如何将这些分子组织到纳米级模式中 体内。 AECM与所有上皮相关，对于动物生活至关重要。 AECM的标志是他们 能够形成以亚微米量表图案的复杂的3D结构。这样的纳米体系结构有 通过电子显微镜在各种系统中描述，但在大多数系统中都缺乏分子 相关。尽管它们的重要性很重要，但AECM的组成和生物发生仍然很差 理解，部分原因是AECM生物学的许多方面只能在体内完全研究。而且， AECM生物学中的批判知识差距是分泌分子在细胞外聚集 以纳米级图案形式形成和维护复杂的3D结构的环境。基本功能 AECM组件的空间和临时定位仍然对 挑战。基因编辑技术的障碍性的提高已使内源性标记 蛋白质（敲蛋白）；加上高级成像，敲击蛋白标记的蛋白质可以严格分析 AECM纳米级结构和生物发生。我们将利用这些技术进步来开发 在秀丽隐杆线虫中表达荧光标记的AECM蛋白的标准化菌株，AECM 工具包’。这些试剂不仅会揭示AECM生物学的新颖方面，而且还将建立原则 用于在其他系统中对AECM进行标记和实时成像。我们的工具包将开放纳米级领域 AECM对分子遗传解剖的构图，并导致对AECM的了解 疾病。我们将使用CRISPR/CAS9基因组编辑生成一组100张敲击菌株，并使我们的 工具包和协议广泛可用，为社区生成关键资源。我们将确定 AECM组件的定位，生物发生和纳米级结构。我们将使用现场成像 确定AECM组件是如何分泌的，并在复杂的3D结构中组装成复杂的3D结构 发展。我们的AECM工具包将阐明AECM组装和动态，并填补关键知识 AECM结构和发展和疾病的动态方面的差距。