A Comprehensive Endogenous Basement Membrane Toolkit to Elucidate how Basement Membranes Stretch on Mechanically Active Tissues and Decline during Aging

一个全面的内源性基底膜工具包，用于阐明基底膜如何在机械活动组织上伸展和衰老过程中的衰退

• 批准号: 10430646
• 负责人: David R Sherwood
• 金额: $ 23.39万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-15 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10430646
• 关键词: Adult; Age; Aging; Alzheimer' s Disease; Animal Model; Animals; Basement membrane; Caenorhabditis elegans; Cardiovascular Diseases; Collagen; Collagen Type IV; Collection; Complex; Data; Databases; Defect; Diabetes Mellitus; Disease; Electron Microscopy; Enzymes; Excision; Experimental Models; Extracellular Matrix; Extracellular Matrix Proteins; Functional disorder; Gene Family; Genes; Genetic; Goals; Growth Factor; Health; Human; In Vitro; Kidney Diseases; Laminin; Mechanical Stress; Mechanics; Mediating; Membrane; Membrane Proteins; Methodology; Methods; Mission; Modeling; Molecular; Morphogenesis; Mus; Mutation; Nidogen; Optics; Organ; Ovulation; Pathology; Peptide Hydrolases; Photobleaching; Pilot Projects; Property; Protein Family; Public Health; RNA Interference; Reagent; Recovery; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Site; Stretching; Therapeutic; Thinness; Time; Tissues; United States National Institutes of Health; Vertebrates; Visual; Visualization; Work; animal tissue; crosslink; directed differentiation; embryonic protein; fluorophore; genetic analysis; genome editing; homologous recombination; human disease; human tissue; improved; in vivo; insight; knock-down; mechanical signal; normal aging; novel strategies; novel therapeutic intervention; perlecan; protein expression; quantitative imaging; reconstitution; therapy development; tissue degeneration; tool

PROJECT SUMMARY Basement membranes (BMs) are thin, dense, supramolecular assemblies of extracellular matrix proteins that surround animal tissues and provide mechanical and signaling support essential for tissue function. Genetic and regulatory defects in BM components underlie numerous diseases, such as diabetes, kidney, and cardiovascular disease, and thickening of BM during aging is thought to be a key driver of tissue decline. Despite the critical importance of BMs to human health, there are currently no animal models that allow comprehensive, real time visualization and experimental manipulation of BM components to study key aspects of BM regulation, which has hindered the development of therapies to treat BM disorders. The overall objective of this proposal is to create a complete toolkit of endogenously fluorescently tagged BM components in C. elegans and use these strains to develop models to experimentally examine two important aspects of BMs: How BMs stretch to support mechanically active tissues, and how BMs accumulate collagen, thicken, and cause tissue decline during aging. C. elegans has single genes encoding most major BM protein components, is optically clear, and has conditional knockdown approaches, which facilitates powerful insight into BM regulation and function. Preliminary work has used Cas9-mediated homologous recombination to insert the mNeonGreen (mNG) fluorophore in-frame with 57 of 98 BM-associated genes, which have been confirmed for protein expression and viability. Homology, genome editing sites, and embryonic protein localization are being cataloged on a newly created database (basementmembraneBASE). Pilot studies have also revealed that during ovulation the BM stretches nearly two- fold to support the spermathecal tissue, and that BMs thicken and type IV collagen levels increase dramatically (~five-fold) within BMs on multiple tissues during C. elegans aging. To complete the objective of finishing a BM toolkit and developing models to study BM stretching and aging, the following specific aims will be pursued: (1) finishing the endogenous tagging of all BM components with mNG and tagging core BM components with mScarlet-I and mEos2 (photoconversion), (2) to use the visual BM toolkit to establish a new model to reveal how BM stretches to support tissue integrity during C. elegans ovulation, and (3) pioneer the first experimental model to study mechanisms of BM collagen accumulation, BM thickening, and tissue decline during aging.The proposed study will powerfully advance our understanding of BM stretching and aging by developing reagents and methodologies to dynamically track BM component presence and levels, determine BM component addition rates through fluorescent recovery after photobleaching (FRAP), and to assess BM removal rates through photoconversion. Preliminary studies have already revealed a unique spermathecal BM composition that likely allows the BM to stretch, and that BM collagen is the only core BM component that increases during aging. The proposed research is significant, as the tools and methods created will provide powerful new approaches to study BMs and establish new models to elucidate key aspects of BM regulation and dysfunction.

项目摘要 地下膜（BMS）是细胞外基质蛋白的薄，密集的，超分子的组件 围绕动物组织，并为组织功能提供机械和信号支持。遗传和 BM组件中的监管缺陷是许多疾病的基础，例如糖尿病，肾脏和心血管 疾病和衰老过程中BM的增厚被认为是组织下降的主要驱动力。尽管很关键 BMS对人类健康的重要性，目前没有动物模型可以全面实时 对BM组件的可视化和实验操作，以研究BM调节的关键方面，这 阻碍了治疗BM疾病的疗法的发展。该提议的总体目的是 在秀丽隐杆线虫中创建一个完整的内生荧光标记的BM组件的工具包，并使用这些 菌株开发模型以实验检查BMS的两个重要方面：BMS如何伸展以支持 机械活性组织，以及BMS如何积聚胶原蛋白，增厚并在衰老过程中导致组织下降。 秀丽隐杆线虫具有编码大多数主要BM蛋白质成分的单个基因，在光学上是清晰的，有条件 敲低的方法，这有助于对BM调节和功能有力见解。初步工作 使用CAS9介导的同源重组，以57 在98个BM相关的基因中，已证实蛋白质表达和活力。同源性，基因组 编辑网站和胚胎蛋白质本地化正在新创建的数据库上分类 （基底膜基键）。试点研究还表明，在排卵过程中，BM伸展近两个 折叠以支撑精子组织，并且BMS增厚和IV型胶原蛋白水平急剧增加 （〜5倍）在秀丽隐杆线虫衰老过程中BMS内的BMS中。完成完成BM的目标 工具包和开发模型以研究BM伸展和衰老，将追求以下特定目标：（1） 用MNG完成所有BM组件的内源性标签，并标记使用的核心BM组件 mscarlet-i和meos2（光转换），（2）使用视觉BM工具包建立一个新模型来揭示如何 BM伸展以支持秀丽隐杆线虫在排卵过程中的组织完整性，（3）先锋第一个实验模型 研究BM胶原蛋白积累，BM增厚和组织下降的机制。 拟议的研究将通过开发试剂来有力地提高我们对BM伸展和衰老的理解 以及动态跟踪BM组件的存在和水平的方法，确定BM组件添加 光漂白后通过荧光恢复（FRAP）的速率，并通过 光转换。初步研究已经揭示了一种独特的精子BM组成 允许BM伸展，并且BM胶原蛋白是在衰老过程中增加的核心BM组件。这 拟议的研究很重要，因为创建的工具和方法将为有力的新方法提供 研究BMS并建立新模型，以阐明BM调节和功能障碍的关键方面。