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: 10580610
• 负责人: David R Sherwood
• 金额: $ 19.33万
• 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/10580610
• 关键词: Adult; Age; Aging; Alzheimer' s Disease; Animal Experiments; 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; Proteins; Public Health; RNA Interference; Reagent; Recovery; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; 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; 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.

项目总结 基底膜(BMS)是由细胞外基质蛋白组成的薄而致密的超分子组合体。 包围动物组织，并提供组织功能所必需的机械和信号支持。遗传和 BM成分的监管缺陷是许多疾病的基础，如糖尿病、肾脏和心血管疾病 疾病和衰老过程中BM的增厚被认为是组织衰退的关键驱动因素。尽管关键的是 BMS对人类健康的重要性，目前还没有一个动物模型能够全面、实时地 BM组件的可视化和实验操作，以研究BM调控的关键方面，其中 阻碍了治疗BM疾病的疗法的发展。这项提议的总体目标是 在线虫中创建一个内源性荧光标记的BM组件的完整工具包，并使用这些 菌株开发模型来实验检查BMS的两个重要方面：BMS如何伸展以支持 机械活动的组织，以及骨髓细胞如何在衰老过程中积累胶原蛋白、增厚和导致组织衰退。 线虫只有一个基因编码大多数主要的BM蛋白成分，是光学透明的，并具有条件性 击倒方法，这有助于对BM监管和功能的强大洞察。前期工作已完成 利用Cas9介导的同源重组将mNeonGreen(MNG)荧光团插入57 在98个与BM相关的基因中，已经证实了蛋白质表达和活性。同源性，基因组 编辑站点和胚胎蛋白质定位正在一个新创建的数据库中编目 (BasementmameBASE)。初步研究还显示，在排卵期间，BM伸展近两个- 折叠以支撑精囊组织，BMS增厚，IV型胶原水平显著增加 在线虫老化过程中，在多个组织上的BMS(~5倍)。完成完成BM的目标 为了研究BM拉伸和老化的工具包和开发模型，将追求以下具体目标：(1) 用MNG完成所有黑石组件的内源标记，用MNG标记核心黑石组件 MScarlet-I和mEos2(光转换)，(2)使用可视化的BM工具包建立新的模型，揭示如何 在线虫排卵期间，BM可以伸展以支持组织完整性，并且(3)开创了第一个实验模型 目的：研究衰老过程中BM胶原堆积、增厚和组织衰退的机制。 拟议的研究将通过开发试剂有力地促进我们对骨髓拉伸和老化的理解 和方法，以动态跟踪BM组件的存在和级别，确定BM组件添加 通过光漂白后的荧光恢复(FRAP)来评估BM的去除率 光转化。初步研究已经揭示了一种独特的受精卵BM成分，很可能 允许BM拉伸，BM胶原蛋白是唯一在老化过程中增加的核心BM成分。这个 拟议的研究具有重要意义，因为创建的工具和方法将提供强大的新途径 研究BMS并建立新的模型来阐明BM调节和功能障碍的关键方面。