The cross-scale biomechanics of tissue morphogenesis

组织形态发生的跨尺度生物力学

• 批准号: 9557529
• 负责人: SCOTT A HOLLEY
• 金额: $ 41.88万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-05 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9557529
• 关键词: Address; Adhesions; Binding; Biological Assay; Biomechanics; Biophysics; Cadherins; Cell Adhesion Molecules; Cell Culture Techniques; Cell-Cell Adhesion; Cells; Chimeric Proteins; Co-Immunoprecipitations; Complex; Cutis Laxa; Cytoskeleton; Data; Deposition; Diffusion; Elastic Fiber; Elasticity; Embryo; Exhibits; Extracellular Matrix; Extracellular Matrix Proteins; Fibronectin Receptors; Fibronectins; Fluorescence; Fluorescence Resonance Energy Transfer; Genetic; Human; In Vitro; Integrin alpha5; Integrin alpha5beta1; Integrin alphaV; Integrin alphaVbeta3; Integrins; Liquid substance; Maps; Mass Spectrum Analysis; Mechanics; Mediating; Mesenchyme; Molecular; Molecular Conformation; Morphogenesis; Mosaicism; Motion; Movement; Mutate; Paraxial Mesoderm; Pattern; Process; Protein Analysis; Proteins; Regulation; Repression; Role; Skin; Spectrum Analysis; Structure; Surface; Systems Analysis; Tertiary Protein Structure; Testing; Tissues; Variant; Zebrafish; cell motility; complement system; experimental study; fibrillogenesis; fibulin; fibulin-4; in vivo; knockout gene; liquid chromatography mass spectrometry; molecular scale; mutant; particle; progenitor; protein complex; protein degradation; receptor; response; spatiotemporal

Dynamic molecular interactions among cell adhesion proteins, the extracellular matrix (ECM) and the cytoskeleton give rise to tissue-level mechanics, but delineating the mechanism of emergence of spatiotemporally regulated tissue mechanics remains a challenge. This proposal addresses this problem utilizing an integrated cross-scale approach that quantifies molecular scale protein movement and protein-protein association as well as tissue mechanics using cell tracking and quantitative metrics from fluid mechanics. Specifically, the assembly of the paraxial mesoderm from motile mesodermal progenitors is examined. The proposal seeks to elucidate a regulatory mechanism in which Fibronectin fibrillogenesis is confined to the surface of the paraxial mesoderm despite the presence of Fibronectin and its primary Integrin receptors throughout the tissue. Fluorescence cross correlation spectroscopy (FCCS) in live embryos revealed a protein complex containing Integrin 5 and Cadherin 2 and genetic experiments indicate that this complex represses Fibronectin matrix assembly within the mesenchyme of the paraxial mesoderm. On the surface of the paraxial mesoderm, Integrin 5 is de-repressed and Fibronectin matrix coats the tissue surface. Aim 1 is to define the role of Integrin V, the other main Fibronectin receptor, in this process using FCCS and genetic mosaics. In addition, fluorescent timer fusion proteins are used to reveal the patterns of adhesion protein turnover in vivo, and a FRET/FLIM assay is used to examine Integrin conformation in live embryos. Aim 2 is to identify other components of this protein complex using co-IP and Mass Spectroscopy. These proteins will be further characterized by in vivo FCCS, gene knockout and systems analysis of cell motion in the mutants.

细胞粘附蛋白和细胞外基质之间的动态分子相互作用 (ECM)和细胞骨架引起组织水平的力学，但描绘 时空调节组织力学的出现机制仍然是一个 挑战.该提案利用集成的跨尺度解决了这个问题 一种量化分子尺度蛋白质运动和蛋白质-蛋白质缔合的方法 以及使用来自流体的细胞跟踪和定量度量的组织力学 力学具体地说，近轴中胚层从运动中胚层的组装 祖细胞进行了检查。建议旨在阐明一个监管机制， 纤维连接蛋白原纤维形成仅限于近轴中胚层的表面，尽管在细胞外基质中存在纤维连接蛋白原纤维。 整个组织中存在纤连蛋白及其主要整合素受体。 荧光交叉相关光谱（FCCS）在活胚胎揭示了蛋白质 含有整合素β 5和钙粘蛋白2的复合物和遗传实验表明， 复合物抑制近轴间充质内的纤连蛋白基质组装 中胚层在近轴中胚层的表面上，整联蛋白β 5被去阻遏， 纤连蛋白基质覆盖组织表面。目的1是定义整合素α V的作用， 其他主要纤连蛋白受体，在此过程中使用FCCS和遗传镶嵌。在 此外，荧光计时器融合蛋白用于揭示粘附蛋白的模式 使用FRET/FLIM测定来检查肝细胞中整合素的构象。 胚胎目的2是使用co-IP鉴定该蛋白质复合物的其他组分， 质谱。这些蛋白质将通过体内FCCS、基因表达谱和免疫印迹法进一步表征。 基因敲除和突变体细胞运动的系统分析。