The cross-scale biomechanics of tissue morphogenesis

组织形态发生的跨尺度生物力学

• 批准号: 9363434
• 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/9363434
• 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、细胞外基质之间的动态分子相互作用 （ECM）和细胞骨架产生组织水平的力学，但描绘了 时空调节的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 量化分子尺度蛋白质运动和蛋白质-蛋白质关联的方法 以及使用细胞跟踪和流体定量指标的组织力学 机械师。具体来说，从运动的中胚层组装近轴中胚层 检查祖细胞。该提案旨在阐明一种监管机制，其中 纤连蛋白原纤维生成仅限于近轴中胚层的表面，尽管 整个组织中存在纤连蛋白及其主要整合素受体。 活胚胎中的荧光互相关光谱（FCCS）揭示了一种蛋白质 含有整合素α5和钙粘蛋白2的复合物和遗传实验表明，这种 复合物抑制近轴间充质内的纤连蛋白基质组装 中胚层。在轴旁中胚层表面，整合素 α5 被去抑制， 纤连蛋白基质覆盖组织表面。目标 1 是定义整合素 V 的作用， 其他主要纤连蛋白受体，在此过程中使用 FCCS 和遗传嵌合体。在 此外，荧光计时器融合蛋白用于揭示粘附蛋白的模式 体内周转，并使用 FRET/FLIM 测定来检查活体中的整合素构象 胚胎。目标 2 是使用共免疫沉淀 (co-IP) 和方法鉴定该蛋白质复合物的其他成分 质谱。这些蛋白质将通过体内 FCCS、基因进一步表征 突变体细胞运动的敲除和系统分析。