The cross-scale biomechanics of tissue morphogenesis

细胞黏附蛋白与细胞外基质之间的动态分子相互作用 (ECM)和细胞骨架产生了组织水平的机制，但描绘了 时空调节组织力学的产生机制仍然是一个 挑战。该提案利用集成的跨规模解决了这一问题 量化分子尺度蛋白质运动和蛋白质-蛋白质结合的方法 以及使用细胞跟踪和来自流体的定量度量的组织力学 机械师。具体地说，从运动性中胚层组装近轴中胚层 对祖先进行了检查。该提案旨在阐明一种监管机制，其中 纤维连接蛋白纤维形成局限于轴旁中胚层的表面，尽管 纤维连接蛋白及其初级整合素受体在整个组织中的存在。 活体胚胎的荧光交叉相关光谱(FCCS)揭示了一种蛋白质 含有整合素5和钙粘附素2的复合体和遗传学实验表明，这 复合体抑制纤维连接蛋白基质在旁轴间充质内的组装 中胚层。在近轴向中胚层表面，整合素5被去抑制并 纤维连接蛋白基质覆盖在组织表面。目标1是定义整合素V的作用 其他主要的纤维连接蛋白受体，在这个过程中使用FCC和遗传马赛克。在……里面 此外，荧光计时器融合蛋白被用来揭示黏附蛋白的模式 体内周转，FRET/FLIM法检测体内整合素构象 胚胎。目标2是使用co-IP和 质谱学。这些蛋白将进一步通过体内的fccs基因进行鉴定。 突变体细胞运动的基因敲除和系统分析。

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.