The Molecular Biophysics and Tissue Biomechanics of Somite Morphogenesis

细胞外基质和相关的上皮细胞之间的相互调节是不可或缺的 发展、动态平衡和疾病。体节是脊椎的节段性前体 通过间充质向上皮样转变形成的柱状和肌肉组织。索姆特 形态发生依赖于纤维连接蛋白ECM，即纤维连接蛋白受体整合素 -5--1、细胞黏附蛋白-2与受体双向信号转导 酪氨酸激酶EphA4及其膜结合配体EPhin-B2a。这些基因/途径 介导细胞-ECM黏附、细胞-细胞黏附和接触介导的细胞排斥，以及我们的 假设体节边界的物理组织活动是通过 差异细胞黏附与细胞外基质限制细胞的特异性时空交织 排斥力。目标1、荧光相关光谱(FCS)和荧光 互相关光谱(FCCS)将被用来定量蛋白质扩散和蛋白质 体内结合常数。这些实验将决定是否隔离 这些细胞表面蛋白通过扩散和捕获或主动动员发生。 此外，整合素5、钙粘素2和ePhrin-b2a在推动这些变化中的作用 亚细胞定位将通过在活的突变胚胎中进行FCCS来阐明。在……里面 目的2，细胞运动的系统分析将被用来量化组织的生物力学 野生型和突变型胚胎的体节形态发生。在目标3中，我们量化相对的 通过细胞质信号的整合素激活水平与通过 ECM。生物机制之间的正反馈和负反馈产生网络效应 它们很难先验地预测，也很难在实验中完全探索。在硅胶模型中 用于系统研究细胞黏附、细胞-细胞外基质之间的关系 黏附和细胞接触在体节形态发生中介导的排斥力 解释更多的资源密集型湿实验室实验并确定优先顺序。

Reciprocal regulation between the ECM and associated epithelial cells is integral to development, homeostasis and disease. Somites are segmental precursors of the vertebral column and musculature that form via a mesenchymal to epithelial transition. Somite morphogenesis is dependent upon a Fibronectin ECM, the Fibronectin receptor Integrin 51, the cell adhesion protein Cadherin 2 and bidirectional signaling via the receptor tyrosine kinase EphA4 and its membrane bound ligand Ephrin-B2a. These genes/pathways mediate cell-ECM adhesion, cell-cell adhesion and contact mediated cell repulsion, and our hypothesis is that the physical organizing activity of the somite boundary emerges via specific spatiotemporal intertwining of differential cell adhesion and ECM constrained cell repulsion. In Aim 1, fluorescence correlation spectroscopy (FCS) and fluorescence crosscorrelation spectroscopy (FCCS) will be used quantify protein diffusion and protein binding constants in vivo. These experiments will determine whether the segregation of these cell surface proteins occurs via diffusion and capture or active mobilization. Additionally, the roles of integrin 5, cadherin 2 and ephrin-b2a in driving these changes in subcellular localization will be elucidated by performing FCCS in live mutant embryos. In Aim 2, a systems analysis of cell motion will be used to quantify tissue biomechanics during somite morphogenesis in wild-type and mutant embryos. In Aim 3, we quantify the relative levels of Integrin activation via cytoplasmic signals versus via positive feedback through the ECM. Positive and negative feedback between biological mechanisms creates network effects that are hard to predict a priori and difficult to fully explore experimentally. in silico modeling will be used to systematically examine the relationships between cell adhesion, cell-ECM adhesion and cell contact mediated repulsion in somite morphogenesis in order to help interpret and prioritize more resource intensive wet-lab experiments.