Identification of Mechanically Sensitive Proteins in Early Development

描述(申请人提供)：胚胎经历戏剧性的细胞和组织重组，这是形态形成和塑造所有动物基因组中编码的各种身体计划所必需的。这些潜在的运动导致了力的产生，胚胎中的其他细胞和组织可以局部和全局地感知这些力。机械转导机制负责感知这些力并将其转化为化学信号。因此，体力可以用来指导和指导早期发展的关键方面。越来越多的证据表明，机械刺激影响基因表达、分化、细胞黏附和形态发生，这一假说得到了支持。细胞和组织对机械力的反应也是各种病理的潜在因素，包括高血压和动脉粥样硬化、肿瘤的发生和转移、骨变性和耳聋。尽管机械转导对发育、正常生理和疾病都很重要，但其中涉及的分子机制仍然知之甚少。围绕机械转导的关键问题之一是关于细胞感知局部应力的机制(S)，这些应力通常是通过涉及钙粘附素、整合素和细胞骨架的粘附性特化来传递的。这些研究要检验的主要假设是，机械感觉涉及参与细胞黏附和相关信号事件的蛋白质构象的力依赖变化。需要的方法不仅将严格检验整个假设，而且将通过识别受以下因素影响的蛋白质类别同时对这一重要的新领域产生重大影响 在生理相关条件下发生的构象变化。我们将应用半胱氨酸鸟枪质谱仪(CSMS)来鉴定从已知对机械敏感的胚胎组织中获得的蛋白质的构象变化。非洲爪哇原肠胚期胚胎的中胚层经历了一种集体形式的细胞迁移，这需要细胞-细胞的凝聚和纤维连接蛋白(FN)底物的参与才能定向移动。最近，我们发现了一种新的机械敏感的钙粘素复合体，它与中间纤维细胞骨架相连，这是组织定向运动所必需的。那里 有两个具体的目标。在目标1中，细胞将被荧光Cys活性染料处理，以标记在对钙粘附素或整合素施加压力后暴露的硫醇基团。标记的蛋白质将被分离，通过标记强度的变化进行鉴定，进行串联质谱分析，并使用生物信息学方法分析序列数据。第二个目标将集中在检测完整中胚层的蛋白质构象变化。还将开发图案化的弹性底物，使钙粘附素和整合素粘连能够同时施加力，以模拟单细胞中组织水平的应力。这些研究将为对力在形态发生、正常组织生物学和疾病中的作用感兴趣的研究人员提供一个候选的机械敏感蛋白质的目录，这些蛋白质将构成一个重要的资源。

DESCRIPTION (provided by applicant): Embryos undergo dramatic cell and tissue rearrangements that are required for morphogenesis and the sculpting of the various body plans encoded within the genomes of all animals. These underlying movements result in the generation of forces that are sensed both locally and globally by other cells and tissues in the embryo. Mechanisms of mechanotransduction are responsible for sensing these forces and converting them to chemical signals. Thus, physical force may serve to instruct and guide key aspects of early development. This hypothesis is supported by mounting evidence that mechanical stimuli influence gene expression, differentiation, cell adhesion and morphogenesis. Cell and tissue responses to mechanical forces are also underlying factors in varied pathologies that include hypertension and atherosclerosis, tumorigenesis and metastasis, bone degeneration, and deafness. Despite the importance of mechanotransduction to development, normal physiology and disease, the molecular mechanisms involved remain poorly understood. One of the key questions surrounding mechanotransduction concerns the mechanism(s) by which cells sense local stresses, which typically are transduced through adhesive specializations involving cadherins, integrins and the cytoskeleton. The primary hypothesis to be tested by these studies is that mechanosensation involves force-dependent changes in the conformations of proteins involved in cell adhesion and related signaling events. Approaches are needed that will not only test rigorously the overall hypothesis, but that will simultaneously make a significant impact on this important new field by identifying classes of proteins subject to conformational changes occurring under physiologically relevant conditions. We will apply cysteine shotgun mass spectrometry (CSMS) to identify conformational changes in proteins obtained from an embryonic tissue known to be mechanosensitive. The mesendoderm of Xenopus laevis gastrula stage embryos undergoes a collective form of cell migration that requires both cell-cell cohesion and engagement of a fibronectin (FN) substrate in order to move directionally. Recently, we discovered a novel mechanosensitive cadherin complex with links to the intermediate filament cytoskeleton that is required for directed motility of this tissue. There are two specific aims. In Aim 1, cells will be treated with fluorescent cys-reactive dyes to label thiol groups exposed following application of stress to cadherins or integrins. Labeled proteins will be separated, identified by changes in labeling intensity, subjected to tandem mass spectrometry and the sequence data analyzed using bioinformatics approaches. The second aim will focus on detecting protein conformational changes in intact mesendoderm. Patterned elastic substrates will also be developed that will enable simultaneous application of force to both cadherin and integrin adhesions in order to mimic tissue-level stresses in single cells. These studies will contribute a catalog of candidate, mechanosensitive proteins that will comprise an important resource for investigators interested in the role of force in morphogenesis, normal tissue biology and disease.