Identification of Mechanically Sensitive Proteins in Early Development

描述（由申请人提供）：胚胎经历戏剧性的细胞和组织重排，这是所有动物基因组内编码的形态发生和各种身体计划的造型所需的。这些潜在的运动导致力的产生，这些力被胚胎中的其他细胞和组织局部和全局地感知。机械传导机制负责感知这些力并将其转化为化学信号。因此，体力可能有助于指导和指导早期发展的关键方面。越来越多的证据支持这一假设，即机械刺激影响基因表达，分化，细胞粘附和形态发生。细胞和组织对机械力的反应也是各种病理学的潜在因素，包括高血压和动脉粥样硬化、肿瘤发生和转移、骨退化和耳聋。尽管机械传导对发育、正常生理和疾病的重要性，但所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涉及细胞感知局部应力的机制，其通常通过涉及钙粘蛋白、整合素和细胞骨架的粘附特化来转导。这些研究要检验的主要假设是，机械感觉涉及参与细胞粘附和相关信号事件的蛋白质构象的力依赖性变化。我们需要的方法不仅要严格检验整个假设，而且要同时对这一重要的新领域产生重大影响，方法是鉴定出受 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法（CSMS），以确定从胚胎组织中获得的蛋白质的构象变化，已知是机械敏感。非洲爪蟾原肠胚阶段胚胎的中内胚层经历集体形式的细胞迁移，其需要细胞-细胞凝聚力和纤连蛋白（FN）底物的接合以定向移动。最近，我们发现了一种新的机械敏感性钙粘蛋白复合物的链接到中间丝细胞骨架，这是需要定向运动的组织。那里 有两个具体目标。在目标1中，将用荧光cys-反应性染料处理细胞以标记在对钙粘蛋白或整联蛋白施加应激后暴露的巯基。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的中内胚层蛋白质构象的变化。还将开发图案化弹性基底，其将能够同时对钙粘蛋白和整合素粘附施加力，以模拟单细胞中的组织水平应力。这些研究将提供一个候选的机械敏感蛋白的目录，这些蛋白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重要的资源。 公共卫生相关性：近年来，越来越明显的是，细胞和组织以多种方式产生和响应物理力，这些方式对胚胎发育，组织再生，正常生理和疾病进展至关重要。细胞对彼此及其细胞外环境施加的拉力和牵引力可以影响细胞行为甚至基因表达谱，然而，人们对细胞用于感知力并将这些刺激转化为细胞内化学信号的机制知之甚少。该项目将使用最先进的方法来发现机械刺激细胞后蛋白质的结构变化，从而使我们能够开始阐明细胞的结构变化。 与力觉有关的细胞机制

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. PUBLIC HEALTH RELEVANCE: In recent years it has become increasingly apparent that cells and tissues generate and respond to physical forces in a number of ways that are important for embryo development, tissue regeneration, normal physiology and disease progression. The pulling and tugging forces that cells exert on one another and their extracellular environments can influence cell behavior and even profiles of gene expression, however, little is known about the mechanisms cells use to sense force and convert these stimuli to chemical signals within the cell. This project will use state-of-the-art approaches to discover structural changes in proteins following mechanical stimulation of cells and thus, will allow us to begin elucidating the cellular mechanisms involved in force sensation.