Identification of Mechanically Sensitive Proteins in Early Development

描述（由申请人提供）：胚胎经历剧烈的细胞和组织重排，这是所有动物基因组中编码的各种身体计划的形态发生和雕刻所必需的。这些潜在的运动导致产生的力被胚胎中的其他细胞和组织局部和全局感知。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，体力可以指导和指导早期发育的关键方面。机械刺激影响基因表达、分化、细胞粘附和形态发生的证据支持了这一假设。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、正常生理和疾病具有重要意义，但其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，这通常是通过涉及钙粘蛋白、整合素和细胞骨架的粘附特化来转导的。这些研究要验证的主要假设是，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变化。需要的方法不仅要严格检验整个假设，而且要同时通过识别受影响的蛋白质类别，对这一重要的新领域产生重大影响

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.