Identification of Mechanically Sensitive Proteins in Early Development

早期发育中机械敏感蛋白的鉴定

• 批准号: 8390270
• 负责人: DOUGLAS W. DESIMONE
• 金额: $ 23.41万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-20 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8390270
• 关键词: Adhesions; Adhesives; Animals; Area; Atherosclerosis; Behavior; Biochemical; Bioinformatics; Biological Assay; Biological Models; Biology; Cadherins; Cataloging; Catalogs; Cell Adhesion; Cell Polarity; Cell-Cell Adhesion; Cells; Chemicals; Chimeric Proteins; Complex; Coupled; Cysteine; Cytoskeleton; DNA Sequence Rearrangement; Data Analyses; Development; Disease; Disease Progression; Disseminated Malignant Neoplasm; Dyes; Embryo; Embryonic Development; Environment; Esthesia; Event; Extracellular Matrix; Fibronectins; Gastrula; Gene Expression; Generations; Genome; Goals; Hereditary Disease; Hypertension; Immigration; Integrins; Intermediate Filaments; Keratin; Label; Link; Mass Spectrum Analysis; Mechanical Stimulation; Mechanical Stress; Mechanics; Metastatic Neoplasm to the Bone; Methods; Molecular Conformation; Morphogenesis; Movement; Natural regeneration; Normal tissue morphology; Pathology; Pattern; Physiological; Physiology; Preparation; Process; Protein Conformation; Proteins; Proteomics; Research Personnel; Resources; Role; Shotguns; Signal Transduction; Site; Staging; Stimulus; Stress; Stretching; Sulfhydryl Compounds; Testing; Tissues; Xenopus; Xenopus laevis; blastomere structure; cell behavior; cell motility; cohesion; deafness; embryo tissue; extracellular; flexibility; in vivo; interest; migration; novel; research study; response; tandem mass spectrometry; tissue regeneration; tissue repair; tumorigenesis

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.

描述（由申请人提供）：胚胎经历戏剧性的细胞和组织重排，这是形态发生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产生力，这些力可以被胚胎中的其他细胞和组织局部和整体感知。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。越来越多的证据表明机械刺激影响基因表达、分化、细胞粘附和形态发生，这一假设得到了支持。细胞和组织对机械力的反应也是多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、骨变性和耳聋。尽管力转导对发育、正常生理和疾病很重要，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，这些机制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。这些研究要测试的主要假设是，机械感觉涉及细胞粘附和相关信号事件中涉及的蛋白质构象的力依赖性变化。需要的方法不仅能够严格检验总体假设，而且能够通过识别受限制的蛋白质类别，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（CSMS）来识别从已知具有机械敏感性的胚胎组织中获得的蛋白质的构象变化。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，需要细胞间的凝聚力和纤连蛋白 (FN) 底物的接合才能定向移动。最近，我们发现了一种新型机械敏感性钙粘蛋白复合物，与该组织定向运动所需的中间丝细胞骨架相连。那里 是两个具体目标。在目标 1 中，将用荧光半胱氨酸反应染料处理细胞，以标记在对钙粘蛋白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。第二个目标将集中于检测完整中内胚层中的蛋白质构象变化。还将开发图案化的弹性基材，该基材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。这些研究将提供候选的机械敏感蛋白质目录，这些蛋白质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物理力并对其做出反应，这对于胚胎发育、组织再生、正常生理和疾病进展非常重要。细胞相互施加的拉力和细胞外环境可以影响细胞行为，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细胞机械刺激后蛋白质的结构变化，从而使我们能够开始阐明 参与力觉的细胞机制。