A toolkit for imaging and photo-manipulation of signaling in zebrafish

斑马鱼信号成像和光操作工具包

• 批准号: 8332584
• 负责人: Klaus M. Hahn
• 金额: $ 64.33万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8332584
• 关键词: Actins; Active Sites; Address; Amino Acids; Animals; Area; Biology; Biosensor; CCI-779; Catalytic Domain; Cell Communication; Cells; Cellular biology; Collaborations; Communities; Cyclic AMP-Dependent Protein Kinases; Data; Development; Developmental Biology; Disease; Engineering; Epithelial; Epithelial Cells; Fishes; Focal Adhesion Kinase 1; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; HRAS gene; Health; Heterodimerization; Image; Kinetics; Laboratories; Leukocytes; Life; Light; Long-Term Effects; MAP Kinase Gene; MAPK1 gene; MEKs; Mammalian Cell; Membrane; Molecular Models; Movement; Mutation; Myosin Light Chain Kinase; Oncogenic; Optics; Organ; Oxygen; Pathogenesis; Peptides; Phosphorylation; Phosphotransferases; Physiological; Physiology; Protein Dynamics; Protein-Serine-Threonine Kinases; Proteins; Reporting; Research; Research Personnel; Resolution; Route; Signal Transduction; Sirolimus; Specificity; System; Techniques; Tertiary Protein Structure; Testing; Tissues; Translating; Tyrosine; Universities; Wisconsin; Work; Zebrafish; analog; base; cell motility; cell type; design; epithelial to mesenchymal transition; in vivo; insight; instrumentation; molecular modeling; new technology; novel; novel strategies; optogenetics; small molecule; src-Family Kinases; success; tool; voltage; zebrafish development

DESCRIPTION (provided by applicant): The goal of the proposed research is to provide new tools to quantify and manipulate signaling in living zebrafish. The use of fluorescent biosensors, and more recently photomanipulation of protein activity, has generated a revolution in cell biology. The challenge has been to apply these tools for use in live animals. Our recent study highlighted the development and application of novel optogenetic techniques to manipulate and analyze leukocyte movements in vivo during zebrafish development, and we have developed new biosensor designs that can report conformational changes and phosphorylation of endogenous proteins. By building on this work, we propose to generate not only specific new tools for the zebrafish research community, but new approaches that can be applied broadly for unprecedented insight into tissue and organ physiology in live animals. We will focus on 1) fluorescent biosensors optimized for living fish, using new approaches enabling high throughput biosensor generation and substantially reduced physiological perturbation 2) the ability to activate or inhibit proteins in specific zebrafish cells with light, and 3) rendering kinases susceptible to small molecules for activation in vivo with high specificity. We will make these tools available to the zebrafish research community by using the Gal4/UAS system optimized for zebrafish. The new technologies will be validated and tested by addressing physiologically relevant questions regarding epithelial to mesenchymal transition (EMT). We propose the following aims: Aim 1. Develop fluorescent biosensors to quantify the spatio-temporal dynamics of protein activity in zebrafish, using novel designs with greatly enhanced sensitivity and reduced physiological perturbation. Aim 2. Develop the ability to regulate protein activity in livig zebrafish with light, enabling localized changes in activity with subcellular and seconds resolution. Aim 3. Develop a broadly applicable approach to render kinases responsive to membrane permeable small molecules or light, with essentially absolute specificity. This work is possible because we are combining diverse expertise from two investigators who have jointly developed this proposal based on their ongoing and productive collaboration. Dr. Huttenlocher has expertise in cell motility, zebrafish biology and imaging, and Dr. Hahn has focused his laboratory on the development of new molecules and approaches to study signaling and motility in living cells. The long term goal of this work is to bring the revolution in cell biology and cel signaling in vivo, thereby enabling application to broad areas of developmental biology and disease pathogenesis. PUBLIC HEALTH RELEVANCE: Dynamic cell signaling contributes to normal health and disease. There has been recent progress in developing tools to image and manipulate cell signaling, however, there has been a gap in translating these tools for use in live animals. The goal of the proposed research is to provide new tools to quantify and manipulate signaling in living zebrafish thereby enabling application to broad areas of developmental biology and disease pathogenesis.

描述（由申请人提供）：拟议研究的目的是提供新工具来量化和操纵斑马鱼中的信号。荧光生物传感器的使用以及近来的蛋白质活性的光持持续性，引起了细胞生物学的革命。挑战是将这些工具应用于活动物中。我们最近的研究强调了新型光遗传技术在斑马鱼发育过程中在体内操纵和分析白细胞运动的发展和应用，我们开发了新的生物传感器设计，可以报告内源蛋白的构象变化和磷酸化。通过在这项工作的基础上，我们建议不仅为斑马鱼研究社区生成特定的新工具，而且还可以广泛应用的新方法，用于对活动物中的组织和器官生理学的前所未有的洞察力。我们将重点关注1）为活鱼优化的荧光生物传感器，采用新方法，实现高吞吐量生物传感器的生成并大大降低生理扰动2）能够激活或抑制光线特定斑马鱼细胞中蛋白质的能力，而3）呈现小分子易于激活的激活具有高特异性的小分子。我们将通过使用针对斑马鱼优化的GAL4/UAS系统来使斑马鱼研究社区可用。新技术将通过解决有关上皮过渡（EMT）的生理相关问题来验证和测试。我们提出以下目的：目标1。开发荧光生物传感器，以量化斑马鱼中蛋白质活性的时空动力学，使用具有大大增强敏感性和生理扰动降低的新型设计。 AIM 2。发展具有光线斑马鱼中蛋白质活性的能力，从而可以通过亚细胞和秒分辨率进行局部活性变化。 AIM 3。开发一种广泛适用的方法来使动力酶响应于具有绝对特异性的膜可渗透的小分子或光。这项工作之所以可能，是因为我们将两位调查人员的多样专业知识结合在一起，他们基于他们持续和富有成效的合作，共同制定了这项建议。 Huttenlocher博士在细胞运动，斑马鱼生物学和成像方面具有专业知识，Hahn博士将实验室重点放在了新分子的发展以及研究活细胞中信号传导和运动性的方法上。这项工作的长期目标是将细胞生物学和CEL信号的革命在体内带入，从而实现了发育生物学和疾病发病机理的广泛领域。 公共卫生相关性：动态细胞信号传导有助于正常健康和疾病。在开发图像和操纵细胞信号传导的工具方面，最近有了进步，但是，将这些工具转换为现场动物的工具存在差距。拟议的研究的目的是提供新工具来量化和操纵斑马鱼中的信号传导，从而使发育生物学和疾病发病机理的广泛领域应用。