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

描述(申请人提供)：拟议研究的目标是提供新的工具来量化和操纵活的斑马鱼的信号。荧光生物传感器的使用，以及最近对蛋白质活性的光操纵，已经在细胞生物学中产生了一场革命。挑战一直是将这些工具应用于活体动物。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操纵和分析体内白细胞运动的开发和应用，并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。通过这项工作，我们建议不仅为斑马鱼研究社区产生特定的新工具，而且还提出可以广泛应用的新方法，以前所未有地深入了解活动物的组织和器官生理学。我们将重点关注1)针对活鱼优化的荧光生物传感器，使用能够高通量产生生物传感器并大幅减少生理扰动的新方法；2)能够用光激活或抑制特定斑马鱼细胞中的蛋白质的能力；以及3)使激酶容易被小分子激活并具有高特异性的体内激活能力。我们将通过使用针对斑马鱼优化的Gal4/UAS系统，将这些工具提供给斑马鱼研究社区。新技术将通过解决与上皮向间充质转化(EMT)有关的生理学相关问题进行验证和测试。我们提出了以下目标：目的1.开发荧光生物传感器来量化斑马鱼蛋白质活性的时空动态，采用新的设计，大大提高了灵敏度，减少了生理扰动。目的2.发展用光调节活体斑马鱼蛋白质活性的能力，使亚细胞和秒分辨率的局部活性变化成为可能。目的3.开发一种广泛适用的方法，使激酶对膜可渗透的小分子或光做出反应，基本上具有绝对的特异性。这项工作之所以可能，是因为我们正在结合两名调查人员的不同专业知识，他们在持续和富有成效的合作基础上共同制定了这项建议。Huttenlocher博士在细胞运动学、斑马鱼生物学和成像方面拥有专业知识，而Hahn博士的实验室致力于开发新分子和方法来研究活细胞中的信号和运动性。这项工作的长期目标是在体内带来细胞生物学和细胞信号转导的革命，从而使其能够应用于发育生物学和疾病发病机制的广泛领域。

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.