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

描述（由申请人提供）：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电操纵，已经在细胞生物学中产生了一场革命。挑战在于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学技术的发展和应用，以操纵和分析斑马鱼发育过程中体内白细胞的运动，我们已经开发了新的生物传感器设计，可以报告内源性蛋白质的构象变化和磷酸化。在这项工作的基础上，我们建议不仅为斑马鱼研究界提供特定的新工具，而且可以广泛应用于对活体动物组织和器官生理学前所未有的深入了解的新方法。我们将专注于1)为活鱼优化的荧光生物传感器，使用新方法实现高通量生物传感器的产生，并大大减少生理扰动2)在特定的斑马鱼细胞中用光激活或抑制蛋白质的能力，以及3)使激酶对小分子敏感，以高特异性在体内激活。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界。新技术将通过解决有关上皮到间充质转化（EMT）的生理学相关问题来验证和测试。我们提出以下目标：目标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，使用新颖的设计，大大提高灵敏度和减少生理扰动。目标2。培养用光调节活斑马鱼蛋白质活性的能力，实现亚细胞和秒分辨率的局部活动变化。目标3。开发一种广泛适用的方法，使激酶对膜渗透性小分子或光有反应，具有绝对的特异性。这项工作是可能的，因为我们结合了两名研究人员的不同专业知识，他们在持续和富有成效的合作的基础上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拥有专业知识，Hahn博士的实验室专注于研究活细胞信号和运动的新分子和方法的开发。这项工作的长期目标是带来细胞生物学和细胞信号在体内的革命，从而使其应用于发育生物学和疾病发病机理的广泛领域。

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.