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

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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.