Dissecting signaling kinetics and regulatory feedback

Dr. Giorgos Pyrowolakis (BIOSS and Institute of Biology I, University of Freiburg)

Regulatory feedback is critically involved in fine-tuning kinetics and biological output of signaling. Our recent work on graded BMP/Smad signaling during Drosophila wing epithelium development identified and characterized a number of conserved feedback regulators that are suggested to adjust the signaling gradient in time and space and to couple signaling output to input. Despite extensive work in the system, the kinetics of Smad activation and the impact of regulatory feedback are practically unexplored: It is unclear whether Smad activation displays adaptation, increases over time, or oscillates, and how regulatory feedback might affect Smad activation kinetics in the context of graded signalling. These shortcomings are mainly due to experimental limitations in temporal resolution.

The advent of optogenetics provides excellent tools to monitor pathway activation in real time and address such long-standing questions. We suggest here establishing in vivo experimental systems in which the activation of the BMP receptor complex is rendered light-inducible. The basic idea is to use light-inducible interaction systems to convert BMP receptor assembly (the first step in BMP signal activation) from ligand-dependent to light-dependent (in collaboration with the Weber group, BIOSS). Pilot experiments in cell culture systems should reveal the optimal set-up and combinations. Selected hybrid receptor constructs will be integrated into the endogenous Drosophila genomic loci by Cas9/CRISPR-based genome editing for expression at physiological levels. These tools will be then combined with live-sensors for BMP signaling activity to monitor light-induced pathway activation in wing disc epithelia in a time resolved manner. Once established, the system will be used to address the role of feedback regulators in vivo. Specifically, we will use genetic tools as well as genomic engineering to manipulate levels and BMP-responsiveness to BMP, respectively, and monitor how this would affect the observed signaling kinetics.