A novel localization based biosensor, SEDAR, visualizes spatio-temporal dynamics of activated Ras in endogenous Drosophila tissues

Abstract

Chemokine signaling via growth factor receptor tyrosine kinases (RTKs) regulates development, differentiation, growth and disease implying that it is involved in a myriad of cellular processes. A single RTK, for example the Epidermal Growth Factor Receptor (EGFR), is used repeatedly for a multitude of developmental programs. Quantitative differences in magnitude and duration of RTK signaling can bring about different signaling outcomes. Understanding this complex RTK signals requires real time visualization of the signal. To visualize spatio-temporal signaling dynamics, a biosensor called SEnsitive Detection of Activated Ras (SEDAR) was developed. It is a localization-based sensor that binds to activated Ras directly downstream of the endogenous activated RTKs. This binding was reversible and SEDAR expression did not cause any detectable perturbation of the endogenous signal. Using SEDAR, endogenous guidance signaling was visualized during RTK mediated chemotaxis of border cells in Drosophila ovary. SEDAR localized to both the leading and rear end of the cluster but polarized at the leading edge of the cluster. Perturbation of RTKs that led to delays in forward migration of the cluster correlated with loss of SEDAR polarization in the cluster. Gliding or tumbling behavior of border cells was a directly related to the high or low magnitude of SEDAR polarization respectively, in the leading cell showing that signal polarization at the plasma membrane provided information for the migratory behavior. Further, SEDAR localization to the plasma membrane detected EGFR mediated signaling in five distinct developmental contexts. Hence SEDAR, a novel biosensor could be used as a valuable tool to study the dynamics of endogenous Ras activation in real time downstream of RTKs, in three-dimensional tissues, at an unprecedented spatial and temporal resolution.