Reconstitution of Ras-PI3Kγ membrane communication and feedback using light-induced signaling inputs

Reactions involving small GTPases and phosphatidylinositol phosphate (PIP) lipids serve essential roles in signal transduction at the plasma membrane. In cells, these distinct classes of molecules are linked through positive and negative feedback loops that give rise to emergent properties such as excitability and polarization. Here, we reconstitute communication and feedback between Ras GTPase and phosphatidylinositol 3-kinase gamma (PI3Kγ)-mediated PIP3 production on supported membranes using purified proteins. We employ light-induced membrane recruitment to rapidly shift steady-state conditions and observe the spatiotemporal response of the signaling module. Alone, the Ras-PI3Kγ module exhibits transient and reversible activation due to global inhibition. The introduction of GEF-mediated positive feedback enables sustained threshold crossing and local amplification of Ras(GTP) and PIP3, resulting in a traveling, bistable wave of activity with characteristics of an excitable network. Spatial coupling between Ras(GTP) and PIP3 lipids depends on lateral diffusion and feedback circuit architecture. This work illuminates the roles activation thresholds, membrane diffusion, and positive feedback play in regulating the dynamics of Ras-PI3Kγ membrane signaling reactions in the presence of global inhibition. Positive feedback loops regulate amplification of membrane signaling reactions. Here, Doerr et al. investigate how feedback circuits, activation thresholds, and diffusion dictate the spatiotemporal dynamics of reconstituted Ras-PI3K membrane signaling using light-induced inputs.