Abstract 1562 Molecular dissection of feedback modules using light-induced signaling inputs

The ability to rapidly and transiently modulate the concentration and spatial distribution of molecules is a hallmark of signal transduction in biology. Following receptor activation at the plasma membrane, small GTPases are activated, and phosphatidylinositol phosphate (PIP) lipids are dynamically interconverted. Communication between these distinct families of signaling molecules is hypothesized to be established through a series of interconnected positive and negative feedback loops, allowing for the efficient generation of steep, precisely-oriented, intracellular concentration gradients. However, the proposed feedback mechanisms that link these membrane signaling reactions are poorly understood. To date, no feedback circuits have been reconstituted using a reductionist biochemistry approach. To tackle this problem, we reconstituted an isolated signaling pathway in which a small GTPase activates phosphatidylinositol 3-kinase (PI3K), thereby triggering the generation of PI(3,4,5)P3 on supported lipid bilayers. Our approach utilizes the iLID-SspB light-inducible protein heterodimerization system as a transient, spatially-controllable ON switch for this pathway. We introduce positive feedback in the form of chimeric guanine nucleotide exchange factors (GEFs) and negative regulation in the form of GTPase activating proteins (GAPs) and phosphatases. By manipulating the feedback architecture of the reconstituted pathway and observing its dynamic responses, we provide evidence for how communication between small GTPases and PIP lipid-modifying enzymes drives signal amplification at the plasma membrane. This research is funded by an NSF CAREER award granted to Scott Hansen.