Rac1 signal multiplexing : a quantitative approach from in-vitro to in-vivo – MULTIPLEX

A challenge in modern biology is to understand how circuits of proteins enable cells to monitor information from their environment and make decisions to mount efficient responses. Small GTPases are among the most prominent actors in cellular pathways, and they are involved in the regulation of virtually all cellular processes. A commonality of all small GTPases is their ability to function as molecular switches by their alternation between an inactive, GDP-bound, and an active, GTP-bound form. This simple mechanism is in reality exceedingly complex, because multiple signals converge towards each small GTPase through their numerous activators (guanine nucleotide exchange factors or GEFs, which stimulate their loading with GTP), which in turn generate a unique response by selecting a specific downstream effector that binds to the activated GTPase. Conversely, the level of active GTPases is balanced by the inhibiting activities of their GTPase-activating proteins (GAPs), which inactivate them by stimulating hydrolysis of GTP. Importantly, all these mechanisms operate at the periphery of membranes, which are thus additional components in GTPases signaling. By analogy to telecommunication networks, small GTPases systems can thus be likened to multiplexed molecular circuits, in which multiple inputs are combined into a single signal (the small GTPase) before they are broken down again into specific outputs. Currently, the kinetics and specificity determinants of the small GTPases signaling in this multiplexed framework have remained mysterious. Our hypothesis is that the specificity of signaling arises from multiscale contributions: differential affinities (proteins), molecular compositions (complexes) and spatial partitioning on membranes (nanoclusters).

This project addresses this issue by quantitative approaches merging in vitro and in cellulo systems, focusing on the activation and signaling kinetics of the small GTPase Rac1. Rac1 is a master regulator of cell motility, endocytosis and cell growth, and is associated to many pathological conditions such as tumorigenesis, nervous system development disorders, cardiac diseases or infections. This small GTPase is regulated by a very large number of GEFs and GAPs and can signal to diverse effectors, and thus can be seen as archetypal of multiplexing. An important step towards understanding the system-level features of Rac1 signaling in cell biology and disease is the reconstitution of its regulation and activity under well-controlled conditions where kinetics parameters of activation (GDP/GTP exchange and GTP hydrolysis) and signaling (effector binding) can be accurately measured. Bringing together the expertise of the Cherfils lab (ENS Paris-Saclay) in the reconstitution of small GTPases in artificial membranes and of the Coppey lab (Institut Curie Paris) in subcellular quantitative optogenetics, our objective is to identify and quantify these parameters by integrating in vitro kinetics assays using purified proteins and controlled artificial membranes (liposome and supported lipid bilayer) with in cellulo biochemical assays in which regulator concentrations are controled by optogenetics. Ultimately, these ground-truth parameters will be integrated into mathematical models of Rac1 circuitry and signaling. Our project should deliver a molecular « instruction manual » of Rac1 on membranes, pave the way for future studies that address the GTPase multiplexing issue and generate conceptual advances that will broaden our understanding of the many facettes of small GTPases. In the longer term, it may inspire system-based molecular strategies in drug discovery for inhibiting GTPase-controled signaling pathways in diseases.