Agata Nawrotek

Laboratoire de Biologie et Pharmacologie Appliquée, Université Paris-Saclay

A comparative study of small GTPase regulation on membrane: from in vitro to in vivo systems

Small GTPases act as molecular switches, functioning as "ON" and "OFF" buttons for many cellular pathways, and together with their multi-protein regulatory networks they guide essential cellular functions. Impressively, each small GTPase has a distinct set of regulators dedicated to specific tasks. Despite the complexity of these networks, the activation level of each small GTPase is precisely controlled. This study addresses key questions: How do regulators and effectors on membranes trigger precise pathways in complex cellular environments? How do atypical and canonical regulators interact and fine-tune activities to prevent deregulation? What key factors influence the specificity of small GTPase activation? To explore these questions, we reconstituted biomimetic systems in vitro with purified proteins and artificial membranes, correlating these experiments with cellular studies. In this project, our focus is on Rac1, a small GTPase involved in cell movement, endocytosis, and growth. Defects in Rac1 signalling are linked to developmental disorders, autoimmune diseases, cancers, and diabetes. Rac1 orchestrates numerous signalling pathways, dependent on the spatial and temporal distribution of its regulatory modules. We quantitatively characterised Rac1 circuits in vitro, including its activators, deactivators, and effectors. Using a fluorescence-based assay on liposomes, we investigated Rac1 activation-deactivation dynamics by its GEF (activators: Tiam1 and PREX1) and GAP (inactivators: RacGAP1). These findings were correlated with GEF recruitment rates and Rac1 activation kinetics on supported lipid bilayers using TIRF microscopy and optogenetics in cells. We describe the competitive interactions between Rac1 regulators and effectors, emphasising the critical role of local protein concentration in maintaining Rac1 activation levels. Our results significantly advance the understanding of Rac1 signalling networks and lay the foundation for future investigations of more complex in vitro reconstituted signalling pathways.