Small GTPases of the Rho family are well known as master regulators of the actin cytoskeleton. Members of the Rac subfamily are primarily involved in the regulation of cell motility, invasiveness and adhesion, and their dysregulation is associated with the pathogenesis of numerous diseases. Rac GTPases primarily promote the formation of actin-driven cell protrusions, but reports on the effects of Rac deficiency in different cell lineages are inconsistent. Similar to mammals, D. discoideum amoebae express three basic Rac isoforms, but the question of their functional redundancy remains unresolved.

Clockwise rotation of a plasma membrane region enriched in the active small GTPase Rac1 (green) and the opposite region enriched in the effector protein DGAP1 (magenta).

We will address this question by performing a comprehensive phenotypic characterization of newly generated mutant cells expressing pairs or only single Rac1 isoforms. We will focus on processes involving the actin cytoskeleton, such as cell growth, phagocytosis, macropinocytosis, migration, adhesion, chemotaxis, cytokinesis and multicellular development. We will also study the behavior of Rac1-deficient cells in three-dimensional matrices, under compression, against flow and on adhesive surfaces. The disruption of signaling pathways leading to the observed phenotypes will be investigated by localization and interaction studies with Rac1 effectors in mutant cells. The activity of the remaining Rac1 isoforms in double mutants will be investigated by monitoring localization of a fluorescent probe specific for Rac1-GTP.

We have recently discovered that Rac1 activity in D. discoideum cells can exhibit oscillatory dynamics. We will develop elementary mass-conserving deterministic and stochastic reaction-diffusion models of Rac1 dynamics that include only activation, deactivation and diffusion. The comparison between the observed and simulated dynamics of single Rac1 isoforms will alow us to draw conclusions about specific regulatory mechanisms that control the recruitment of Rac1 to, and the release from, the membrane, and their coupling with the activation/deactivation cycle.