Optogenetics reveals Cdc42 local activation by scaffold-mediated positive feedback and Ras GTPase

Abstract

AbstractThe small GTPase Cdc42 is critical for cell polarization. Scaffold-mediated positive feedback regulation was proposed to mediate symmetry-breaking to a single active zone in budding yeast cells. In rod-shaped fission yeast S. pombe cells, active Cdc42-GTP localizes to both cell poles, where it promotes bipolar growth. Here, we implement the CRY2-CIBN optogenetic system for acute light-dependent protein recruitment to the plasma membrane, which allowed to directly demonstrate positive feedback. Indeed, optogenetic recruitment of constitutively active Cdc42 leads to co-recruitment of the GEF Scd1, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is completely bypassed and positive feedback restored by an engineered interaction between the GEF and a Cdc42 effector, the Pak1 kinase. Remarkably, such re-wired cells are viable and grow in a bipolar manner even when lacking otherwise essential Cdc42 activators. Interestingly, these cells reveal that Ras1 GTPase plays a dual role in localizing and activating the GEF, thus potentiating the feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, is minimally required for rod-shape formation.