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

Abstract

The 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 scaffoldmediated positive feedback, gated by Ras activity, is minimally required for rodshape formation.

Testing models of cell cortex wave generation by Rho GTPases

“…For example, binding of the Cdc42 GAP Bem2 to the septin scaffold was shown to provide negative feedback to Cdc42 activity in the context of budding yeast polarization 46 . The results presented here also bring into sharper focus the notion of flux of Rho between the cytoplasm and the plasma membrane as a result of the combined action of positive and negative feedback loops, an idea previously only tested for Cdc42 in budding and fission yeast [47][48][49] . Our results suggest that, in the simplified system exploited here, the molecular circuit established by the expression of the GEF Ect2 and the GAP RGA-3/4 is sufficient to drive such flux.…”

Optogenetic modulation of guanine nucleotide exchange factors of Ras superfamily proteins directly controls cell shape and movement

“…All of these alterations could be directly induced, by recruiting either full length GEF proteins or only their GEF domains, within a matter of minutes without allowing signaling or cytoskeletal networks to readjust. Thus, optical control of signaling is an effective approach for spatiotemporal control of cellular signaling (Schwechter et al, 2013;Toettcher et al, 2013;Guntas et al, 2015;Valon et al, 2017;Yang et al, 2018;Lamas et al, 2020;Inaba et al, 2021;De Belly et al, 2023). Opto-SOS, a recruitable RasGEF, induced ERK signaling in Drosophila and uncovered the role of this pathway in promoting endodermal differentiation (Toettcher et al, 2013;Krueger et al, 2019;McFann et al, 2021).…”