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

Abstract

Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffoldmediated positive feedback was proposed to break symmetry of budding yeast cells and produce a single zone of Cdc42 activity, the existence of similar regulation has not been probed in other organisms. Here, we address this problem using rod-shaped cells of fission yeast Schizosaccharomyces pombe, which exhibit zones of active Cdc42-GTP at both cell poles. We implemented the CRY2-CIB1 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 guanine nucleotide exchange factor (GEF) Scd1 and endogenous Cdc42, in a manner dependent on the scaffold protein Scd2. We show that Scd2 function is dispensable when the positive feedback operates through an engineered interaction between the GEF and a Cdc42 effector, the p21-activated kinase 1 (Pak1). Remarkably, this rewired positive feedback confers viability and allows cells to form 2 zones of active Cdc42 even when otherwise essential Cdc42 activators are lacking. These cells further revealed that the small GTPase Ras1 plays a role in both localizing the GEF Scd1 and promoting its activity, which potentiates the positive feedback. We conclude that scaffold-mediated positive feedback, gated by Ras activity, confers robust polarization for rod-shape formation.

Cdc42 GTPase Activating Proteins (GAPs) Maintain Generational Inheritance of Cell Polarity and Cell Shape in Fission Yeast

“…3B). An attractive candidate for this function is the scaffold Scd2, a component of the polarisome, that has an important role in Cdc42 autoamplification (Lamas et al, 2020). Scd2 average distribution correlates with Cdc42 levels and cell growth at the tips: during interphase, Scd2-GFP localizes asymmetrically in monopolar wild-type cells, whereas in bipolar wild-type cells Scd2-GFP is distributed more symmetrically ( Fig.…”

Clustering-mediated activation of Cdc42 GTPase antagonized by GAPs in fission yeast

“…GTP-loading is promoted by two GEFs, Scd1 and Gef1. Scd1, which localizes to cell poles, receives information from the upstream Ras1 GTPase signal and mediates feedback control through the scaffolding activity of Scd2 Kelly and Nurse, 2011;Lamas et al, 2020). For this, Scd1 forms a quaternary complex with Cdc42-GTP, the Pak1 kinase effector and Scd2 (Chang et al, 1994;Endo et al, 2003), which leads in vivo to the positive feedback activation of other Cdc42 molecules, as shown in our recent work using optogenetic strategies (Lamas et al, 2020).…”

“…Scd1, which localizes to cell poles, receives information from the upstream Ras1 GTPase signal and mediates feedback control through the scaffolding activity of Scd2 Kelly and Nurse, 2011;Lamas et al, 2020). For this, Scd1 forms a quaternary complex with Cdc42-GTP, the Pak1 kinase effector and Scd2 (Chang et al, 1994;Endo et al, 2003), which leads in vivo to the positive feedback activation of other Cdc42 molecules, as shown in our recent work using optogenetic strategies (Lamas et al, 2020). The second GEF, Gef1, which localizes to cell poles only in some conditions, promotes Cdc42 activation in response to stress and becomes essential only in absence of Scd1 (Coll et al, transduction (Maxwell et al, 2018;Remorino et al, 2017;Zhang et al, 2001).…”

“…In this work, we used an artificial optogenetic strategy to induce the recruitment and clustering of Cdc42 at the plasma membrane of fission yeast cells. We built on our recent work that used the CRY2-CIB1 optogenetic system to probe the positive feedback of Cdc42 (Lamas et al, 2020). The CRY2-CIB1 system relies on the blue light-induced binding of CRY2PHR (simply denoted CRY2 below) to the Nterminal part of CRY2-binding partner CIB1 (CIBN) (Kennedy et al, 2010).…”

A toolbox of Stable Integration Vectors (SIV) in the fission yeastSchizosaccharomyces pombe

“…However, we refrain from using different constructs to compare between conditions as genomic position effects may become evident only in certain backgrounds (Allshire and Ekwall, 2015). While we present the full vector series for the first time here, some plasmids have been used in published studies (Billault-Chaumartin and Martin, 2019;Gerganova et al, 2019;Lamas et al, 2019;Marek et al, 2019;Vještica et al, 2018) which illustrates their application in fission yeast research.…”