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

PLoS Biol. 2020 Jan 24;18(1):e3000600. doi: 10.1371/journal.pbio.3000600. eCollection 2020 Jan.

Abstract

Local activity of the small GTPase Cdc42 is critical for cell polarization. Whereas scaffold-mediated 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Polarity / genetics
  • Feedback, Physiological / physiology
  • Nuclear Matrix / physiology*
  • Optogenetics
  • Organisms, Genetically Modified
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / metabolism
  • Schizosaccharomyces* / genetics
  • Schizosaccharomyces* / metabolism
  • cdc42 GTP-Binding Protein / genetics
  • cdc42 GTP-Binding Protein / metabolism*
  • ras Proteins / physiology*

Substances

  • Schizosaccharomyces pombe Proteins
  • cdc42 GTP-Binding Protein
  • ras Proteins

Grants and funding

This work was supported by an ERC Consolidator grant (CellFusion) and a Swiss National Science foundation grant (310030B_176396) to SGM. AV was funded by the EMBO long-term fellowship ALTF 740-2014. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.