Identifying genes involved in the fission yeast actin integrity stress response

Abstract

All cells must be able to sense environmental insults (stressors) to be able to mount a response that ensures cell survival in such adverse conditions. Conventional stressors include changes in physical and chemical conditions (redox potential, temperature, pressure, pH) and nutrient availability. The response to most stresses across unicellular organisms and individual cells in multicellular organisms is well characterised. It mainly comprises reprogramming of gene expression, which is mediated by highly conserved mitogen-activated protein (MAP) kinase cascades.

However, how cells sense most environmental insults is much less understood. In the fission yeast Schizosaccharomyces pombe (S. pombe), treatment with the actin-depolymerising drug Latrunculin A (LatA) activates Sty1, the MAP kinase in the stress-activated protein kinase (SAPK) pathway. LatA-induced Sty1 activation is dependent on actin depolymerisation, which suggests the existence of an “actinintegrity stress response” (AISR), a pathway by which S. pombe cells can sense and respond to perturbations in their actin cytoskeleton. Nothing is known about AISR either in S. pombe or any other model system.

In the first part of this project, a fluorescence-activated cell sorting (FACS) based genetic screen was developed to identify genes that are involved in “actin-integrity sensing” (AIS) in S. pombe. Two fluorescence reporters of Sty1 activation were constructed by fluorescent-tagging of the proteins Lsd90 and Ddr48, both of which are upregulated in response to a variety of stresses. These reporters enable robust separation of stress and unstressed cells by FACS. These reporters were then used in a two-step FACS-based screen, first isolating cells that do not respond to LatA, and from those, isolating cells that do respond to KCl treatment (salt stress). Isolation of LatA-nonresponding, KCl-responding mutants should enable isolation of AIS mutants, while eliminating any mutations in the “core SAPK pathway”.

In the second part of this project, this approach was used to isolate putative AIS mutants. Fourteen LatA-nonresponding, KCl-responding isolates were isolated. In ten of these isolates, LatA treatment did not lead to actin depolymerisation, which is likely the cause of the LatA-nonresponding, KCl-responding phenotype. From the remaining five mutants, mutations in genes crm1, csx1, and rxt2, were identified as causing the mutant phenotype. These findings lay the groundwork for further understanding of AIS.