Cell Regulation Group - Stress Responses in Fission Yeast
We use fission yeast as a model system for studying stress responses since there appears to be remarkable conservation involving similar signalling pathways and the mobilisation of closely related transcription factors. All cells sense and react to changes in their environment. Single-celled organisms, in particular, must contend with fluctuations in nutrients, pH, temperature and external osmolarity, as well as exposure to UV irradiation and a range of potentially toxic environmental compounds. Appropriate responses to these environmental stresses must be induced for cell survival and proliferation. A comprehensive characterisation of these responses - the mechanisms involved in sensing stress, the signalling pathways transmitting this information within the cell, and the resulting compensatory changes in physiology and gene expression - is essential to understand how cells adapt and survive under non-ideal conditions. Exposure to low levels of stress often triggers an adaptive response resulting in a transient resistance to higher levels of the same stress. This adaptation to stress can also lead to increased resistance (or cross-protection) to other types of stress. The adaptive response is short-lived and requires new protein synthesis, indicating that changes in gene expression are critical. The phenomenon of cross-protection suggests either that different stress conditions can activate similar defence mechanisms or, more broadly, that there is a general stress response that can confer a basic level of protection.
In the fission yeast Schizosaccharomyces pombe , the Sty1 protein kinase pathway is involved in the regulation of numerous stress responses (Figure 1). The Sty1 kinase is phosphorylated and activated by different stress stimuli and inactivation of the kinase results in pleiotropic stress sensitivity. Thus, Sty1 is predicted to play a key role in mediating a general stress response. Components of this MAPK cascade are homologous to components of the Hog1 osmosensing MAPK pathway in S. cerevisiae and to the mammalian and Drosophila JNK and p38 stress-activated protein kinase cascades.
Two bZip transcription factors, Atf1 and Pap1, co-ordinate many of the changes in gene expression following stress. Atf1 is similar to the mammalian ATF2 factor and Pap1 is similar to cJun. Our studies are focussed on how these factors are regulated, in particular how phosphorylation regulates Atf1 (Figure 5), what target genes they control and how they are differentially mobilised depending upon the nature and magnitude of the stress signals.
Figure 5 - Structure of Atf1
In collaboration with Jurg Bahler at the Sanger Centre in Cambridge , we carried out a comprehensive global microarray analysis of the transcriptional response to oxidative, heat, heavy metal, osmotic and DNA damage stress (Figure 6). This analysis provided a comprehensive overview of cellular responses to environmental stress and insights into how the cell integrates information concerning the state of the environment and orchestrates the expression of the appropriate set of genes. More detailed analysis of the transcriptional changes following oxidative stress has illustrated the need to respond appropriately to the level as well as the nature of the stress. Very different sets of genes are activated at low and high H2O2 stress, coordinated through the differential mobilisation of the Pap1 and Atf1 factors.
Figure 6 - Microarray data showing the core envionmental stress response in fission yeast
Δsty1 and Δpap1 cells are also sensitive to a variety of drugs, probably through their regulation of multidrug resistance-associated transporters. Multidrug resistance is the main mechanism by which many cancers demonstrate resistance to chemotherapeutic drugs and is a major factor in failure of chemotherapy treatment. In many cases the mechanisms involved in developing resistance are poorly understood and accordingly we are using fission yeast as a model system to identify new pathways that could modulate the sensitivity of cells to a range of chemotherapeutic drugs. A screen for drug sensitivity has led to the isolation of a number of mutants that show sensitivity to a variety of drugs.
These mutants are being characterised and are clearly identifying novel pathways that regulate drug resistance. One such mutant led us to identify a kinase that appears to regulate potassium transporters and therefore the plasma membrane potential. The accumulation of the chemotherapeutic drug doxorubicin can be clearly seen in the mutant cells where it accumulates in the vacuoles (Figure 7). In contrast the drug cannot enter wild type cells. Future work will focus on the detailed characterisation of these pathways and investigating whether similar pathways are important in drug resistance in human cells.
Figure 7- Visualisation of drug permeability of the hal4-1 mutant