Decoding
cross-talk between HOG and mating pathways in yeast through mathematical modeling
and single-cell analysis

To survive and replicate, cells must
respond to changing extracellular conditions. Cells achieve this by sensing
external stimuli at the membrane and then activating a chain of reactions
through which cells ultimately generate a transcriptional and/or metabolic
response to adapt to the changing environment. Mitogen-activated protein kinase
(MAPK) pathways are an important subset of signaling pathways commonly seen in
eukaryotes. MAPK pathways are made up of 3 sequentially activated kinases where
each kinase is phosphorylated and thus activated by an upstream kinase. MAPK
pathways may have components that are shared between multiple pathways. When
cells are subjected to external stimulus that activates a pathway that shares
components with another pathway, there is a possibility that the signal from that
pathway will ‘leak’ into the other one if the shared components activate the
downstream components of the adjacent pathway. This event of two pathways
interacting and external stimulus of a certain pathway activating a different
pathway is called ‘cross-talk’. Saccharomyces cerevisiae has been well
studied in previous literature as a model organism due to the simplicity of its
pathways and the availability of well-established tools to study them. It is
possible to study the differences in cross-talk between genetically modified
yeast strains in order to understand more about the mechanisms of cross-talk. The
two most well studied signaling pathways in yeast in the context of cross-talk are
the high osmolarity glycerol (HOG) and mating pathways. The HOG pathway is
activated in hyperosmolar conditions and leads to an increase in intracellular glycerol
volume to counteract the cell shrinkage due to the hyperosmolarity. The mating
pathway is activated in the presence of pheromone in the extracellular
environment and leads to shmoo formation. Both pathways share a MAPKKK
(mitogen-activated protein kinase kinase kinase) Ste11. There have been some
studies on the cross-talk between these two pathways and different mechanisms
and components have been suggested to facilitate cross-talk (McClean,
Mody, Broach, & Ramanathan, 2007) (Patterson, Evguenia, & Thorner,
2010). However, a detailed mathematical model to specifically study cross-talk covering
all possible cross-talk mechanisms and cross-talk nodes is yet to be developed.
We have developed a mathematical model incorporating all suggested cross-talk
nodes using a minimal number of estimated parameters to simulate the temporal
concentration profiles of different pathway components. We have studied the
effect of different cross-talk mechanisms and different stress environments on
single cell dynamics using the model. We also performed fluorescence in situ
hybridization (FISH) experiments to investigate the activation of the HOG and
mating pathways in single cells. For our FISH experiments, cells were subjected
to both individual and simultaneous stress. We also varied the stimulus
intensity and, in some cases, examined how the activation of each pathway
changes over time. In each case, the number of transcripts that are strongly
induced by the HOG and mating pathways (namely STL1 and FUS1) in each cell were
measured. Finally, experimentally measured transcript numbers were used to
calibrate and validate the model. Our work shows which of the hypothesized
cross-talk nodes and mechanisms seem to be the most significant. This model allows
for a better understanding of how relatively small changes between strains
allow for different crosstalk properties. It also enables us to better
understand how crosstalk can be context dependent (seen in one condition and
not another) or dependent on the dynamics of stimuli presentation or of
chemical reactions within the pathway.

References

McClean,
M. N., Mody, A., Broach, J. R., & Ramanathan, S. (2007). Cross-talk and
decision making in MAP kinase pathways. Nature Genetics, 409-414.

Patterson,
J. C., Evguenia, K. S., & Thorner, J. (2010). Single-cell analysis reveals
that insulation maintains signaling specificity between two yeast MAPK pathways
with common components. Science signaling.