McClean:Research

Signal Processing by the Yeast Hyperosmotic Response Pathway
How quickly and accurately information propagates through a signaling pathway determines how it functions within the cell. We characterized information propagation through the hyperosmotic response pathway. The osmotic stress signal propagates through the pathway by a series of kinase-dependent phosphorylation reactions, which ultimately lead to the phosphorylation of the Hog1 MAP kinase. In its phosphorylated form, Hog1 is active and localizes to the nucleus where it interacts with various transcription factors and activates the cellular transcriptional response to osmotic stress.

We developed a microfluidic device capable of oscillating input to the pathway over a range of frequencies. At input frequencies up to a critical frequency, called the pathway bandwidth, the pathway activity faithfully reports the input stimulus. At frequencies higher than the bandwidth, the pathway can no longer faithfully transmit the input stimulus. By measuring the frequency-response of pathway activity, as measured by localization of fluorescently tagged Hog1 protein, we were able to characterize the speed at which signals propagate through the pathway. By measuring the signaling bandwidth of this pathway we were able to put bounds on the rates of all biochemical reactions in the cascade. We also studied signal propagation through the pathway in different pathway mutants to understand the contribution of pathway architecture to the speed and function of the pathway. In particular, we found that signaling through one branch of the pathway is twice as slow as the alternate branch.

Measurement of Bandwidth from Transcriptional Responses
We are developing a microfluidic technique, which coupled with fluorescence microscopy, will allow measurement of the bandwidth of the transcriptional response to hyperosmotic stress. Transcription of genes that are upregulated in response to hyperosmotic stress requires transcription factors that are activated by nuclear localized Hog1. How interaction with these transcription factors processes the incoming Hog1 localization signal can be understood by measuring the bandwidth of transcription and translation in response to oscillating input signals. It is interesting to speculate that perhaps the bandwidth of the transcriptional response is smaller than that of Hog1 localization because there is an additional level of signal filtering that occurs downstream of Hog1. Additionally, if pathway bandwidth can be measured on the order of seconds by looking at the relatively slow processes of transcription and translation, then this method for measuring signal processing in vivo can be applied to pathways in which a fast localization reporter does not exist. Consequently, this technique may be useful for understanding temporal ordering of promoter activation and differential regulation at distinct or duplicate promoters. In the near future, we would also like to use these experimental techniques to amplify and identify undetected cross-talk between signaling pathways.

Mutual Inhibition as a Mechanism for Signaling Specificity
Two well-characterized MAP kinase pathways in haploid Saccharomyces cerevisiae yeast are those that mediate cellular response to hyperosmotic pressure and mating pheromone. Despite sharing the same protein and numerous homologous components, the two pathways show specific responses to their given stimuli. Signal insulation by incorporation of pathway components into distinct macromolecular complexes could provide this specificity. On the other hand, mutual inhibition between the pathways could lead to signaling specificity by eliminating unwanted interactions.

We developed two models of signaling specificity in these pathways, one using an insulation mechanism and the other using mutual inhibition between pathway components. This modeling predicted that the mechanisms of mutual inhibition and insulation could be distinguished from one another by measuring the input-output characteristics of the pathways in response to simultaneous mating pheromone and hyperosmotic pressure. Simultaneous input produces output from both pathways if the pathways are insulated from one another (i.e. via scaffolding) and bimodal switch-like output if mutual inhibition mechanisms account for this specificity.

Using cell lines with fluorescently tagged pathway-responsive promoters we performed single-cell measurements that allowed us to distinguish between the two mechanisms of specificity using the model predictions. Cells exposed to both inputs exhibit bimodal and bistable response over a range of inputs. These results indicate that mutual inhibition, and not insulation, is the mechanism for signaling specificity. Furthermore, we showed that destroying the mutual inhibition between the pathways destroys the bistable response.