The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisae Phillip Samayoa 20.309 – Paper Presentation October 9, 2008 The Frequency Dependence of Osmo-Adaptation in Saccharomyces cerevisae Jerome T. Mettetal, Dale Muzzey, Carlos Gómez-Uribe, Alexander van Oudenaarden

Introduction Objective Approach Significance Determine the dominant processes of yeast’s response to osmotic shock Approach Systems-engineering methods Back out a predictive model of signaling dynamics Significance Improved understanding of MAPK’s role in osmotic regulation New approach to developing cellular models Know feedback loops are involved, but their role in signaling dynamics is poorly understood Don’t have to incorporate all reaction of a system to find the dominant ones. Suffers from missing parameters/interactions

High Osmolarity Response Less glycerol export (FPS protein) HOG1 MAPK is transported to the nucleus indication of osmotic stress transcription Measurement of Osmotic Stress HOG1 tagged with YFP Nuclear Protein tagged with RFP (<YFP>nuclear/<YFP>cell)population

Input Stimulus & Measure Output Looking at the cell’s level of stress allows us to see how an input is processed by a cell . Need to figure out the black box that transforms the signal

Fourier Analysis Extracts a Predictive Model \ Example (RC circuit): Input = Vo*sin(wt) Vout = sin(wt)*Vo(1 + iwRC)-1 Transformed data into frequency space after allowing it to reach steady state (shake black box with multiple freqs -> amplitudes) Looking at the data, fit a second order linear time invariant function to model A(w). Found the (complex) parameters that described the model

The Backed out Model can Predict the Cell’s Step Response Shows a good fit to the phase difference as well

The LTI Model can be Converted into a Molecularly defined Model Using linear algebra to pull out a two state differential equation model. Two feedback loops. Now mutant strain with reduced Pbs2 expression to determine relative strength of feedbacks.

Mutant Strain Showed Delayed Short-Term Response Dynamics But known biological details indicate that Hog-1 dependent response by synthesizing glycerol producing proteins is over much longer time scales (~30 min.) -> lack of understanding of MAPK role in osmotic regulation is incomplete (short term component)

Gene Expression Mediates Response Over Longer Time Scales Cells can synthesize proteins Cells can’t synethsize proteins Each subsequent pulse recovered from faster without cyclohexamide. More intense shocks slower also. In the short run, as predicted before, the transcription response isn’t a big factor and cells can respond still.

Summary Engineering principles to predict response of a system Moving Forward Measure state-space variables Cellular networks In more complex systems to better understand structure of network: Such as x, or glycerol concentration