The mating response of the yeast Saccharomyces cerevisiae provides an ideal experimental system for studying many fundamental aspects of cell biology, including signal transduction, transcriptional regulation, polarity establishment, and gradient sensing. The response is initiated when a mating-type specific pheromone binds to and activates a G-protein coupled receptor (GPCR) on a cell of opposite mating type. The signal is then propagated by a mitogen activated protein kinase (MAPK) cascade. A key function of the MAPK is to initiate the genetic program required for successful mating. In eukaryotic cells, MAPKs mediate responses to growth factors, cytokines, hormones, cell adhesion, stress and nutrients that determine a wide range of cellular decision processes. The MAPK mediated alterations in transcription induced by pheromone are not sufficient for efficient mating. Cells also must orient growth toward a pheromone gradient emanating from a mating partner, a process referred to as chemotropism. Chemotropism requires that cells establish a front (i.e., polarize) and that this front is maintained during directed growth. Polarity establishment and maintenance are required for migration and differentiation in all eukaryotes, and often become dysregulated in diseases, such as cancer. By combining microfluidics with novel fluorescent reporters, we have developed an experimental system that allows us to track both changes in gene expression and localization of key polarity factors in single cells exposed to time-dependent pheromone concentrations. Integrating our microfluidic platform with mathematical modeling will enable a systems-level understanding of the signaling pathways that regulate transcription, cell cycle arrest, and polarity establishment.
Aim 1 combines computational modeling and experimental analyses using temporally periodic pheromone concentrations to quantify the relative contributions of the multiple signaling motifs that regulate transcription. Quantitative measurements of reporter gene expression in wild-type and mutant backgrounds that perturb regulation will be used to validate or refute the underlying hypotheses of the model.
The aim also tests whether transcriptional persistence following loss of pheromone signal is a form of memory to advance cell cycle arrest and polarization in a second pheromone encounter.
Aim 2 extends this integrated research strategy to dissect the feedback loops that regulate the spatiotemporal dynamics of polarity establishment. Again the experiments will be designed to inform and validate our mathematical models. The ultimate goals of our investigations are to generate truly predictive models of in vivo cellular processes and provide a roadmap for extending research strategies to signaling networks in human cells, allowing the rationale design of new approaches for treating disease.

Public Health Relevance

The mating response of the yeast is an ideal system for studying many fundamental aspects of cell biology, including signal transduction, cell fate decisions, and gradient sensing. In human cells, dysregulation of these processes has been implicated in many diseases, such as cancer and Type II diabetes. By combining computational approaches with the experimental tractability of yeast, this project formulates a strategy for developing and validating predictive models of in vivo cellular processes. If successful, these studies will provide a framework for the rationale design of novel strategies for treating disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM079271-09A1
Application #
8815612
Study Section
Modeling and Analysis of Biological Systems Study Section (MABS)
Program Officer
Lyster, Peter
Project Start
2006-09-30
Project End
2018-08-31
Budget Start
2014-09-30
Budget End
2015-08-31
Support Year
9
Fiscal Year
2014
Total Cost
$439,182
Indirect Cost
$118,071
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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