Signaling by the Ras/ERK pathway controls cell proliferation, migration, and differentiation. Proper function of this pathway is essential for human development and homeostasis. Sporadic mutations in the pathway play a central role in many cancers, while hereditary mutations cause a series of congenital syndromes, termed RASopathies, which result in impaired cognitive development, cardiac malformations, and increased risk of cancer. A major unanswered question is what differentiates normal from pathological Ras/ERK signaling. It is known that the dynamic pattern of ERK activity - including the strength, frequency, and duration of its activity - are essential to proper signaling. However, the standard methods for measuring ERK activity lack the single-cell precision needed to resolve these essential details. In this project, we will use live-cell imaging, which allows nearl continuous monitoring of thousands of cells simultaneously, to collect data on mutant-driven ERK signaling that is far more accurate and detailed than was previously available. We will focus on the mutations found in the RASopathies, where their role as a single gene driving pathological effects in development is more clearly defined than in cancer, where mutations in many other genes are a complication. We will use our imaging platform to compare for the first time the changes in ERK signaling resulting from disease-causing mutations at the single cell level. Using multiple in vitro systems to replicate cellular processes involved in development, we will determine how these changes modify cell proliferation, migration, and differentiation. We will then dissect the mechanisms underlying these phenotypic changes at the level of gene expression, using a new class of reporters that are integrated directly into the genomes of human cells. At the levels of kinase kinetics, gene expression, and cell behavior, we will quantify how mutant cells respond to multiple Ras pathway inhibitors, which are now being considered as treatments for the RASopathies. This work will have several important outcomes. First, it will reveal the quantitative boundaries of signal behavior that are compatible with normal function, allowing us to understand how Ras pathway mutations lead to disease, and why some mutations are more severe than others. Secondly, it will allow us to make rational choices about which drugs to give to patients with different mutations, so that treatment can be personalized to best normalize each individual's specific signaling patterns. Finally, it will result in a mathematical model of the link between kinase activity and downstream gene expression programs that will allow us to better understand developmental programs and engineer desired cellular responses using existing drugs that target kinase activity.

Public Health Relevance

Human cells use a central communication line known as the Ras pathway to coordinate how they move and divide during development. Severe errors in this pathway are part of the complex genetics of cancer; while a newly discovered set of more moderate errors cause 8 hereditary syndromes termed 'RASopathies'. In this project, we will use a highly sensitive imaging technique that allows us to track Ras activity in single cells to closely examine these moderate communication errors and how they change cellular behavior. This will allow more precise targeting when we use drugs to repair the signaling problems in both cancer and RASopathies.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular and Integrative Signal Transduction Study Section (MIST)
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Melillo, Amanda A
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University of California Davis
Anatomy/Cell Biology
Schools of Medicine
United States
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Albeck, John G; Pargett, Michael; Davies, Alexander E (2018) Experimental and engineering approaches to intracellular communication. Essays Biochem 62:515-524
Pargett, Michael; Albeck, John G (2018) Live-Cell Imaging and Analysis with Multiple Genetically Encoded Reporters. Curr Protoc Cell Biol 78:4.36.1-4.36.19
Gillies, Taryn E; Pargett, Michael; Minguet, Marta et al. (2017) Linear Integration of ERK Activity Predominates over Persistence Detection in Fra-1 Regulation. Cell Syst 5:549-563.e5