At the restriction point (R), mammalian cells irreversibly commit to division and will go on to divide even if growth factors are removed. R has mostly been viewed as a point in late G1 just before DNA replication when growth factors activate the kinase ERK to trigger a positive feedback loop of Cdk2 activity that makes progression through the cell cycle irreversible. However, recent single-cell studies cast doubt on this model by placing R much earlier in the cell cycle just after mitosis or even within the previous cell cycle. We developed a single cell assay to find that in primary cells passage through R occurs in G1 at the first passing of a threshold level of Cdk2 activity. While our data identify the threshold for R, we do not understand how growth factor signaling determines the rate of cell cycle progression through G1. Indeed, our preliminary data and reading of the literature suggests the presence of at least one additional, currently uncharacterized, control point (R0) in early G1. Current understanding of the impact of growth factor signaling on R is limited by the fact that only a handful of dynamic profiles of growth factors, such as a single ste increase or a single step decrease or pulse, have previously been investigated. To overcome technical limitations of low-throughput manual media exchange, we have created a microfluidics platform that integrates fluorescence imaging with sharp automated temporal control of the extracellular environment. We will use our microfluidics platform to examine cell cycle progression in mouse primary cells containing a variety of fluorescent reporters for cell cycle progression. This will allow us to test specific hypotheses for the molecular basis of R0 and the rate of progression through G1 to R.
Specific Aims : (1) Use microfluidics to determine how primary cells respond to dynamic proliferation signals, (2) Determine how dynamic mitogen activated protein kinase (MAPK) signals control G1, and (3) Determine how dynamics of cell cycle inhibitors control G1. Cancer relevance: Our proposed systematic investigation of the proliferative response to dynamic growth factor signals will reveal distinct points of regulation within G1 beyond the previously characterized restriction point. This will give insight into normal proliferative control and its misregulation in cancer.

Public Health Relevance

At the restriction point (R), mammalian cells irreversibly commit to division and will go on to divide even if growth factors are removed. We aim to deploy microfluidics and quantitative fluorescence imaging to test specific hypotheses for the molecular basis of R and the rate of progression through G1.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM092925-07
Application #
9133401
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Melillo, Amanda A
Project Start
2010-08-01
Project End
2019-07-31
Budget Start
2016-08-01
Budget End
2017-07-31
Support Year
7
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Stanford University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
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Atay, Oguzhan; Skotheim, Jan M (2017) Spatial and temporal signal processing and decision making by MAPK pathways. J Cell Biol 216:317-330
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Atay, Oguzhan; Doncic, Andreas; Skotheim, Jan M (2016) Switch-like Transitions Insulate Network Motifs to Modularize Biological Networks. Cell Syst 3:121-132
Treutlein, Barbara; Lee, Qian Yi; Camp, J Gray et al. (2016) Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq. Nature 534:391-5
Zatulovskiy, Evgeny; Skotheim, Jan M (2015) Mitosis is swell. J Cell Biol 211:733-5
Doncic, Andreas; Atay, Oguzhan; Valk, Ervin et al. (2015) Compartmentalization of a bistable switch enables memory to cross a feedback-driven transition. Cell 160:1182-95
Atay, Oguzhan; Skotheim, Jan M (2014) Modularity and predictability in cell signaling and decision making. Mol Biol Cell 25:3445-50
Doncic, Andreas; Skotheim, Jan M (2013) Feedforward regulation ensures stability and rapid reversibility of a cellular state. Mol Cell 50:856-68

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