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Onset of CDK2 activity determines passage through the restriction point in primary cells [electronic resource] / Amy Johnson.



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Johnson, Amy.
Publication date:
  • Book, Thesis
  • 1 online resource.
Submitted to the Department of Biology.
Ph.D. Stanford University 2014
The restriction point (R) is crossed when mammalian cells become refractory to stimulatory or inhibitory signals. While it is fundamental to both development and disease, we currently lack an agreement on the molecular basis of the restriction point. Here, we review our current understanding of commitment in yeast and mammals and perform single-cell studies to determine both the timing and molecular mechanism of the restriction point. We show that three cell lines commit to division in previous cell cycles, while two types of primary cells commit solely in G1. Next, we determine the timing and order of molecular events in primary cells using live cell sensors for CDK2 and APC/CCdh1 activity, antibodies for G1 regulators, and a novel live cell E2F1 transcriptional reporter. Our results suggest that while the timing of the restriction point is variable from cell to cell, the first onset of a relatively low level of CDK2 activity is sufficient to commit cells to division, rather than full activation of the positive feedback loop. We demonstrate that CDK2 activation and the onset of E2F1 promoter activity occur at approximately the same time. Taken together, our results support a positive feedback model of commitment where upstream signals drive CDK activity, thus traversing a threshold where positive feedback becomes self-sustaining. Our studies establish a quantitative framework that defines the molecular foundation for G1 restriction point control and will permit further investigation into altered cellular states, such as cancer.
Skotheim, Jan, 1977- primary advisor.
Ferrell, James Ellsworth, advisor.
Morrison, Ashby J., advisor.
Stearns, Tim, advisor.
Stanford University. Department of Biology.

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