We have been studying cell cycle regulation in Xenopus laevis eggs and extracts, carrying out a combination of quantitative experiments and modeling to understand how this biological oscillator works and what features it shares with other regulatory systems. The oscillator circuit includes a positive feedback-based bistable trigger, which opens the possibility that Cdk1 activity might spread through cytoplasm by what are termed trigger waves: self-regenerating activity waves, where some active Cdk1 diffuses a small distance and brings about the activation of the inactive Cdk1 in that volume through the positive feedback, which then diffuses locally and repeats the process. Action potentials are trigger waves; so are calcium waves, as is the spread of a fire through a field or the spread of a joke on the internet. We recently showed that both mitosis and apoptosis spread through Xenopus cytoplasm via trigger waves. We plan to build upon this, and determine: What mechanism underpins the ability of interphase Xenopus cytoplasm to self-organize into cell-like structures? This was an unexpected observation we made in the course of our apoptosis studies, and it is a fascinating phenomenon to try to understand. Is the apoptotic control circuit a bistable system? Our discovery of apoptotic trigger waves suggests that it is, but others have argued that it may not be. We plan to settle the issue with direct experiments. Saltatory vs. continuous mitotic waves. We plan to see whether Cdk1 activation propagates differently close to vs. far from the centrosome, where various pro-mitotic proteins concentrate. Can bistability restrict signals to certain compartments? Theory tells us that a trigger wave may be blocked at the junction between a small tube and a big tube, like a dendrite/axon junction or the base of a primary cilium. We plan to carry out experiments with apoptotic trigger waves in microfluidics devices to test whether this does occur. Do innate immune responses spread via trigger waves? The innate immune system includes positive feedback loops, such as interferon-induced interferon release. This could allow cellular defenses to spread ahead of an infection in a sheet of cells. We plan to test this possibility experimentally. Is there a second mitotic switch? We have found that bistability persists when Cdk1 activity is forced to be graded. Our working hypothesis is that double-negative feedback loops centered on PP2A-B55 are responsible for this bistability. We are testing this hypothesis with hysteresis experiments in extracts. How to proteins that work together stay coordinated as temperature changes, especially in cold-blood organisms? Do enzymes that work together have similar activation energies and Q10 values? And/or has evolution selected particular circuits that tolerate high degrees of parameter variation?

Public Health Relevance

We believe that going beyond describing biological processes to understanding them will require a combination of quantitative experiments and computations. We are taking these approaches to explore how cell regulatory processes can be made robust, switch-like and decisive, and how cellular responses can spread reliably over long distances within big cells and tissues. The biology we are focusing on is cell cycle regulation, programmed cell death, and antiviral defense, but the hope is that the principles that underpin these processes may shed light on many others.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Unknown (R35)
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Special Emphasis Panel (ZRG1)
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Melillo, Amanda A
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Stanford University
Schools of Medicine
United States
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