In Vitro Reconstitution of the Minimal Eukaryotic Cell Cycle Oscillator

Cell growth and division are the basis of all forms of life.

In eukaryotes, the clock-like cycles of genome duplication and cell division are orchestrated by a complex set of biochemical reactions.

Theory suggests that a negative feedback loop regulating the cyclin-dependent kinase 1 generates the cell cycle oscillations; however, because the putative cell cycle clock is tightly interlinked with numerous other biological networks, experimental validation has proven to be a challenge.

To date, several questions key to understanding how cell cycle oscillations arise remain unanswered: Do the identified core components indeed give rise to oscillations? Which components are necessary and sufficient? And what makes the cell cycle clock oscillate with varying frequency? To unravel what makes the cell cycle tick, we will reconstitute the core cell cycle oscillator in vitro.

Specifically, combining state-of-the-art biochemistry, including the powerful frog egg extract system, with synthetic and computational biology, we will (1) develop fluorescent-based sensors to monitor key enzymatic activities of the cell cycle oscillator, (2) use computational models to identify operational conditions for the reconstitution of the oscillator, (3) reconstitute and analyse the individual reactions of the predicted negative feedback loop, and (4) assemble the complete cell cycle oscillator in vitro and establish its capacity to oscillate, the set of necessary components, and which elements of the oscillator define its frequency.

Using in vitro reconstitution as a radically new approach to interrogating the cell cycle, CellCycleInVitro will answer longstanding questions about the cell cycle clock.

But more than this, in developing a novel in vitro experimental platform and biosensors for the study of cell division, we will provide powerful new tools with applications ranging from synthetic biology to the development of new therapeutics targeting uncontrolled cell proliferation.