In Vitro Reconstitution of the Minimal Eukaryotic Cell Cycle Oscillator

Descripción del proyecto

Plataforma de biosensores «in vitro» para el estudio del reloj del ciclo celular

La validación experimental de la regulación de las oscilaciones o el reloj del ciclo celular por la cinasa ciclinodependiente 1 constituye todo un reto. El equipo del proyecto CellCycleInVitro, financiado con fondos europeos, se propone responder a las preguntas acerca del reloj del ciclo celular mediante la reconstitución «in vitro» como método experimental innovador. La combinación de la biología computacional y sintética con la bioquímica más avanzada aplicada al sistema de extracto de huevo de rana recreará el oscilador completo del ciclo celular «in vitro» y establecerá los elementos del oscilador que definen su frecuencia.El desarrollo de una novedosa plataforma de biosensores «in vitro» para el estudio de la división celular proporcionará nuevas herramientas para aplicaciones que abarcan desde la biología sintética hasta el desarrollo de nuevas terapias.

Objetivo

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.