Periodic Reporting for period 1 - CellCycleInVitro (In Vitro Reconstitution of the Minimal Eukaryotic Cell Cycle Oscillator)
Periodo di rendicontazione: 2023-01-01 al 2025-06-30
From a fertilized embryo to a fully developed human body cells divide about 32 trillion times – not accounting for the additional 30 billion cell divisions that cells undergo every single day in order to replenish blood and other tissues. Errors during these cell divisions can result in the gain or loss of genomic information and drive disease development, such as cancer.
Over the last 50 years, researchers have gained incredible insights into the process of cell division. However, the complexity of cell division can be overwhelming. Hundreds – if not thousands – of proteins are involved in ensuring error-free cell division. Until now, studying the cell cycle has often meant to investigate the consequences of removing or interfering with certain parts of this highly complex network and inferring the underlying network structures and functions. However, the various layers of complexity obstruct our view onto the fundamental processes underlying cell cycle regulation.
Our current knowledge suggests that the most basic (and probably most ancient) cell division cycle – stripped of all additional regulatory layers – comprises three essential components – a protein called cyclin B, a protein called cyclin-dependent kinase (Cdk) and a large multi-subunit protein complex called the anaphase-promoting complex/cyclosome (APC/C). These three regulators build a negative feedback loop. Cyclin synthesis activates the cyclin-dependent kinase. The cyclin-dependent kinase activates the APC/C. And finally, the APC/C inactivates cyclin by flagging cyclin for destruction. Thereby the system is reset for a new round of the cell cycle. It is thought, that this time-delayed negative feedback constitutes the core cell cycle clock in every eukaryotic cell and drives cell cycle-dependent processes in an orderly temporal manner. However, whether this simple biological design is indeed capable of producing sustained oscillations has never been tested.
Therefore, we aim to engineer a functional core cell cycle oscillator using the minimal necessary building blocks. Combining state-of-the-art biochemistry, including the powerful frog egg extract system, with synthetic and computational biology we aim to
(1) design novel fluorescence assays that will enable us to follow and optimize the enzymatic reactions of the cell cycle clock in real-time.
(2) develop computational models of the minimal cell cycle clock to efficiently identify the right experimental conditions to make the clock ‘tick’.
(3) eventually purify and assemble all necessary parts of the cell cycle clock to achieve sustained oscillations.
If we can indeed demonstrate that it is possible to build the core cell cycle oscillator from isolated components in the test tube, it will open up numerous possibilities to subsequently carefully construct additional layers of regulation and directly study their impact on the characteristics of cell cycle oscillations, e.g. amplitude, frequency, and robustness, thereby answering 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.
Over the last 50 years, researchers have gained incredible insights into the process of cell division. However, the complexity of cell division can be overwhelming. Hundreds – if not thousands – of proteins are involved in ensuring error-free cell division. Until now, studying the cell cycle has often meant to investigate the consequences of removing or interfering with certain parts of this highly complex network and inferring the underlying network structures and functions. However, the various layers of complexity obstruct our view onto the fundamental processes underlying cell cycle regulation.
Our current knowledge suggests that the most basic (and probably most ancient) cell division cycle – stripped of all additional regulatory layers – comprises three essential components – a protein called cyclin B, a protein called cyclin-dependent kinase (Cdk) and a large multi-subunit protein complex called the anaphase-promoting complex/cyclosome (APC/C). These three regulators build a negative feedback loop. Cyclin synthesis activates the cyclin-dependent kinase. The cyclin-dependent kinase activates the APC/C. And finally, the APC/C inactivates cyclin by flagging cyclin for destruction. Thereby the system is reset for a new round of the cell cycle. It is thought, that this time-delayed negative feedback constitutes the core cell cycle clock in every eukaryotic cell and drives cell cycle-dependent processes in an orderly temporal manner. However, whether this simple biological design is indeed capable of producing sustained oscillations has never been tested.
Therefore, we aim to engineer a functional core cell cycle oscillator using the minimal necessary building blocks. Combining state-of-the-art biochemistry, including the powerful frog egg extract system, with synthetic and computational biology we aim to
(1) design novel fluorescence assays that will enable us to follow and optimize the enzymatic reactions of the cell cycle clock in real-time.
(2) develop computational models of the minimal cell cycle clock to efficiently identify the right experimental conditions to make the clock ‘tick’.
(3) eventually purify and assemble all necessary parts of the cell cycle clock to achieve sustained oscillations.
If we can indeed demonstrate that it is possible to build the core cell cycle oscillator from isolated components in the test tube, it will open up numerous possibilities to subsequently carefully construct additional layers of regulation and directly study their impact on the characteristics of cell cycle oscillations, e.g. amplitude, frequency, and robustness, thereby answering 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.
We have successfully developed two new biosensors, that enable us to follow the changes of the enzymatic activity of one of the major components of the cell clock, CDK1, in real-time. These new biosensors will allow us to efficiently optimize the biochemical reactions involved in activating CDK1 at the right time during the in vitro cell cycle. Furthermore, we have optimized the synthesis of the CDK1-activator cyclin B and can now produce active cyclin B-CDK1 complex in the test tube. This is a major step forward in eventually building the cell cycle clock. Finally, we have purified several other components that are important to make the cell cycle clock work and have made progress in developing a fluorescence assay to monitor the activity of the anaphase-promoting complex/cyclosome - the other crucial player in making the cell cycle clock tick.
If we can demonstrate that the developed biosensors for CDK1 activity can also be used in different cell lines and different organisms beyond the frog egg extract, I expect these to have a significant impact on cell cycle research as they would make the real-time observation of this central enzyme easier and cheaper (e.g. less expensive microscope set-ups and easier image analysis) and therefore more accessible to a large number of laboratories. We anticipate that our efforts in reconstituting the cell cycle clock in the test tube will reveal the detailed mechanism of cell cycle control and open up several new avenues in cell cycle research and beyond.