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Design principles and variability control in a central eukaryotic network

Final Report Summary - SYNTHEYEAST (Design principles and variability control in a central eukaryotic network)

The following section is identical to that submitted in the periodic report, as both reports are being submitted simultaneously due to the early termination of the Marie Curie Career Integration Grant after only 9 months. This early termination (31st of December 2012) results from the obtention of an ERC Starting Grant by the researcher, which started on January 1, 2013.

The Syntheyeast project is based on a synthetic biology approach to eukaryotic cell cycle control using fission yeast as a model system. We set out to study two major questions. First, we aim at uncovering the key processes that constitute core cell cycle regulation; in other words, the minimal set of events that are necessary and sufficient to drive robust cell cycle progression. We proposed to investigate this by generating and characterising a number of minimal synthetic fission yeast strains in which the CDK system, the engine of the cell cycle, is highly simplified (e.g. expression, activity, localisation). Furthermore, we plan to use single-cell analysis and artificial alteration of cell cycle dynamics in our synthetic strains to uncover the importance of such dynamics in sustaining a faithful and reproducible cell cycle. These approaches will allow us to implement mathematical models of this system and extract more general principles linking the architecture of a control circuit and the robustness of the sequence of events it regulates.
Second, we are interested in understanding the mechanisms underlying the buffering of cell-to-cell variability in cell cycle progression, an observation that is common to a large number of eukaryotic systems. By comparing the behaviour of single cells in the various synthetic strains described above and then associating altered variability with specific changes in the control network, we aim at identifying both the major sources of variability inherent to the architecture of the circuit and the regulations that may prevent phenotypic heterogeneity. We also plan to study a potential link between CDK activity dynamics and variability buffering by artificially controlling these activity dynamics using a specific, dose-dependent and reversible method of inhibition. This studies will provide new insights into the eukaryotic systems that have evolved to limit phenotypic heterogeneity.

During the 9 months of support provided by the Marie Curie Career Integration Grant, we have first established the technological platforms that are essential for these projects. We have installed a microfluidic platform within the laboratory and we are now able to design the channel network, produce our microfluidic chips, and then use these devices in live-cell imaging experiments. This involved setting up a standardised workflow, including mask design, photolithography, chip production, cell injection and time-lapse microscopy. We are currently implementing the last aspects of this technology to make this platform optimal for the proposed experiments: microfluidic-based temperature control, testing of other materials for the microfluidic chip that are more compatible with our CDK inhibitor, precise control of the medium flow in the chambers and fast medium switching. Dr. Tong Chen, a post-doctoral fellow in the laboratory, is responsible for these technological developments.
Second, we have reengineered the initial synthetic strain (Coudreuse and Nurse, Nature 2010) in order to optimise our system: we have removed all auxotrophies and have integrated the synthetic CDK module at the cyclin locus in order to make this strain as similar to wild type as possible, except for the CDK control network. Starting with this new background, we are now altering the CDK circuit as planned, deleting additional regulators, implementing an inducible re-localisation system based on the interaction between FRB and FKBP domains (instead of the constitutive re-localisation by direct fusion of the CDK module to targeting domains as initially proposed), and changing the control of the expression of the CDK system. This work is performed by Feriel Baidi, a graduate student in the laboratory. We expect to generate a number of critical strains within the next few months and characterise their phenotypes in detail.
Finally, we have redesigned our strategy to generate a FRET biosensor that will allow us to monitor in real-time and in vivo the activity of the synthetic CDK systems. The standard kinase FRET biosensor we initially planned to use (based on the folding of the sensor upon phosphorylation) presents two major caveats: 1) its specificity to the CDK module may not be sufficiently exclusive and 2) it actually measures the total output of CDK and phosphatase activities rather than CDK activity alone. Furthermore, initial tests with a variety of biosensors we generated (using several FRET pairs) did not provide a sufficient difference in FRET signal between fully active and fully inhibited CDK. We therefore decided to rather follow the activation of the CDK module by monitoring conformational changes that result from its modifications when it is active (phosphorylation of the CDK T-loop and dephosphorylation of an inhibitory tyrosine). This involves inserting the pair of FRET fluorophores appropriately within the CDK module to allow changes in FRET signal upon activation. The design of this system is being investigated in collaboration with Aude Echalier (CBS, Montpellier), who has significant expertise in CDK structure.

Altogether, these projects will bring an unique understanding of the way eukaryotic cells control their cell cycle, taking a synthetic biology approach in fission yeast. Given the high conservation of the core systems involved, our results will be relevant for higher eukaryotes as well. Such questions could not be addressed using standard strategies, and we believe that our work will have important implications for processes that strongly rely on the control of cell proliferation, both in normal situations such as embryonic development and in pathological situations such as cancer.

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