New sensors for an old switch; G1-S CDK kinase sensors for higher plants

Cell division in all eukaryotes is triggered and controlled by cyclin-dependent kinases (CDKs) and the molecular switch at cell cycle entry (G1-S phase transition) is conserved between plants and animals. It is based on the inactivation of the tumor suppression protein Retinoblastoma-Related protein during G1 (RBR) by cumulative CDK-mediated hyperphosphorylation. Phosphorylated RBR is unable to sequester transcription factors, such as E2Fs, and mediators of chromatin accessibility. CDK activity is regulated at multiple levels and represents the key convergence point for multiple signals including abiotic stresses, which block cell division causing growth to stop with profound implications for crop yields.
Detailed analysis of the cellular parameters of growth and division and its responses to stress signals requires an in vivo kinase sensor, but none is available for higher plants. We will develop and test novel sensors based on nuclear import and Fluorescence Resonance Energy Transfer (FRET) to monitor RBR phosphorylation in real time by exploiting our knowledge on RBR phosphorylation and its interactions with E2F. We can capitalise on our new analytical confocal system with Fluorescence Lifetime imaging (FLIM) for FRET detection and light sheet imaging for time courses for live monitoring.
Three strategies for in vivo reporting of the G1-S switch will provide synergising information and project fail-safes through monitoring RBR phosphorylation, RBR-E2F interaction and E2F transcriptionalactivation . (1) Using FRET-FLIM, phosphorylation on conserved CDK-target sites in RBR will be monitored on the pocket domain linker (PA-PB) and structural domain linker (PA-RBNB) that induce conformational change and intramolecular interactions. (2) Human RBR fragment-CFP and E2F-YFP pairs will report on RBR-E2F interaction. Non-functional fragments will be used to avoid perturbing the plant controls. (3) An artificial reporter based on nuclear relocation of factors upon phosphorylation during the G1 phase. You will use the Arabidopsis genetic model system for proof of principle, and facilitate introduction into a monocot crop species.

This project is particularly suited to a motivated life scientist with a keen interest in synthetic biology, advanced imaging and molecular engineering.