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

Duration/Funding of the PhD: 4 years

Start date of the PhD: 1st October 2015

Main Supervisor: Professor J. Murray and Dr Walter Dewitte (School of Biosciences, Cardiff University)

Second Supervisor: Dr Pete Watson and Professor P. Borri (School of Biosciences, Cardiff University)

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 Fluorescence Resonance Energy Transfer (FRET) sensors to monitor RBR phosphorylation in real time by exploiting our knowledge on RBR phosphorylation and its interactions with E2F, and capitalise on our new analytical confocal system with Fluorescence Lifetime imaging (FLIM) for FRET detection.

Three strategies for in vivo reporting of the G1-S switch will provide synergising information and project failsafes through monitoring RBR phosphorylation, RBR-E2F interaction and E2F transcriptional activation.

(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 human fragments will be used to avoid perturbing the plant controls.

(3) An artificial promoter-reporter monitoring transcriptional activation by E2Fs using E2F binding sites to monitor transcriptional activation by E2Fs. Sensors will be validated in lines with controllable CDK activity, and outputs used to instruct an ODE based mathematical model for cell cycle progression developed in the lab. During the first rotation in the Murray & Dewitte team the student will use quantitative confocal imaging and FRAP analysis of existing fluorescent reporters of the cell cycle. During the second rotation project (Watson & Borri) the student will develop automated image capture and analysis pipelines for cellular markers, across multiple microscopy systems. Both rotations will contribute to the skill set for the PhD project.

Year 1: Engineer genetically encoded CDK sensors and introduce into Arabidopsis.
Year 2: Validate sensors using quantitative imaging.
Year 3: Responses of sensors to stresses; Mathematical modelling of outputs.

Applications are invited from graduates who possess at least 2.1 Honours or Master’s degree in biology, biochemistry, biomedical, genetics or other relevant discipline.

To apply, please email your CV, 2 referees and relevant academic qualifications along with a covering letter to Professor Jim Murray at [Email Address Removed]' AND also submit an online application at the following University's online portal by selecting 'Doctor of Philosophy (Biosciences - BBSRC funded) (October Start): http://www.cardiff.ac.uk/regis/general/applyonline/biosipgr.html

Informal enquiries are also encouraged: please contact Prof Jim Murray at [Email Address Removed]' . For further information, please visit:
http://www.cardiff.ac.uk/biosi/research/molecularbiosciences/index.html