Phosphodiesterase involvement in Primary Cilium Formation and Function Driving Ciliopathies

Primary cilia are ubiquitous organelles fundamental for vision, hearing, smell perception, breathing, excretion, reproduction and development and their disruption or loss leads to several diseases (ciliopathies). Primary cilia act as cellular antenna that receive, integrate, and transmit extracellular signals to intracellular compartments via modulation of signalling cascades, including Ca2+ and cAMP pathways. Polycystin-1 (PC1) and polycystin-2 (PC2) are required for induction of calcium transients in response to ciliary bending, however the precise function of the PC complexes on the cilium is a hotly debated and unresolved issue and there are fundamental questions outstanding as to whether changes in Ca2+ and cAMP in the cilia compartment can have the observed global cytoplasmic effects associated with PC1/PC2 mutations. Mutations in PC1 and PC2 cause autosomic dominant polycystic kidney disease (ADPKD), a condition where a complex involving PC1/2: PKA: Akap150: PDE4C has been suggested to drive cyst formation.
The student will use genetically encoded FRET reporters for cAMP that are targeted to specific subcellular sites (including cilia, basolateral membrane and endoplasmic reticulum) and mutagenesis or knock down (siRNA) of PC1/2, in combination with selective pharmacological manipulation of cAMP levels in order to establish how different PC domains modulate compartmentalised cAMP signalling and how this affects cellular function. The impact on compartmentalised cAMP signalling of manipulation of PDE4 activity will be investigated.
The research will be conducted predominantly in the Department of Physiology, Anatomy and Genetics at the University of Oxford and partly in the Mironid laboratories at Strathclyde University, central Glasgow.
The student will be trained in the development of novel targeted cAMP sensors, in their validation and calibration using 'in cell' approaches (for quantitative analysis of cAMP dynamics), as well as in real-time FRET imaging. In addition, the students will gain skills in isolation of primary cells, tissue culture, cell proliferation essays , fluid excretion (MDCK cysts formation essay, gene expression and knock down (siRNA) and general molecular biology and biochemistry techniques (quantitative PCR, immunoprecipitations and western blotting, immunocytochemistry). During the placement with Mironid Ltd. the training will include an understanding of the principles of drug discovery (structure activity relationships, translational relevance of bench science and pharmacokinetic properties required for drug development).