The role of inositol lipids and inositol phosphates in cellular function. (360G-Wellcome-079194_Z_06_Z)

We plan to improve our knowledge of the physiological functions of inositides by building on what we have learned during the current grant. (i) For inositol lipids we will investigate the localisation, regulation, molecular partners and physiological function of all three of the Type II PtdIns5P 4-kinases (PIPkins) using a combination of cell imaging, immunocytochemistry, biochemistry and knock-in tags in DT-40 cells. (ii) For inositol phosphates we will explore the function of Ins(1,4,5)P3 3-ki nase A and B in post-dendritic spines of hippocampal neurones and in astrocytes respectively, by a range of molecular techniques, principally by transfection with enzyme mutants lacking specific regulatory functions, by imaging their movement and proteolysis, and by calcium imaging either with dyes or with spine-targeted Ca2+ sensors. We will isolate specific physiological interacting proteins for GAP1IP4BP and GAP1m using knock-in tags in DT-40 cells. We will also explore the physiological cont ribution made by Ins(1,4,5)P3 3-kinase to InsP6 synthesis.